RESUMEN
OBJECTIVE: To determine the effect of endothelin-1 and endothelin-4 on human monocyte production of cytokines. DESIGN: Previous work from our laboratory has shown that endothelin-1 activates leukocytes. Endothelin-1 and endothelin-3 are principally produced by vascular endothelium. However, epidermal cells in gut mucosa, lung, and kidney produce endothelin-2 and endothelin-4, which differ by a single amino acid. While structurally similar to endothelin-1, endothelin-2 and endothelin-4 may affect gut smooth muscle and other tissues differently. The effect of endothelin-1 and endothelin-4 was examined on monocyte production of interleukins (IL) and neutrophil activation factors. SETTING: A clinically-oriented basic science laboratory in a Veterans Administration Hospital and Medical Center. SUBJECTS: Healthy volunteer adult male/female medical students, researchers, and hospital workers. INTERVENTIONS: Human peripheral blood mononuclear cells were separated on density gradients and cultured in media, with or without the addition of bacterial endotoxin or varying molar concentrations of endothelin-1 and endothelin-4. Supernatants were harvested at 10 mins, and at 1, 6, 12, 24, and 48 hrs, and enzyme-linked immunosorbent assays were performed to determine the presence of tumor necrosis factor-alpha, IL-1 beta, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor. MEASUREMENTS AND MAIN RESULTS: Endothelin-1 and endothelin-4 were potent stimuli for monocyte production of tumor necrosis factor-alpha, IL-8, and granulocyte-macrophage colony-stimulating factor. They also caused IL-1 beta and IL-6 production. CONCLUSIONS: Endothelin-1 and endothelin-4 may activate leukocytes after shock or gut ischemia, resulting in further injury to reperfused tissues and distant injury to lungs and other organs.
Asunto(s)
Endotelina-1/fisiología , Endotelinas/fisiología , Interleucina-8/biosíntesis , Interleucinas/biosíntesis , Monocitos/metabolismo , Adulto , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelina-1/farmacología , Endotelinas/farmacología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Interleucina-6/biosíntesis , Masculino , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
PURPOSE: Intimal hyperplasia caused by smooth muscle cell (SMC) proliferation is the major cause of infrainguinal graft failure within the first 12 months. Tobacco smoking is associated with a twofold increase in graft failure within the first year of extremity bypass surgery, but the mechanism is not clearly understood. This study evaluated the effect of nicotine and its major stable metabolite cotinine on vascular SMC proliferation in vitro. METHODS: SMC were harvested from human arteries and grown in culture with standard methods. Cells were seeded at a density of 1.8 x 10(4) cells/well in 24 multiwell dishes and cell cycle-synchronized. Subsequently the SMC were incubated with media containing 0.1% or 15% fetal bovine serum and nicotine or cotinine at concentrations ranging from 10(-9) mol/L to 10(-6) mol/L. Control samples were incubated with corresponding media but without the drugs. SMC proliferation was determined at 4 days with a cell counter. DNA synthesis was assessed at 24 hours with 3H-thymidine uptake. The results were expressed as a percentage change compared with the control samples (mean +/- SEM). Results were analyzed by analysis of variance and t tests. RESULTS: In the presence of serum both nicotine and cotinine at concentrations of 10(-7) and 10(-8) mol/L were mitogenic for SMC in vitro (p < 0.05). A weak mitogenic effect was observed at a low serum concentration for cotinine but not nicotine. Cotinine at a concentration of 10(-9) mol/L, a level seen among passive smokers, was a statistically significant stimulus for DNA synthesis in both minimum serum and serum-supplemented media. At high concentrations both substances were toxic for the cells. CONCLUSION: We have demonstrated a potential role for nicotine and cotinine in the development of intimal hyperplasia and ultimately failure of the vascular reconstruction.
Asunto(s)
Cotinina/efectos adversos , Mitógenos/efectos adversos , Músculo Liso Vascular/efectos de los fármacos , Nicotina/efectos adversos , Análisis de Varianza , Animales , Aorta/citología , Bovinos , Recuento de Células , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , ADN/biosíntesis , ADN/efectos de los fármacos , Sangre Fetal , Supervivencia de Injerto , Humanos , Hiperplasia , Arteria Ilíaca/citología , Músculo Liso Vascular/citología , Fumar/efectos adversos , Timidina/metabolismo , Tritio , Túnica Íntima/citología , Túnica Íntima/efectos de los fármacos , Procedimientos Quirúrgicos VascularesRESUMEN
Endothelin is produced by injured or ischemic endothelium and causes monocyte production of interleukins-6 and 8 in vitro. Endothelin levels increase in patients with burn injuries, and we asked whether interleukin-6 and 8 levels increased in patients with burn injuries concurrently with endothelin. Fourteen patients with more than 20% body surface area burns were resuscitated to maintain urine output of 0.5 to 1.0 ml/kg/hr. Blood was drawn on admission and at 12, 24, and 48 hours. Endothelin was measured by radioimmunoassay, interleukins-6 and 8 were measured by enzyme-linked immunosorbent assay. Endothelin levels increased to 6.1 +/- 2.3 fmol on admission, 5.7 +/- 2.1 at 12 hours, 6.9 +/- 2.7 at 24 hours, and 6.4 +/- 2.7 at 48 hours (vs 0.5 in healthy controls). Interleukin-6 increased to 243 +/- 220 pg/ml on admission, 276 +/- 198 at 12 hours, 400 +/- 282 at 24 hours, and 379 +/- 274 at 48 hours (vs less than 50 in healthy controls). Interleukin-8 increased to 504 +/- 309 pg/ml on admission, 483 +/- 263 at 12 hours, 575 +/- 306 at 24 hours, and 698 +/- 667 at 48 hours (vs less than 50 in controls). Endothelin-1 and interleukin-6 and 8 levels increase in patients with burn injuries. Endothelin-mediated activation of monocytes that cause cytokine production may have clinical relevance in patients with burn injuries.
