RESUMEN
OBJECTIVES: The primary objective of this research was to assess the activation level of circulating monocytes in patients with unstable angina. BACKGROUND: Markers of systemic inflammatory responses are increased in patients with unstable coronary syndromes, but the activation state and invasive capacity of circulating monocytes have not been directly assessed. METHODS: Peripheral blood mononuclear cell (MC) activation in blood samples isolated from patients with stable and unstable coronary artery disease was measured in two studies. In study 1, a modified Boyden chamber assay was used to assess spontaneous cellular migration rates. In study 2, optical analysis of MC membrane fluidity was correlated with soluble CD14 (sCD14), a cellular activation marker. RESULTS: Increased rates of spontaneous monocyte migration (p < 0.01) were detected in patients with unstable angina (UA) (Canadian Cardiovascular Society [CCS] angina class IV) on comparison to patients with acute myocardial infarction (MI), stable angina (CCS angina classes I to III) or normal donors. No significant increase in lymphocyte migration was detected in any patient category. Baseline MC membrane fluidity measurements and sCD14 levels in patients with CCS class IV angina were significantly increased on comparison with MCs from normal volunteers (p < 0.001). A concomitant reduction in the MC response to activation was detected (p < 0.05). CONCLUSIONS: Using two complementary assays, activated monocytes with increased invasive capacity were detected in the circulation of patients with unstable angina. This is the first demonstration of increased monocyte invasive potential in unstable patients, raising the issue that systemic inflammation may both reflect and potentially drive plaque instability.
Asunto(s)
Angina Inestable/sangre , Angina Inestable/inmunología , Activación de Linfocitos/inmunología , Monocitos/inmunología , Análisis de Varianza , Angina Inestable/clasificación , Angina Inestable/tratamiento farmacológico , Biomarcadores/sangre , Estudios de Casos y Controles , Membrana Celular/inmunología , Movimiento Celular/inmunología , Quimiotaxis de Leucocito/inmunología , Humanos , Inmunohistoquímica , Inflamación , Receptores de Lipopolisacáridos/sangre , Receptores de Lipopolisacáridos/inmunología , Fluidez de la Membrana/inmunología , Infarto del Miocardio/sangre , Infarto del Miocardio/inmunología , Índice de Severidad de la EnfermedadRESUMEN
The serine proteinase granzyme B is crucial for the rapid induction of target cell apoptosis by cytotoxic T cells. Granzyme B was recently demonstrated to enter cells in a perforin-independent manner, thus predicting the existence of a cell surface receptor(s). We now present evidence that this receptor is the cation-independent mannose 6-phosphate/insulin-like growth factor receptor (CI-MPR). Inhibition of the granzyme B-CI-MPR interaction prevented granzyme B cell surface binding, uptake, and the induction of apoptosis. Significantly, expression of the CI-MPR was essential for cytotoxic T cell-mediated apoptosis of target cells in vitro and for the rejection of allogeneic cells in vivo. These results suggest a novel target for immunotherapy and a potential mechanism used by tumors for immune evasion.
Asunto(s)
Apoptosis/efectos de los fármacos , Receptor IGF Tipo 2/metabolismo , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/farmacología , Linfocitos T Citotóxicos/inmunología , Animales , Unión Competitiva/efectos de los fármacos , Trasplante de Células , Células Cultivadas , Citotoxicidad Inmunológica/efectos de los fármacos , Endocitosis/efectos de los fármacos , Citometría de Flujo , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Granzimas , Humanos , Etiquetado Corte-Fin in Situ , Células Jurkat , Riñón/inmunología , Células L , Manosafosfatos/metabolismo , Manosafosfatos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Unión Proteica/efectos de los fármacos , Receptor IGF Tipo 2/antagonistas & inhibidoresRESUMEN
In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin-induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death.
