RESUMO
Several studies indicate that cell-mediated immune responses, i.e., macrophage (MPhi) cytokine release capacities, myosin heavy chain (MHC) class II (Ia) expression, etc., are suppressed after trauma-hemorrhage in male mice. Testosterone has been shown to be responsible for the depression of MPhi cytokine responses in males after trauma-hemorrhage. Antigen presentation via MHC class II plays a key role in initiating and maintaining cell-mediated and humoral immune responses. It remains unknown, however, whether testosterone has any effect on MHC class II after trauma-hemorrhage. To study this, male C3H/HeN mice were castrated or sham castrated 2 wk before trauma (midline laparotomy) and hemorrhage (Hem; blood pressure 35 +/- 5 mmHg for 90 min and resuscitation) or sham operation. Four hours thereafter, MHC class II (Ia) expression was measured using flow cytometry. The results indicate that MHC class II (Ia) expression on peritoneal and splenic MPhi was significantly suppressed in male mice after trauma-hemorrhage. Prior castration, however, prevented the depression in MHC class II (Ia) expression on peritoneal and splenic MPhi after trauma-hemorrhage. Castration did not affect MHC class II (Ia) expression in MPhi from sham-castrated mice. Thus testosterone depresses MHC class II (Ia) expression on peritoneal and splenic MPhi after trauma-hemorrhage in males. Because MHC class II is necessary for an adequate immune response, our results suggest that depletion of male sex steroids or blockade of androgen receptors using agents such as flutamide might prevent immunosuppression via maintaining MHC class II (Ia) expression after trauma and severe blood loss.
Assuntos
Hemorragia/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Macrófagos Peritoneais/imunologia , Orquiectomia , Ferimentos e Lesões/imunologia , Antagonistas de Androgênios/farmacologia , Antagonistas de Receptores de Andrógenos , Animais , Apresentação de Antígeno , Antígeno CD11b/metabolismo , Flutamida/farmacologia , Hemorragia/patologia , Terapia de Imunossupressão , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Receptores Androgênicos/efeitos dos fármacos , Testosterona/fisiologia , Ferimentos e Lesões/patologiaRESUMO
Intact rat soleus muscles or hemidiaphragms were incubated at 37 degrees C for 1 h in 1.0 ml of Krebs-HCO3 buffer containing 10 mM glucose, 5 mM (8-14C)-labelled ATP and (alpha-32P)-labelled ATP together with 5 mM MgCl2 under an atmosphere of 95% O2-5% CO2. Samples of the incubation medium and tissue extract were subjected to electrophoretic separation and the radioactivity present as adenine nucleotides was counted. Extensive degradation of the added nucleotides was observed in the presence of both tissues. The concentrations of 14C-labelled and 32P-labelled ATP found in the muscle and diaphragm indicated that ATP was present within the muscle and diaphragm cells. By maintaining higher concentrations of ATP in the medium, ATP uptake in both tissues exhibited a saturation-type kinetics. These results provide further evidence for intracellular uptake of ATP and also suggest that the transport of ATP into the cell could be a carrier-mediated process.
Assuntos
Trifosfato de Adenosina/metabolismo , Músculos/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Permeabilidade da Membrana Celular , Diafragma/citologia , Diafragma/metabolismo , Inosina Monofosfato/metabolismo , Músculos/citologia , RatosRESUMO
Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Although upregulation of adrenomedullin (ADM), a novel potent vasodilatory peptide, plays an important role in producing cardiovascular responses during the progression of sepsis, it remains unknown whether the clearance of this peptide is altered under such conditions. To determine this, male adult rats were subjected to sepsis by cecal ligation and puncture (CLP) followed by fluid resuscitation. At 5 h (i.e., the hyperdynamic phase of sepsis) or 20 h (the hypodynamic phase) after CLP, the animals were injected with 125I-labeled ADM through the jugular vein. Blood and tissue samples (including the lungs, kidneys, gastrointestinal tract, pancreas, spleen, mesentery, liver, brain, skeletal muscle, heart, and skin) were harvested 30 min after the injection and the radioactivity was determined. The results indicate that there were no significant alterations in tissue [125I]ADM distribution at 5 h after CLP compared to shams. At 20 h after CLP, however, there was a significant decrease in radioactivity in the lungs. In contrast, a significant increase of radioactivity was observed in all other organs except the liver and kidneys. The pulmonary distribution of [125I]ADM was found to be far greater than in any other organs tested, irrespective of the effect of sepsis. In separate groups of animals, injection of [125I]ADM into the left ventricle resulted in a significant decrease in radioactivity in the lungs of both sham and septic animals at 20 h after surgery. These results suggest that the lungs are the primary site of ADM clearance, which is significantly diminished during the late stage of sepsis. The decreased clearance of ADM by the lungs may play an important role in maintaining the sustained levels of plasma ADM under such conditions.