Asunto(s)
Quemaduras/metabolismo , Endotelina-1/análisis , Interleucina-6/análisis , Interleucina-8/análisis , Adulto , Anciano , Análisis de Varianza , Quemaduras/patología , Endotelina-1/metabolismo , Humanos , Puntaje de Gravedad del Traumatismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Persona de Mediana Edad , Pronóstico , Radioinmunoensayo , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Endothelin-1 (ET-1), a powerful vasoconstrictor, is a 21 amino acid peptide produced by endothelium. It negatively affects pulmonary, cardiac, hepatic, and renal function. It also constricts bronchial and gut smooth muscle. This peptide also stimulates monocytes to produce prostaglandin E2 (PGE2), tumor necrosis factor, interleukin-6 and 8, and substances that stimulate neutrophil superoxide production. Plasma levels of ET-1 also increase in shock, low flow states, ischemia, and sepsis. STUDY DESIGN: Fourteen patients between the ages of seven and 72 years were admitted to the Bridgeport Hospital Burn Unit and resuscitated with a modified Parkland formula. Plasma was drawn on admission, at 12, 24, and 48 hours. Endothelin-1 and PGE2 were measured by radioimmunoassay. RESULTS: Endothelin-1 levels increased ten- to 20-fold in all patients. Prostaglandin E2 levels increased five- to 40-fold in all patients. There was no correlation between plasma ET-1 or PGE2 levels with either size of burn, inhalation injury, patient age, organ dysfunction, or survival in this small study of early burn injury. CONCLUSIONS: The increased plasma ET-1 response in patients with burns may have a role in the genesis of the systemic response to burns. This peptide may also activate monocyte production of PGE2 and other mediators of the systemic inflammatory response syndrome. This study was done to measure ET-1 and PGE2 levels in patients with burns greater than 20 percent of the body surface area on admission, at 12, 24, and 48 hours. The correlations between severity of burn, ET-1 levels, and PGE2 production were also assessed.
Asunto(s)
Quemaduras/sangre , Dinoprostona/sangre , Endotelinas/sangre , Adolescente , Adulto , Factores de Edad , Anciano , Presión Sanguínea/fisiología , Superficie Corporal , Quemaduras/patología , Quemaduras/fisiopatología , Quemaduras/orina , Quemaduras por Inhalación/sangre , Niño , Humanos , Persona de Mediana Edad , Monocitos/metabolismo , Tasa de Supervivencia , Factores de TiempoRESUMEN
OBJECTIVE: To define whether endothelin-1, a peptide produced by injured/ischemic endothelium, has any effect on monocyte intracellular calcium and the production of interleukin (IL)-1 and IL-6. DESIGN: Prospective controlled laboratory study. Human monocytes from healthy donors were assayed for intracellular calcium by fluorometry and were stimulated for 24 hrs in tissue culture with purified endotoxin or endothelin. SETTING: Veterans Affairs Medical Center surgical critical care basic science laboratory. MEASUREMENTS AND MAIN RESULTS: Endothelin-1 increased the intracellular calcium concentration in fura-2 loaded human monocytes to a mean value of 37 +/- 4 nmol. Phytohemagglutinin increased intracellular calcium in control monocytes to a mean value of 97 +/- 12 nmol (n = 15; p < .001). Endothelin had no effect on neutrophil or lymphocyte intracellular calcium. Monocytes incubated with 10(-9) M endothelin significantly increased IL-6 production to values nearly as high as the lipopolysaccharide controls, but did not increase IL-1 production (n = 8; p < .01). CONCLUSION: Endothelin-1 increased intracellular calcium in monocytes and caused production of IL-6.
