RESUMEN
Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.
Asunto(s)
Apoptosis , Permeabilidad de la Membrana Celular , Membrana Celular/química , Granzimas/química , Complejos Multiproteicos/química , Perforina/química , Anticuerpos Neutralizantes/química , Membrana Celular/metabolismo , Humanos , Células Jurkat , Necrosis/metabolismo , Transporte de ProteínasRESUMEN
Granzymes (gzms) are key components of T-killer (Tc) cells believed to mediate pro-apoptotic activities. Recent evidence suggests that gzms also possess non-cytotoxic activities that contribute to host defense. In this study, we show that Tc cells from lymphocytic choriomeningitis virus (LCMV)-infected wild-type (wt) and gzm A/B-deficient mice express similar levels of gzmK protein, with both mouse strains efficiently controlling infection. GzmK, in recombinant form or secreted by ex vivo-derived LCMV-immune gzmAxB(-/-) Tc cells, lacks pro-apoptotic activity. Instead, gzmK induces primary mouse macrophages to process and secrete interleukin-1ß, independent of the ATP receptor P2X(7). Together with the finding that IL-1Ra (Anakinra) treatment inhibits virus elimination but not generation of cytotoxic Tc cells in wt mice, the data suggest that Tc cells control LCMV through non-cytotoxic processes that involve gzmK.
Asunto(s)
Infecciones por Arenaviridae/inmunología , Granzimas/metabolismo , Virus de la Coriomeningitis Linfocítica , Linfocitos T Citotóxicos/inmunología , Animales , Infecciones por Arenaviridae/enzimología , Granzimas/deficiencia , Granzimas/genética , Mediadores de Inflamación/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1beta/metabolismo , Virus de la Coriomeningitis Linfocítica/efectos de los fármacos , Virus de la Coriomeningitis Linfocítica/genética , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Linfocitos T Citotóxicos/enzimologíaRESUMEN
The objective of this work was to determine if Escherichia coli methionine bioassay characteristics were influenced by selective media amended with antibiotics and the antifungal compound cycloheximide. Bacterial cells were grown in minimal media with increasing concentrations of methionine and were incubated at 37 degrees C with vigorous agitation for 6 hours. Addition of antistatic agents to the media did not change the growth kinetic response (P>0.05) to methionine concentration (3.4 to 26.8 microM). This supports the utility of this strain as a methionine bioassay organism for feed and other environmental sources.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Cicloheximida/farmacología , Escherichia coli/crecimiento & desarrollo , Metionina/metabolismo , Alimentación Animal , Bioensayo , Medios de Cultivo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , CinéticaRESUMEN
The antitumor effect of T cells is executed either through CD95 or Perforin (PFN)/Granzyme B (GrB) pathways. Induction of apoptosis by either mode requires activation of caspase family members. However, recent studies have suggested that cell death can proceed in the absence of caspase induction and apoptotic events. We investigated the contribution of CD95 and PFN/GrB-mediated cytotoxicity to apoptotic and necrotic mechanisms of cell death in human renal cell carcinoma. Although freshly isolated and cultured tumors expressed CD95 on their surface, they were resistant to CD95-mediated apoptosis. CD95 resistance coincided with decreased levels of FADD protein and diminished caspase-3-like activity. In contrast, we demonstrated that tumor cell death mediated by PFN/GrB can be achieved in the absence of functional caspase activity and is accompanied by a dramatic accumulation of nonapoptotic necrotic cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis/efectos de los fármacos , Carcinoma de Células Renales/patología , Linfocitos T Citotóxicos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/metabolismo , Proteínas Portadoras/metabolismo , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Resistencia a Antineoplásicos , Activación Enzimática/efectos de los fármacos , Proteína Ligando Fas , Proteína de Dominio de Muerte Asociada a Fas , Granzimas , Humanos , Células Jurkat , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacología , Necrosis , Perforina , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidasas/farmacología , Células Tumorales Cultivadas , Receptor fas/inmunología , Receptor fas/metabolismoRESUMEN
HSV-1 inhibits apoptosis of infected cells, presumably to ensure that the infected cell survives long enough to allow completion of viral replication. Because cytotoxic lymphocytes kill their targets via the induction of apoptosis, protection from apoptosis could constitute a mechanism of immune evasion for HSV. Several HSV genes are involved in the inhibition of apoptosis, including Us5, which encodes glycoprotein J (gJ). Viruses deleted for Us5 showed defects in inhibition of caspase activation after Fas ligation or UV irradiation. Transfected cells expressing the Us5 gene product gJ were protected from Fas- or UV-induced apoptosis, as measured by morphology, caspase activation, membrane permeability changes, or mitochondrial transmembrane potential. In contrast, caspase 3 activation in mitochondria-free cell lysates by granzyme (gr)B was inhibited equivalently by Us5 deletion and rescue viruses, suggesting that gJ is not required for HSV to inhibition this process. However, mitochondria-free lysates from transfected cells expressing Us5/gJ were protected from grB-induced caspase activation, suggesting that Us5/gJ is sufficient to inhibit this process. Transfected cells expressing Us5/gJ were also protected from death induced by incubation with purified grB and perforin. These findings suggest that HSV has a comprehensive set of immune evasion functions that antagonize both Fas ligand- and grB-mediated pathways of CTL-induced apoptosis. The understanding of HSV effects on killing by CTL effector mechanisms may shed light on the incomplete control of HSV infections by the immune system and may allow more rational approaches to the development of immune modulatory treatments for HSV infection.