Assuntos
Peptídeos/farmacocinética , Sepse/fisiopatologia , Vasodilatadores/farmacocinética , Adrenomedulina , Animais , Ceco/cirurgia , Ventrículos do Coração , Injeções Intravenosas , Radioisótopos do Iodo , Veias Jugulares , Pulmão/metabolismo , Masculino , Peptídeos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Distribuição Tecidual , Vasodilatadores/administração & dosagemRESUMO
The sympathetic-adrenal system is rapidly activated in response to sepsis or hemorrhagic shock, resulting in an increase in circulating levels of catecholamines. Although it has been shown that the occurrence of hepatocellular dysfunction under such conditions is associated with an early and sustained increase in plasma norepinephrine (NE), it remains unknown whether the increased NE per se plays any role in producing hepatocellular dysfunction. To study this, exogenous NE was administered, by implantation of a peritoneal mini-osmotic pump (consistently releasing NE), to produce a plasma level of NE similar to that observed after sepsis or hemorrhage. At 24 h after the pump implantation, cardiac output was determined by dye dilution technique and hepatocellular function [i.e., the maximal velocity (V(max)) and the efficiency of the transport (K(m)) of in vivo indocyanine green clearance) was assessed without blood sampling. In addition, tissue perfusion in various organs was determined using laser Doppler flowmetry. Plasma levels of interleukin-6 were measured by bioassay and liver enzymes were assayed enzymatically. The results indicate that sustained (24 h) elevation of plasma levels of NE caused a decrease in cardiac output and microvascular blood flow in the liver, spleen, and small intestine. In addition, the increase in plasma NE produced significant depression in hepatocellular function as evidenced by reduced V(max) and K(m). This was associated with elevated plasma levels of liver enzymes, indicating hepatocyte injury. In addition, plasma levels of interleukin-6 increased significantly. These findings suggest that sustained elevated levels of NE, observed after sepsis or hemorrhagic shock, may play an important role in producing hepatocellular dysfunction and altered hepatocyte integrity.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Fígado/efeitos dos fármacos , Norepinefrina/farmacologia , Agonistas alfa-Adrenérgicos/administração & dosagem , Agonistas alfa-Adrenérgicos/sangue , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Peso Corporal , Hemodinâmica/efeitos dos fármacos , Infusões Parenterais , Interleucina-6/sangue , Fluxometria por Laser-Doppler , Fígado/enzimologia , Fígado/fisiopatologia , Masculino , Norepinefrina/administração & dosagem , Norepinefrina/sangue , Ratos , Ratos Sprague-Dawley , Simpatomiméticos/administração & dosagem , Simpatomiméticos/farmacologia , Fator de Necrose Tumoral alfa/análiseRESUMO
Although studies have shown that endothelium-derived nitric oxide (NO) release is depressed during endotoxic shock or polymicrobial sepsis, it remains unknown whether the decreased release of endothelium-derived NO during the hyperdynamic stage of sepsis is due to downregulation of endothelial NO synthase. To study this, adult rats were subjected to sepsis by cecal ligation and puncture (CLP). At 10 h after CLP (i.e., hyperdynamic sepsis) or sham operation, the aorta was removed and a monoclonal antibody against endothelial (constitutive) NO synthase (E-NOS) was used to determine the immunohistochemical presence and electron microscopic localization of E-NOS in rat aortic endothelial cells. Image analysis was used to quantify aortic E-NOS. In additional groups of animals, the aorta was isolated at 10 h after CLP and the vascular responses to an endothelium-dependent vasodilator, acetylcholine, and an endothelium-independent vasodilator, nitroglycerine, were determined. The results indicate that the number of E-NOS negative endothelial cells increased from 7% in shams to 22% in septic animals. E-NOS densely labeled endothelial cells were significantly reduced from 20% to 8% at 10 h after CLP. The E-NOS positive area in aortic endothelial cells was reduced from 26.1 +/- 1.0 microm2/standard frame in sham to 22.3 +/- 0.9 microm2/standard frame in septic animals. Moreover, acetylcholine-induced but not nitroglycerine-induced vascular relaxation was significantly depressed at 10 h after the onset of sepsis. These results, taken together, indicate that the decreased E-NOS in the vascular endothelial cell is at least in part responsible for endothelial cell dysfunction (i.e., the reduced endothelium-derived NO release) observed during the early, hyperdynamic stage of polymicrobial sepsis.