Asunto(s)
Calcio/metabolismo , Endotelinas/farmacología , Interleucina-6/biosíntesis , Monocitos/metabolismo , Células Cultivadas , Escherichia coli , Fura-2/farmacología , Humanos , Interleucina-1/biosíntesis , Lipopolisacáridos/farmacología , Linfocitos/metabolismo , Neutrófilos/metabolismo , Fitohemaglutininas/farmacología , Estudios Prospectivos , RadioinmunoensayoRESUMEN
Endothelin is a peptide with 21 amino acids that is produced by ischemic or injured endothelial cells. As indicated by in vitro and animal studies, endothelin is also a potent constrictor for renal mesangial and coronary vessels, a bronchoconstrictor, and an endocrine regulator. Our laboratory has shown that endothelin is an agonist for monocyte production of interleukins-1, -6, and -8, prostaglandin E2, and substances that trigger superoxide production. Systemic endothelin levels increase in patients with hypoperfusion and sepsis, indicating that endothelin may be yet another important cytokine in critical illness. Though endothelin has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high endothelin levels. (We hypothesize that this might be due to prostaglandin E2-mediated vasodilation predominating over endothelin-mediated vasoconstriction.) We questioned whether any changes occur in systemic endothelin levels in patients with major burns (> 20%). Ten hemodynamically stable patients resuscitated by a modified Parkland formula to a urine output greater than 30 ml/hr had endothelin levels drawn on admission and at 1, 6, 12, 24, and 48 hours after admission. Endothelin levels were measured by radioimmunoassay. Mean endothelin levels were elevated at 205 +/- 28 fmol/ml at all time points in contrast to levels of 39 +/- 9 fmol/ml in the healthy control group. In summary, systemic endothelin levels increase significantly in patients with major burns. Endothelin may be yet another cytokine playing a significant role in the immune, inflammatory, and multiorgan dysfunction observed with major burns.
Asunto(s)
Superficie Corporal , Quemaduras/sangre , Endotelinas/sangre , Adulto , Anciano , Endotelinas/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , RadioinmunoensayoRESUMEN
Endothelin-1 (ET-1) is a vasoconstrictive peptide released by ischemic/injured endothelium which increases intracellular ionized calcium [Ca2+]i in vascular smooth muscle. Previous work from this lab has shown that ET-1 also increases human peripheral blood monocyte [Ca2+]i, and that 24 h incubation of monocytes with 10(-9) M ET-1 causes production of prostaglandin E2 and interleukin-6. In these studies, ET-1-stimulated monocyte supernatants were evaluated for their effect on neutrophil superoxide production. While ET-1 alone had no direct effect, incubation of neutrophils for 20 min in ET-1-stimulated monocyte supernatants resulted in a 10-fold increase in superoxide production over basal levels, 44% as much superoxide production as induced by peptide N-formyl-methionyl-leucyl-phenylalanine (N = 6, p < .001). Monocyte supernatants were analyzed for interleukin-8 (IL-8 or neutrophil activation protein) content by radioimmunoassay. ET-1-stimulation resulted in production of 54% as much IL-8 as lipopolysaccharide controls (N = 6, p < .001). While a number of monokines can activate neutrophils, IL-8 has been shown to be a potent neutrophil activator as well as a superoxide primer. Therefore, ET-1-treated monocytes probably upregulate neutrophil superoxide production via a mechanism which includes IL-8.
Asunto(s)
Endotelinas/metabolismo , Endotelinas/farmacología , Monocitos/metabolismo , Neutrófilos/metabolismo , Superóxidos/metabolismo , Grupo Citocromo c/análisis , Grupo Citocromo c/metabolismo , Escherichia coli/química , Humanos , Interleucina-8/farmacología , Lipopolisacáridos/farmacología , Monocitos/química , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Factores de TiempoRESUMEN
Impaired blood flow in shock and ischemia results in significant organ dysfunction and failure of critical cellular functions. Although some cellular function can proceed via anaerobic mechanisms, a point is reached at which restoration of blood flow and oxygen delivery does not result in restoration of function ("refractory shock" or the "no-reflow phenomenon"). But even if blood flow is restored after shock or ischemia, a second mechanism of cellular injury is initiated: monocytes and neutrophils are activated, resulting in an inflammatory response. Current evidence suggests that the activation of inflammatory cells is triggered by substances from ischemic or injured vascular endothelium that cause leukocyte adherence, activation, and further injury. This review summarizes the current literature on endothelial cell, monocyte, and neutrophil interactions in reperfusion injury after shock or ischemia and suggests how a recently described peptide from the vascular endothelium may play an important role in the cascade.
Asunto(s)
Endotelio Vascular/fisiología , Isquemia/fisiopatología , Leucocitos/fisiología , Reperfusión , Choque/fisiopatología , Animales , HumanosRESUMEN
The activation and proliferation of a prostaglandin E2 (PGE2) producing monocyte/macrophage may play a major role in down-regulating immune function after injury. But the mechanism by which monocyte or macrophage activation occurs in injury is unknown. Endothelin is a 21-amino acid peptide produced by vascular endothelium in response to ischemia, injury, or endotoxin. Prior work from our laboratory has shown that endothelin increases intracellular calcium in monocytes and causes production of interleukins-6 and -8. In the data reported in this paper, 10(-9) M endothelin stimulated human monocytes to produce 1050 +/- 63 pg/ml of PGE2 at 6 hr and 1328 +/- 47 pg/ml at 24 hr. This was nearly as much PGE2 production as that by bacterial lipopolysaccharide (endotoxin) stimulation (1295 +/- 47 pg/ml at 6 hr and 1506 +/- 94 at 24 hr). Endothelin had no effect on production of leukotriene B4. Endothelin may play an important initiating role in post-traumatic immunosuppression.