RESUMEN
The potential biochemical mechanisms that mediate the 'shedding' of soluble IL-6 RI (80-kDa) receptor fragments in populations of adherent macrophages were explored. Stimulated macrophages displayed proportional increases in both the expression of membrane-associated IL-6 RI (80-kDa) and the release of soluble receptor fragments. The use of protease inhibitors implicated thiol/cysteine and carboxyl/aspartate proteases in this process. Cathepsin-D depleted membrane-associated IL-6 RI (80-kDa) complexes and generated soluble receptor fragments. A carboxyl/aspartate protease from activated macrophages isolated utilizing pepstatin-A affinity chromatography, was also found to affect membrane-associated IL-6 RI (80-kDa) complexes and generate soluble receptor fragments. Most likely, this fraction corresponded to cathepsin-D based upon its origin, pepstatin-A binding avidity, Hb-PAGE zymography, and hydrolysis of an enzyme-specific substrate. We conclude that cathepsin-D can generate soluble fragments of IL-6 RI (80-kDa) expressed by both macrophages and vascular endothelium.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Catepsina D/antagonistas & inhibidores , Endotelio Vascular/metabolismo , Leucina/análogos & derivados , Macrófagos/metabolismo , Receptores de Interleucina-6/metabolismo , Animales , Tampones (Química) , Calcimicina/farmacología , Bovinos , Adhesión Celular , Membrana Celular/metabolismo , Citratos , Inhibidores de Cisteína Proteinasa/farmacología , Femenino , Leucina/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Pepstatinas/farmacología , Citrato de SodioRESUMEN
The aim of this investigation was to identify the potential biochemical mechanisms that alter the integrity of membrane-associated IL-1 RII (decoy) receptor complexes expressed by populations of adherent macrophages and vascular endothelium. The initial research strategy utilized to achieve this objective involved delineating the ability of macrophage activation or exposure of macrophages and vascular endothelium to a spectrum of enzyme proteases to influence the expression of membrane-associated IL-1 RII (decoy) or generate soluble fragments of this receptor complex. Results from these investigations revealed that stimulated macrophages displayed proportional increases in both the expression of membrane-associated IL-1 RII (decoy) and release of soluble receptor fragments. Exposure of macrophages and vascular endothelium to the reference proteases discovered the ability of cathepsin-D to biochemically deplete membrane-associated IL-1 RII (decoy) in addition to generating soluble fragments of this receptor complex. Complementary investigations isolated a carboxyl/aspartate protease from activated macrophages utilizing pepstatin-A affinity chromatography. Exposure of vascular endothelium to pepstatin-A binding proteins resulted in a detectable depletion of membrane-associated IL-1 RII (decoy) and generation of soluble receptor fragments. Evaluation of pepstatin-A binding proteins by SDS-PAGE identified a primary protein fraction with a molecular mass of 47-52 kDa that closely correlates with the known molecular size of leukocyte cathepsin-D fractions. Macrophage pepstatin-A binding protein fractions evaluated by nondenaturing haemoglobin-substrate PAGE (Hb-PAGE) analysis detected a lucent proteolytic band at 47-52 kDa. Macrophage pepstatin-A binding proteins also hydrolyzed a synthetic enzyme-specific substrate that selectively recognizes cathepsin-D biochemical activity. In conclusion, the leukocyte carboxyl/aspartate protease cathepsin-D can biochemically alter the integrity and generate soluble fragments of membrane-associated IL-1 RII (60-kDa decoy) receptor complexes expressed by macrophages and vascular endothelium.
Asunto(s)
Endotelio Vascular/fisiología , Macrófagos/inmunología , Receptores de Interleucina-1/metabolismo , Animales , Catepsina D/farmacología , Bovinos , Adhesión Celular , Membrana Celular/metabolismo , Células Cultivadas , Endopeptidasas/farmacología , Endotelio Vascular/efectos de los fármacos , Femenino , Sustancias Macromoleculares , Macrófagos/efectos de los fármacos , Pepstatinas/metabolismo , Inhibidores de Proteasas/farmacologíaRESUMEN
Moose (Alces alces) found dead (FD) and hunter-killed (HK) in 1995 on the north slope of Alaska (USA) in the Colville River Drainage were evaluated for heavy metal and mineral status. Compared to previous reports for moose and domestic cattle, and data presented here from Alaska moose outside the Colville River area, levels of Cu were determined to be low in hoof, hair, liver, kidney, rumen contents, and muscle for these north slope moose. Iron (Fe) was low in muscle as well. These findings, in conjunction with evidence of poor calf survival and adult mortality prompted investigation of a mineral deficiency in moose (serum, blood, and hair) captured in the spring of 1996 and 1997. Captured males had higher Ca, Zn and Cu levels in hair than captured females. Female moose hair samples were determined to be low (deficient) in Cu, Ca, Fe, and Se with mean levels (ppm) of 2.77, 599.7, 37.4, and 0.30, respectively. Serum Cu level was low, and to a lesser degree Zn was deficient as well. Whole blood (1997 only) was marginally deficient in Se and all animals were deficient in Cu. Based on whole blood, sera and hair, Cu levels were considered low for moose captured in spring 1996 and 1997 in the Colville River area as compared to published data and other populations evaluated in this study. Low levels of ceruloplasmin activity support this Cu deficiency theory. Evidence indicates that these moose are deficient in Cu and other minerals; however, the remote location precluded sufficient examination of animals to associate this apparent deficiency with direct effects or lesions. Renal levels of Cd increased with age at expected levels.