Assuntos
Aorta Torácica/enzimologia , Óxido Nítrico Sintase/metabolismo , Sepse/metabolismo , Animais , Regulação para Baixo , Endotélio Vascular/metabolismo , Técnicas In Vitro , Masculino , Óxido Nítrico/análise , Ratos , Ratos Sprague-Dawley , VasodilataçãoRESUMO
Since administration of ATP-MgCl2 following trauma and hemorrhagic shock improves tissue perfusion as well as cell and organ function, the aim of this study was to determine whether this agent has any salutary effects on the hepatic rate-controlling enzymes specific for gluconeogenesis, such as phosphoenolpyruvate carboxykinase (PEPCK), and for glycolysis, such as pyruvate kinase (PK), under such conditions. To study this, rats underwent a 5-cm midline laparotomy (i.e., trauma-induced) and were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of maximum bleed out volume was returned in the form of Ringer's lactate (RL). The animals were then resuscitated with 3 times the volume of shed blood with RL over 45 min, followed by 2 times RL with ATP-MgCl2 (50 micromol/kg body wt.) or an equivalent volume of normal saline over 95 min. Hepatic PEPCK, PK and glucokinase activities were determined 4 h after the completion of resuscitation. The mRNA levels of PEPCK and PK in the isolated hepatocytes were determined by Northern blot analysis. The results indicate that glucokinase activity was not significantly altered after hemorrhage, irrespective of ATP-MgCl2 treatment. Although the mRNA levels of PEPCK were similar in all groups, PEPCK activity decreased significantly after hemorrhage. ATP-MgCl2 treatment, however, restored PEPCK activity. Hemorrhage resulted in an increase in PK activity and its mRNA. ATP-MgCl2 treatment significantly decreased PK activity and the mRNA. Thus, up-regulation of the gluconeogenic enzyme, PEPCK, and down-regulation of the glycolytic enzyme, PK, by ATP-MgCl2 may be responsible, in part, for the beneficial effects of this agent following trauma-hemorrhage and resuscitation.
Assuntos
Trifosfato de Adenosina/farmacologia , Fígado/enzimologia , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Piruvato Quinase/metabolismo , Ressuscitação , Choque Hemorrágico/enzimologia , Ferimentos e Lesões/enzimologia , Animais , Citosol/enzimologia , Glucoquinase/metabolismo , Gluconeogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Although circulating levels of adrenomedullin (ADM), a newly reported vasodilatory peptide with 52 amino acid residues in the human and 50 amino acid residues in the rat, are elevated during the early and late stages of sepsis, ADM levels in cardiovascular tissues and its precise localization remain to be determined. To study this, rats were subjected to sepsis by cecal ligation and puncture (CLP), followed by administration of 3 ml/100 g b.wt. normal saline to these and sham-operated animals. The heart and thoracic aorta were harvested at 5 h (i.e. the early stage of sepsis) and 20 h (late sepsis) after CLP. Tissue levels of ADM were determined by radioimmunoassay. The localization of ADM in the left ventricle and thoracic aorta was examined by using immunohistochemistry and electron microscopy techniques. The results indicated that ADM levels in the heart and thoracic aorta increased significantly at 5 h after CLP and remained elevated at 20 h after the onset of sepsis. Immunohistochemistry findings showed that ADM immunoreaction products were localized in the cytoplasm of the cardiac myocytes and aortic endothelial cells. Using electron microscopy, ADM immunoreaction products were found in the cytoplasmic matrixes. The immunostainings were also associated with the outer membranes of mitochondria and vesicles of the myocytes as well as vascular endothelial cells. It appears that the cardiovascular tissues, among other organ systems, contribute to the increased levels of plasma ADM under those conditions. Since ADM is localized in different cell populations in the heart and the large blood vessel (i.e. myocytes versus vascular endothelial cells), this peptide may play a differential role in regulating cardiac and vascular functions during sepsis as an autocrine and/or paracrine mediator.