Asunto(s)
Apoptosis , Inhibidores de Caspasas , Serina Endopeptidasas/farmacología , Simplexvirus/patogenicidad , Proteínas del Envoltorio Viral/farmacología , Receptor fas/fisiología , Animales , Chlorocebus aethiops , Activación Enzimática , Granzimas , Herpesvirus Humano 1/patogenicidad , Herpesvirus Humano 2/patogenicidad , Humanos , Células Jurkat , Cinética , Transfección , Células Vero , Proteínas del Envoltorio Viral/genéticaRESUMEN
Cytotoxic lymphocytes may induce apoptosis in their target cells by the FasL (Fas ligand) pathway or the perforin/granzyme B pathway. It has been shown that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-mediated apoptosis. The aims of this study were to determine whether CC cells are also resistant to perforin/granzyme B and whether the FasL resistance lies upstream of caspase-3 activation. The resistance of the Fas-expressing rat CC531s cells to the FasL pathway was confirmed by treating them with recombinant human soluble FasL, using rat hepatocytes as a positive control. The intracellular delivery of granzyme B by sublytic concentrations of perforin, on the other hand, resulted in many features of apoptosis (chromatin condensation, nucleus fragmentation, loss of microvilli and internucleosomal DNA fragmentation) within 3 h. Since both the FasL and perforin/granzyme B pathways converge at caspase-3, we measured caspase-3 activity to learn whether the FasL resistance was due to failure to activate this crucial executioner. Caspase-3 activation occurred in CC531s cells after perforin/granzyme B treatment, but not after the addition of recombinant FasL. Furthermore, we showed that caspase-3 activity is involved in the execution of perforin/granzyme-B-induced apoptosis in CC531 s cells, since the cell-permeable caspase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these results suggest that CC cells are sensitive to perforin/granzyme-B-induced apoptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias del Colon/patología , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacología , Serina Endopeptidasas/farmacología , Bencimidazoles , Caspasa 3 , Caspasas/metabolismo , Neoplasias del Colon/metabolismo , ADN de Neoplasias/análisis , Resistencia a Antineoplásicos , Proteína Ligando Fas , Granzimas , Humanos , Perforina , Proteínas Citotóxicas Formadoras de Poros , Propidio , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
BACKGROUND: Cutaneous disease is commonly encountered in primary care. The frequency of patients presenting to primary care physicians with skin disease and their eventual disposition is not well studied. OBJECTIVE: The purpose of this study was to determine the prevalence of patients seen with skin disease in a primary care setting and the likelihood of their referral to a dermatologist. The impact the primary care provider had on the quality of skin care was also examined. METHODS: A retrospective chart review was performed of patients seen during a 2-year period at a general medicine clinic within the University of Miami and upon referral to a University of Miami dermatology office. Data were obtained on the prevalence of skin disease, dispositions of referral, diagnoses made, and procedures performed. RESULTS: During a 2-year period, 36.5% of patients who presented to their primary care physician had at least one skin problem. Of 208 patients with skin disease, in 58.7% (122/208) it was their chief complaint. A wide range of diagnoses were made by the primary care physician, with a limited number of diagnostic procedures performed. Of the 37.5% of patients referred to a dermatologist, 68% were referred on initial evaluation. Diagnoses made by the primary care physician were concordant with that made by the dermatologists 57% of the time. CONCLUSION: Patients frequently see their primary care physician for skin disease. A large percentage are referred to dermatologists, often for a biopsy of a suspect lesion, to confirm a suspected diagnosis, or to establish a diagnosis of lesions of unknown origin.