Asunto(s)
Bovinos/fisiología , Cobre/análisis , Ciervos/metabolismo , Cabello/química , Metales Pesados/análisis , Minerales/análisis , Alaska , Animales , Calcio/análisis , Calcio/deficiencia , Bovinos/metabolismo , Enfermedades de los Bovinos/diagnóstico , Causas de Muerte , Ceruloplasmina/análisis , Cobre/deficiencia , Femenino , Estado de Salud , Hierro/análisis , Deficiencias de Hierro , Riñón/química , Hígado/química , Masculino , Músculo Esquelético/química , Selenio/análisis , Selenio/deficiencia , Factores Sexuales , Distribución Tisular , Zinc/análisis , Zinc/deficienciaRESUMEN
Adherent macrophage populations derived from monocytes isolated from peripheral blood were evaluated for their ability to "shed" the membrane-associated receptor for TNF-alpha (TNFR) following exposure to a calcium ionophor (A23187) and a synthetic chemotactic peptide (fMLP) reagent. A soluble fraction of TNFR was detected in "cell-free" supernatant produced by stimulated macrophage populations applying 125I-TNF-alpha and biotinylated TNF-alpha ligand-binding analysis (96-well format) in combination with conventional autoradiographic techniques. Approximate molecular weight of the shed TNFR glycoprotein fraction was estimated to be 75 kDa based on interpretation of nondenaturing PAGE gels transferred laterally onto sheets of nitrocellulose membrane subsequently probed by ligand-binding analysis applying 125I-TNF-alpha and biotinylated TNF-alpha as detection modalities. Immunorecognition techniques were also employed to detect TNFR fragments shed from macrophages using biotinylated anti-TNFR Type II (75 kDa) monoclonal antibody in combination with conjugated strepavidin:HRPO and a chemiluminescent substrate reagent. In an effort to identify the class of enzyme directly mediating TNFR Type II (75 kDa) shedding, a spectrum of carboxyl- (e.g., aspartate), hydroxyl- (e.g., serine), thiol (e.g., cysteine), and metalo- (e.g., Ca2+, Mg2+) protease-inhibiting agents were evaluated. Experimental findings implied that a carboxy (aspartate) peptidase, and possibly to a lesser extent, serine (hydroxyl), and thiol (cysteine) peptidases participate in macrophage TNFR Type II (75 kDa) shedding phenomena. Subsequent investigations demonstrated that the carboxy (aspartate) peptidase cathepsin-D promoted liberation of TNFR Type II (75 kDa) in unactivated populations of adherent macrophages. In an effort to complement these observations, a protein fraction with presumed carboxy (aspartate) protease activity was isolated from the cell-free supernatant generated by activated populations of adherent macrophages using immobilized pepstatin-A beaded agarose. Exposure of unstimulated populations of adherent macrophages to the partially purified pepstatin-A binding protein fractions resulted in the liberation of a soluble TNFR Type II (75 kDa) fragment based on interpretation of ligand-binding and immunorecognition analysis of samples developed by SDS-PAGE/PAGE format and transferred onto sheets of nitrocellulose membrane. The molecular weight of the macrophage pepstatin-A binding protein fraction was estimated to be 47-52 kDa with lesser bands also visible at approximately 26-32 kDa, and 100 kDa based on SDS-PAGE analysis. Nondenaturing hemoglobin-PAGE substrate gel analysis of protein fractions possessing pepstatin-A binding-avidity detected a protease with a molecular weight of approximately 47-52 kDa that proteolytically digested hemoglobin, in addition to a synthetic cathepsin-D specific peptide substrate. Collective interpretation of these experimental findings directly corresponds with many of the physical (molecular) and functional (biochemical) characteristics known to be associated with the leukocyte carboxy (aspartate) peptidase cathepsin-D, which is a non-metaloprotease known to exert relatively limited proteolytic activity.