Assuntos
Aorta/metabolismo , Miocárdio/metabolismo , Peptídeos/metabolismo , Sepse/metabolismo , Adrenomedulina , Animais , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Sepse/sangue , Sepse/patologia , Regulação para CimaRESUMO
Although hepatocellular function is depressed early after trauma and hemorrhage (which are associated with low flow conditions and tissue hypoxemia), it remains unknown whether hypoxemia without blood loss, produces hepatocellular dysfunction and, if so, whether IL-6 and PGE2 are associated with this dysfunction. To study this, rats were placed in a plastic box which was flushed with a gas mixture containing 6.3% O2:93.7% N2 or room air for 60 min, followed by their return to room air. At 0 and 4 h after hypoxemia, hepatocellular function (i.e., maximum velocity of indocyanine green clearance (Vmax) and the efficiency of the transport (Km)) was measured using an in vivo hemoreflectometer. Cardiac output was assessed by dye dilution technique. Tissue microvascular blood flow was determined by laser Doppler flowmetry. Plasma IL-6 and PGE2 were measured by bioassay and radioimmunoassay, respectively. The results indicate that hypoxemia produced a depression in hepatocellular function (i.e., decreased Vmax by 44-50% and Km by 55-68%) despite stable cardiac output and hepatic microcirculation at 0 and 4 h after hypoxemia. Moreover, hypoxemia resulted in a significant increase in plasma IL-6 (by 372%-389%) as well as PGE2 (by 38% at 0 h post-hypoxemia). Thus, hypoxemia observed after trauma and hemorrhagic shock appears to be responsible for producing hepatocellular dysfunction possibly through the up-regulation of IL-6 and PGE2. In view of this, long-lasting hypoxemia in trauma victims should be avoided, perhaps by early intubation and ventilation so that the potential additional proinflammatory cytokine and PGE2 release can be prevented.
Assuntos
Dinoprostona/biossíntese , Hipóxia/fisiopatologia , Interleucina-6/biossíntese , Fígado/fisiopatologia , Animais , Velocidade do Fluxo Sanguíneo , Pressão Sanguínea , Débito Cardíaco , Hipóxia/etiologia , Ratos , Choque Hemorrágico/complicações , Regulação para CimaRESUMO
Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Although studies have suggested that endothelins (ETs) contribute to the development of shock after a bolus injection of endotoxin, little is known about the role of ETs in the transition from the hyperdynamic phase to the hypodynamic phase of sepsis. To study this, male adult rats were subjected to sepsis by cecal ligation and puncture (CLP) followed by fluid resuscitation. Plasma levels of ET-1 and ET-2 were measured by radioimmunoassay at 2, 5, 10 h (i.e. the early stage of sepsis), and 20 h (late stage) following CLP or sham operation. Tissue levels of ET-1 and ET-2 were determined in the heart, lungs, small intestine, and spleen at 5 h after CLP or sham operation. In addition, preproendothelin-1 (precursor of ET-1) gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) at 5 h in the heart, lungs, small intestine, spleen, and liver. The results indicate that plasma levels of ET-1 and ET-2 were not different from values of sham groups at 2 and 20 h, but were significantly higher than the sham values at 5 and 10 h after CLP. While there were no significant increases in tissue levels of ET-1 and ET-2 at 5 h post-CLP, RT-PCR analysis indicates a significant upregulation of preproendothelin-1 gene expression in the heart, spleen, and liver (but not in the lungs or small intestine) at 5 h after the onset of sepsis. These results indicate that the heart, spleen, and liver appear to be important ET-producing organs during the early stage of sepsis. The lack of significant increases in tissue ET levels could be due to the possibility that the newly converted peptide is quickly transferred to the bloodstream. Since the hyperdynamic phase of sepsis occurs at 2-10 h and the hypodynamic phase occurs at 20 h after CLP, the increased plasma levels of ET at 5 and 10 h suggest that mediators other than ETs (such as adrenomedullin) are responsible for producing the biphasic hemodynamic responses during the progression of polymicrobial sepsis.
Assuntos
Endotelinas/metabolismo , Hemodinâmica , Sepse/metabolismo , Animais , Ceco/microbiologia , Endotelina-1 , Endotelinas/genética , Masculino , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sepse/microbiologia , Fatores de TempoRESUMO
The effects of exogenous administration of 1 mM [8-14C]ATP-MgCl2 and adenosine-MgCl2 on intracellular accumulation of adenine nucleotides were examined in isolated, perfused rat kidneys. The kidneys were made filtering or non-filtering by increasing the colloid oncotic pressure of the perfusate solution in order to assess the relative contributions of the glomerular and peritubular routes in the uptake of the nucleotides. The results indicate that: although labeled ATP is undetectable in the perfusate after 20 min, there is a significant accumulation of labeled ATP in the tissue and although labeled adenosine-MgCl2 administration also leads to labeled intracellular ATP, the total intracellular ATP is much less than with ATP-MgCl2 administration.