Asunto(s)
Apoptosis/fisiología , Endocitosis , Glicoproteínas de Membrana/fisiología , Serina Endopeptidasas/fisiología , Linfocitos T Citotóxicos/metabolismo , Adenoviridae/fisiología , Animales , Transporte Biológico , Células COS/efectos de los fármacos , Células COS/metabolismo , Compartimento Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Endosomas/metabolismo , Proteínas de Unión al GTP/fisiología , Granzimas , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Células Jurkat/citología , Células Jurkat/metabolismo , Glicoproteínas de Membrana/farmacología , Microinyecciones , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Perforina , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/farmacología , Linfocitos T Citotóxicos/citología , Transfección , Células Tumorales Cultivadas , Proteínas de Unión al GTP rab5RESUMEN
Both thrombotic and inflammatory responses to arterial injury have been implicated in atherosclerotic plaque growth. Calreticulin is a ubiquitous calcium-binding protein with antithrombotic activity and, in addition, is associated with leukocyte activation. We are investigating calreticulin as a potential vascular regulatory protein. The development of intimal hyperplasia was studied at sites of balloon injury in iliofemoral arteries from 91 rats. Calreticulin was infused directly into the artery immediately before balloon injury, and plaque growth was then assessed at 4 weeks' follow-up. Parallel studies of the effects of each calreticulin domain as well as a related calcium-binding protein, calsequestrin, were examined. The effects of calreticulin on platelet activation, clot formation, and mononuclear cell migration were also studied. When infused before balloon injury in rat iliofemoral arteries, calreticulin, or its high-capacity Ca(2+)-binding C domain, significantly reduces plaque development, whereas calsequestrin, a related calcium-binding protein that lacks the multifunctional nature of calreticulin, does not decrease plaque area (saline: 0.037 +/- 0.007 mm2, calsequestrin: 0.042 +/- 0.021 mm2, calreticulin: 0.003 +/- 0.002 mm2, n = 46, P < .04). The N domain and more specifically the P domain, a low-capacity, high-affinity calcium-binding domain in calreticulin, do not reduce intimal hyperplasia (N + P domain: 0.038 +/- 0.012 mm2, C domain: 0.003 +/- 0.002 mm2, n = 45 rats, P < .0001). Calreticulin reduces macrophage and T cell staining in the arterial wall after injury but has no direct effect on monocyte migration in vitro (percent medial area staining positive for macrophage 24 hours after injury (N + P: 4.06 +/- 1.42, calreticulin: 0.273 +/- 0.02; n = 26, P < .009). Calreticulin does, however, reduce platelet-dependent whole blood clotting time, in vitro (baseline: 78.23 +/- 2.04 seconds, calreticulin: 113.5 +/- 1.95 seconds; n = 5, P < .002). We conclude that calreticulin significantly reduces intimal hyperplasia after arterial injury, potentially acting as a vascular regulatory protein.
Asunto(s)
Proteínas de Unión al Calcio/farmacología , Arteria Femoral/lesiones , Arteria Ilíaca/lesiones , Ribonucleoproteínas/farmacología , Túnica Íntima/efectos de los fármacos , Angioplastia de Balón/efectos adversos , Animales , Coagulación Sanguínea/efectos de los fármacos , Proteínas de Unión al Calcio/química , Calreticulina , Calsecuestrina/análisis , Quimiotaxis de Leucocito/efectos de los fármacos , Arteria Femoral/efectos de los fármacos , Arteria Femoral/patología , Humanos , Hiperplasia , Arteria Ilíaca/efectos de los fármacos , Arteria Ilíaca/patología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Monocitos/efectos de los fármacos , Monocitos/patología , Fragmentos de Péptidos/farmacología , Activación Plaquetaria/efectos de los fármacos , Conejos , Ratas , Ratas Sprague-Dawley , Ribonucleoproteínas/química , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/patología , Trombosis/prevención & control , Túnica Íntima/lesiones , Túnica Íntima/patologíaRESUMEN
BACKGROUND: Recurrent atherosclerotic plaque growth, restenosis, is a significant clinical problem after interventional procedures. Initiation of restenosis involves activation of inflammatory and thrombotic cascades, which are regulated by serine proteinase enzymes and inhibitors. We have investigated the use of a viral serine proteinase inhibitor, SERP-1, to reduce plaque development after primary balloon angioplasty. This is the first experimental report of the use of a viral anti-inflammatory protein for the prevention of atherosclerosis. METHODS AND RESULTS: Seventy-four cholesterol-fed rabbits were treated with either local or systemic infusions of SERP-1 protein (or control solutions) after balloon-mediated injury. Sites of SERP-1 infusion in rabbits had dramatically reduced plaque compared with control infusions at the 4-week follow-up. At low-dose infusions (30 to 300 pg), only the primary infusion site had a demonstrable decrease in plaque, whereas at higher-dose infusions (> 3000 pg), a generalized reduction in plaque development was detected. An associated decrease in mononuclear cell infiltration of the arterial wall was detected after SERP-1 infusion within the first 24 hours. Infusion of an active-site mutant of SERP-1 (P1-P1', ala-ala) lacking serine proteinase inhibitory activity failed to prevent plaque growth. CONCLUSIONS: Purified SERP-1, a virus-encoded secreted glycoprotein, reduces plaque growth after primary balloon-mediated injury. Plaque development is decreased by inhibition of serine proteinase activity and is associated with a focal reduction in macrophage infiltration immediately after injury. Investigation of serine proteinase inhibitors may provide new insight into the regulation of arterial responses to injury.