Assuntos
Trifosfato de Adenosina/metabolismo , Adenosina/farmacologia , Rim/metabolismo , Magnésio/farmacologia , Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Técnicas In Vitro , Inosina/metabolismo , Inosina Monofosfato/metabolismo , Rim/efeitos dos fármacos , Cinética , Cloreto de Magnésio , Masculino , Perfusão , RatosRESUMO
Previous studies have shown that adrenomedullin (AM), a potent vasodilatory peptide, is upregulated during sepsis. However, it remains unknown whether the increased AM observed under such conditions is solely due to the elevated levels of circulating lipopolysaccharide (LPS). To determine this, an Alzet micro-osmotic pump, containing a low dose of Escherichia coli LPS or vehicle (sterile normal saline), was implanted in the peritoneal cavity of the normal male adult rat. At 10 h after the pump implantation, samples of blood and small intestine were harvested for the determination of AM by radioimmunoassay. In additional groups, rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). LPS binding agent polymyxin B was administrated intramuscularly at 1 h prior to as well as 5 h after the onset of sepsis. At 10 h after CLP or sham-operation, blood and intestinal samples were harvested and levels of AM were then determined. Plasma levels of LPS were also measured by Limulus amebocyte lysate assay. The results indicate that administration of a low dose of LPS via the peritoneal cavity in normal animals (which did not significantly alter cardiac output, blood pressure or heart rate) markedly increased plasma and intestinal levels of AM. In addition, plasma and tissue levels of AM increased significantly at 10 h after CLP. Administration of polymyxin B, however, attenuated the increase in AM levels under such conditions. Similarly, the increased plasma levels of LPS was significantly reduced by polymyxin B during sepsis. These results, taken together, suggest that the upregulated AM observed during polymicrobial sepsis is at least in part due to the increase in circulating levels of endotoxin.
Assuntos
Lipopolissacarídeos/farmacologia , Peptídeos/metabolismo , Sepse/metabolismo , Adrenomedulina , Animais , Antibacterianos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Débito Cardíaco/efeitos dos fármacos , Ceco/metabolismo , Ceco/microbiologia , Frequência Cardíaca/efeitos dos fármacos , Injeções Intraperitoneais , Lipopolissacarídeos/sangue , Masculino , Peptídeos/análise , Peptídeos/sangue , Polimixina B/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
To determine the effects of glucocorticoids on sugar uptake, xylose uptake by isolated rat soleus muscle of bilaterally adrenalectomized animals was studied. The results indicate that in vitro addition of 10-4 M hydrocortisone, dexamethasone or hydrocortisone sodium succinate had no inhibitory effect on basal xylose uptake. In the presence of both low and high medium insulin, the above steroids failed to inhibit insulin-stimulated uptake. When the concentration of hydrocortisone sodium succinate was increased to 10-2 M, insulinstimulated uptake was decreased. The results thus indicate that glucocorticoids at concentrations observed under physiological or pathological conditions do not inhibit basal or insulin-stimulated sugar uptake.
Assuntos
Dexametasona/farmacologia , Hidrocortisona/farmacologia , Músculos/metabolismo , Xilose/metabolismo , Animais , Transporte Biológico Ativo , Membro Posterior , Insulina/farmacologia , Cinética , Masculino , Músculos/efeitos dos fármacos , RatosRESUMO
Although a burst of immunoresponsiveness may occur during the early stage of sepsis, late sepsis is characterized by severe immunodepression. In addition, although studies have shown that stimulation of macrophage beta-adrenoceptors results in an increase in cAMP and an associated reduction in macrophage phagocytic activity, it remains unknown whether Kupffer cell beta-adrenoceptor characteristics and cAMP levels are altered during polymicrobial sepsis. To study this, Sprague-Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 h (i.e., the early stage of sepsis) or 20 h (late sepsis) after CLP or sham operation, the liver was perfused with collagenase solution and Kupffer cells were isolated. beta-Adrenoceptor characteristics of the isolated Kupffer cells were determined using [125I]iodopindolol, and basal levels of cAMP were measured by radioimmunoassay. The results indicate that while maximum binding capacity (Bmax) of Kupffer cell beta-adrenoceptors was not altered at 5 h, it increased significantly at 20 h after CLP. Similarly, basal levels of cAMP in Kupffer cells did not change at 5 h but increased markedly at 20 h after the onset of sepsis. In contrast, the dissociation constant (Kd, 1/affinity) of Kupffer cell beta-adrenoceptors was not significantly affected by sepsis at both 5 h and 20 h after CLP. Thus, upregulation of beta-adrenoceptors and increase in cAMP levels in Kupffer cells occur during the late stage of polymicrobial sepsis, and this may contribute to the depression of macrophage phagocytic function under such conditions.
Assuntos
AMP Cíclico/análise , Células de Kupffer/metabolismo , Receptores Adrenérgicos beta/metabolismo , Sepse/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Ceco/cirurgia , Masculino , Ratos , Ratos Sprague-Dawley , Sepse/etiologia , Regulação para CimaRESUMO
Although previous studies have demonstrated that plasma levels of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) increase during early sepsis, the precise mechanism responsible for its upregulation remains to be elucidated. Since recent studies have shown that the gut is an important source of norepinephrine (NE) release during early sepsis and enterectomy prior to the onset of sepsis attenuates TNF-alpha production, we hypothesized that gut-derived NE plays a major role in upregulating TNF-alpha via the activation of alpha(2)-adrenoceptors on Kupffer cells. To confirm that NE increases TNF-alpha synthesis and release, Kupffer cells were isolated from normal rats and incubated with NE (20 or 50 nM) or another alpha(2)-adrenergic agonist clonidine (50 nM) without addition of Escherichia coli endotoxin. Supernatant levels of TNF-alpha were then measured. In additional animals, intraportal infusion of NE (20 microM) with or without the specific alpha(2)-adrenergic antagonist yohimbine (1 mM) at a rate of 13 microl/min was carried out for 2 h. Plasma and Kupffer cell levels of TNF-alpha were assayed thereafter. Moreover, the effects of NE and yohimbine on TNF-alpha production was further examined using an isolated perfused liver preparation. The results indicate that both NE and clonidine increased TNF-alpha release by approximately 4-7-fold in the isolated cultured Kupffer cells. Similarly, intraportal infusion of NE in vivo or in isolated livers increased TNF-alpha synthesis and release which was inhibited by co-infusion of yohimbine. Furthermore, the increased cellular levels of TNF-alpha in Kupffer cells after in vivo administration of NE was also blocked by yohimbine. These results, taken together, suggest that gut-derived NE upregulates TNF-alpha production in Kupffer cells through an alpha(2)-adrenergic pathway, which appears to be responsible at least in part for the increased levels of circulating TNF-alpha observed during early sepsis as well as other pathophysiologic conditions such as trauma, hemorrhagic shock, or gut ischemia/reperfusion.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Células de Kupffer/efeitos dos fármacos , Norepinefrina/farmacologia , Receptores Adrenérgicos alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Agonistas alfa-Adrenérgicos/administração & dosagem , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Células Cultivadas , Clonidina/farmacologia , Células de Kupffer/metabolismo , Masculino , Norepinefrina/administração & dosagem , Perfusão , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa/metabolismo , Fator de Necrose Tumoral alfa/análise , Ioimbina/farmacologiaRESUMO
Major burn injury induces T-lymphocyte dysfunction. Previous studies suggest that prostaglandin (PG) E2, which is elevated post-burn, is the causative factor via a cyclic AMP-dependent process. The present study was conducted to elucidate the mechanism by which cAMP induces T-lymphocyte dysfunction following burn injury. Splenocytes were isolated from mice 7 days after receiving a scald burn covering 25% of their total body surface or sham procedure. ConA-induced proliferation by splenocytes from burned mice was significantly suppressed. Macrophage depletion of the splenocyte cultures abrogated the suppression. Concanavalin A-stimulated proliferation by macrophage-depleted splenocytes was suppressed by PGE2 and dibutyryl cAMP in both groups. The IC50 of these cAMP-elevating agents, however, was approximately 100-fold lower for cells from burned mice, indicating an increased sensitivity to cAMP. PGE2 did not suppress PMA/Ca2+ ionophore-induced T-lymphocyte activation. Addition of PMA to ConA-stimulated cultures prevented the suppression of proliferative responses by PGE2, whereas Ca2+ ionophore had no effect. Thus, the suppression of T-lymphocyte activation following burn injury is macrophage-dependent, related to an increased sensitivity to cAMP and due to an uncoupling of cell surface receptors from protein kinase C activation.
Assuntos
Queimaduras/imunologia , AMP Cíclico/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Proteína Quinase C/metabolismo , Linfócitos T/efeitos dos fármacos , Animais , Queimaduras/etiologia , Concanavalina A/antagonistas & inibidores , Dinoprostona/farmacologia , Feminino , Terapia de Imunossupressão , Interleucina-2/biossíntese , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologiaRESUMO
Although studies have shown that the gut is capable of being a cytokine-producing organ and that the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 are upregulated following the onset of sepsis, it remains unknown whether the gut is indeed the major source of the increased cytokine production under such conditions. To determine this, male rats were subjected to cecal ligation and puncture (CLP, a model of polymicrobial sepsis) or sham operation followed by the administration of normal saline solution subcutaneously (i.e., fluid resuscitation). Systemic and portal blood samples were taken simultaneously at 2, 5, 10, or 20 h after CLP or sham operation. Plasma levels of TNF-alpha, IL-1beta, and IL-6 were determined using an enzyme-linked immunosorbent assay. In additional animals, the small intestine was harvested at 10 h after CLP or sham operation and examined for TNF-alpha, IL-1beta, and IL-6 gene expression by RT-PCR. The results indicate that the levels of TNF-alpha, IL-1beta, and IL-6 in both systemic and portal blood samples were significantly elevated during sepsis with the exception that the increase in IL-1beta was not significant at 2 h after CLP. However, there were no significant differences in the levels of those proinflammatory cytokines between systemic and portal blood at any points after the onset of sepsis. Moreover, there were no significant alterations in the proinflammatory cytokine gene expression in the small intestine at 10 h after CLP. Since the levels of TNF-alpha, IL-1beta, and IL-6 were not significantly increased in portal blood as compared to systemic blood and since there was no upregulation of gene expression for these cytokines, it appears that organs other than the gut are responsible for the upregulated proinflammatory cytokines during polymicrobial sepsis.
Assuntos
Citocinas/metabolismo , Intestino Delgado/metabolismo , Sepse/metabolismo , Animais , Citocinas/sangue , Expressão Gênica , Interleucina-1/sangue , Interleucina-1/metabolismo , Interleucina-6/sangue , Interleucina-6/metabolismo , Masculino , Punções , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/etiologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Proteasomes are essential components of the cellular protein degradation machinery. They are nonlysosomal and their participation is critical for (1) the removal of short lived proteins involved in metabolic regulation and cell proliferation, (2) the control of the activities of regulators involved in gene transcription, such as nuclear factor-kappa B (NF-kappa B) and signal transducer and activator of transcription (STAT1), and (3) processing of antigenic peptides for MHC class I presentation. Trauma-hemorrhage induces profound immunosuppression which is characterized by reduced splenocyte proliferation, interleukin (IL)-2 and interferon (IFN)-gamma productive capacity, increased activation of transcription factors NF-kappa B and STAT1 in splenic T lymphocytes, reduced macrophage antigen presentation capacity and inordinate release of proinflammatory cytokines, such as IL-6 and tumor necrosis factor-alpha. Furthermore, it appears that the activity of several regulatory proteins involved in immune function is altered by trauma-hemorrhage. Since proteasomes are involved in regulation and removal of regulatory proteins, we hypothesized that trauma-hemorrhage alters proteasomal activity in splenic lymphocytes. The data showed that activities of 26s proteasome from CD3+CD4+ and CD3+CD8+ splenic T lymphocytes were enhanced following trauma-hemorrhage which was associated with increased expression of NF-kappa B and STAT1. On the other hand, trauma-hemorrhage attenuated the activity of 26s proteasome from splenic B lymphocytes which was restored upon IFN-gamma stimulation and correlated with increased expression of NF-kappa B. These studies indicate a potential role for proteasomes in the regulation of signal transduction in splenic T and B lymphocytes following trauma-hemorrhage, and also suggest them as potential therapeutic targets for attenuation of immune suppression associated with this form of injury.
Assuntos
Linfócitos B/metabolismo , Hemorragia/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Animais , Proteínas de Ligação a DNA/biossíntese , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Hemorragia/etiologia , Janus Quinase 1 , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , NF-kappa B/biossíntese , Proteínas Tirosina Quinases/biossíntese , Fator de Transcrição STAT1 , Transativadores/biossíntese , Ferimentos e LesõesRESUMO
The elevation of soluble Fas (sFas) in the sera of patients with liver disease suggests a role for sFas in the disease process; whether it is protective or not is controversial. To determine the effects of sFas on Fas-induced liver apoptosis, we manipulated mice to produce sFas by transfecting them in vivo with different amounts of an adenovirus that produces mouse sFas driven by the CMV promoter (AdsFas). Fas-mediated apoptosis was induced by administration of anti-mouse Fas (Jo2; 10 microg/mouse) one week later. The administration of AdsFas (10(3), 10(7), or 10(9) pfu/mouse), which was associated with only minimal side-effects, resulted in a significant reduction in the liver transaminase levels and mortality of the mice on challenge with Jo2, as compared to control mice treated with AdLacZ. However, the protective effect of AdsFas was not complete. The possibility that Jo2-induction of TNF-alpha in the Kupffer cells of the liver contributes to the pathology was therefore tested. Although administration of soluble TNF receptor (sTNFRI) alone did not protect the mice from the lethal effects of Jo2, administration of sTNFRI (200 microg/mouse) after infection with AdsFas (10(9) pfu/mouse) resulted in 100% survival of the mice on challenge with Jo2. To confirm that the production of TNF-alpha by Kupffer cells produce the lethal effects of Jo2 that remained after treatment with AdsFas, these cells were selectively ablated by treatment of the mice with gadolinium chloride prior to challenge with Jo2. This treatment greatly reduced early mortality and hepatocellular damage as well as TNF-alpha production 6 h after injection of Jo2. These results indicate that: (1) AdsFas prevents Jo2-induced apoptosis of hepatocytes; (2) In addition to mediating Fas-mediated apoptosis of hepatocytes, Jo2 can separately induce TNF-alpha production by Kupffer cells resulting in early mortality, and (3) Optimal protection from Jo2-induced mortality can be achieved by protection of liver cells by pretreatment with both AdsFas and sTNFRI.
Assuntos
Apoptose , Terapia Genética , Hepatócitos/metabolismo , Células de Kupffer/metabolismo , Fígado/patologia , Receptor fas/genética , Receptor fas/fisiologia , Animais , Anticorpos/imunologia , Feminino , Gadolínio/metabolismo , Hemorragia/etiologia , Hemorragia/prevenção & controle , Hepatite/etiologia , Hepatite/prevenção & controle , Imuno-Histoquímica , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Solubilidade , Fator de Necrose Tumoral alfa/biossíntese , Receptor fas/sangue , Receptor fas/imunologiaRESUMO
Despite significant advances in the management of trauma victims, traumatic injury with the ensuing sepsis and multiple organ failure remains the leading cause of death between the ages of 18 and 44 in the USA. Recently, interest in the clinically and experimentally observed gender dimorphic response to traumatic injury has led to the possibility of modulating cell and organ functions following trauma and hemorrhagic shock by the administration of sex steroids. Here, we review the effects of the adrenal steroid dehydroepiandrosterone (DHEA), a precursor of sex steroid synthesis, on organ and immune functions following trauma-hemorrhage, and its potential as a novel therapy for improving the depressed cell and organ functions in trauma patients.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Desidroepiandrosterona/uso terapêutico , Hemorragia/tratamento farmacológico , Ferimentos e Lesões/tratamento farmacológico , Humanos , Masculino , Modelos Biológicos , Ferimentos e Lesões/imunologiaRESUMO
Studies have shown that cell-mediated immunity is markedly suppressed after thermal injury. T lymphocyte dysfunction and macrophage hyperactivity have been implicated as causative factors. Previous studies have primarily examined the effects of thermal injury on alphabeta T lymphocytes; however, the role of gammadelta T lymphocytes in the immune response after thermal injury is unclear. Therefore, wild-type mice and mice lacking the TCR delta gene (TCR delta-/-) were subjected to a third-degree scald burn and cell-mediated immune responses assessed at 7 days post-injury. TCR delta-/- mice had 75% mortality after burn injury compared with 25% mortality in the wild-type group. Plasma interleukin-6 (IL-6) levels were significantly elevated at 2, 4, and 18 h post-injury, whereas no difference was observed in tumor necrosis factor alpha (TNF-alpha) and prostaglandin E2 (PGE2) plasma levels. Plasma levels of these inflammatory mediators were similar in wild-type and TCR delta-/- mice post-injury. Splenic macrophage PGE2, IL-6, TNF-alpha, and IL-10 production was significantly increased in wild-type mice at 7 days post-injury, whereas macrophages from injured TCR delta-/- mice had a significantly attenuated capacity to produce IL-6 and TNF-alpha. In contrast, the increased release of PGE2 and IL-10 by macrophages post-injury was not reduced in TCR delta-/- mice. These results implicate a dual role for gammadelta T lymphocytes in the immunopathogenic response to burn injury: (1) they contribute to survival from the insult; and (2) they mediate the induction of a pro-inflammatory macrophage phenotype at 7 days post-injury. Thus, gammadelta T lymphocytes, in part through the modulation of macrophage activity, appear to contribute to the immune dysfunction after thermal injury.