Asunto(s)
Atención Primaria de Salud/estadística & datos numéricos , Enfermedades de la Piel/diagnóstico , Enfermedades de la Piel/terapia , Adulto , Dermatología , Femenino , Humanos , Masculino , Derivación y Consulta , Estudios RetrospectivosRESUMEN
Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Apoptosis/inmunología , Citotoxicidad Inmunológica , Transducción de Señal/inmunología , Antígenos de Diferenciación de Linfocitos T/toxicidad , Apoptosis/efectos de los fármacos , Grupo Citocromo c/metabolismo , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/inmunología , Membranas Intracelulares/metabolismo , Células Jurkat , Células Asesinas Naturales/inmunología , Lípidos de la Membrana/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/inmunología , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaAsunto(s)
Citometría de Flujo , Glicoproteínas de Membrana/metabolismo , Proyectos de Investigación , Membrana Celular/química , Permeabilidad de la Membrana Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Resistencia a Medicamentos , Humanos , Líquido Intracelular/química , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Células K562/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/farmacología , Modelos Biológicos , Perforina , Proteínas Citotóxicas Formadoras de Poros , Unión Proteica , Manejo de Especímenes , Coloración y Etiquetado , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
P-glycoprotein (P-gp) is an ATP-dependent drug pump that confers multidrug resistance (MDR). In addition to its ability to efflux toxins, P-gp can also inhibit apoptosis induced by a wide array of cell death stimuli that rely on activation of intracellular caspases for full function. We therefore hypothesized that P-gp may have additional functions in addition to its role in effluxing xenotoxins that could provide protection to tumor cells against a host response. There have been a number of contradictory reports concerning the role of P-gp in regulating complement activation. Given the disparate results obtained by different laboratories and our published results demonstrating that P-gp does not affect cell death induced by another membranolytic protein, perforin, we decided to assess the role of P-gp in regulating cell lysis induced by a number of different pore-forming proteins. Testing a variety of different P-gp-expressing MDR cell lines produced following exposure of cells to chemotherapeutic agents or by retroviral gene transduction in the complete absence of any drug selection, we found no difference in sensitivity of P-gp(+ve) or P-gp(-ve) cells to the pore-forming proteins complement, perforin, or pneumolysin. Based on these results, we conclude that P-gp does not affect cell lysis induced by pore-forming proteins.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Supervivencia Celular/fisiología , Glicoproteínas de Membrana/fisiología , Anticuerpos/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/toxicidad , Resistencia a Múltiples Medicamentos , Humanos , Células K562 , Cinética , Leucemia de Células T , Glicoproteínas de Membrana/farmacología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores de IgG/fisiología , Receptores de Transferrina , Proteínas Recombinantes/metabolismo , Rubidio/farmacocinética , Transfección , Células Tumorales Cultivadas , Vincristina/toxicidadRESUMEN
MCF-7, a breast cancer-derived cell line, is deficient of caspase 3 and relatively insensitive to many chemotherapeutic agents. To study the association of caspase 3 deficiency and chemotherapeutic resistance, we reconstituted caspase 3 in MCF-7 cells and characterized their apoptotic response to doxorubicin and etoposide. Western blots demonstrated that caspase 3 was constitutively expressed in the reconstituted MCF-7 cells. Both morphological observation and survival assays showed that caspase 3 reconstitution significantly sensitized MCF-7 cells to both drugs. Remarkably increased activation of caspases 3, 6, and 7, cleavage of cellular death substrates, and DNA fragmentation were detected in the reconstituted MCF-7 cells after drug treatment. Together, these data demonstrated a specific role for caspase 3 in chemotherapy-induced apoptosis and in activation of caspases 6 and 7. Our results also suggest that caspase 3 deficiency may contribute to chemotherapeutic resistance in breast cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/enzimología , Caspasas/fisiología , Doxorrubicina/farmacología , Etopósido/farmacología , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/fisiología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Caspasa 3 , Caspasas/biosíntesis , Caspasas/genética , Caspasas/metabolismo , Núcleo Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática , Humanos , Concentración 50 Inhibidora , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To investigate the cartilage-degrading capacity of granzyme B and the presence of granzyme B-positive cells at sites of erosion in the rheumatoid synovium. METHODS: Granzyme B was added to [(3)H]proline/[(35)S]sulphate-labelled cartilage matrices and to cartilage explants. Proteoglycan degradation was assessed by the release of (35)S and glycosaminoglycans into the medium and collagen degradation was assessed by the release of (3)H and hydroxyproline and by measuring the fraction of denatured collagen. Granzyme B expression was studied at the invasive front of the synovium by immunohistochemistry. RESULTS: Granzyme B induced loss of both newly synthesized, radiolabelled proteoglycans in cartilage matrices and resident proteoglycans of the cartilage explants. No effect on collagen degradation was found. Granzyme B-positive cells were present throughout the synovium and at the invasive front. CONCLUSION: The presence of granzyme B-positive cells at the invasive front of the synovium together with its ability to degrade articular proteoglycans supports the view that granzyme B may contribute to joint destruction in rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide/enzimología , Cartílago/enzimología , Proteoglicanos/metabolismo , Serina Endopeptidasas/metabolismo , Membrana Sinovial/enzimología , Animales , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Cartílago/metabolismo , Cartílago/patología , Bovinos , Células Cultivadas , Condrocitos/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Granzimas , Humanos , Articulación Metacarpofalángica/enzimología , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Granzyme B (GrB) is the primary molecular mediator of apoptosis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It is a unique mammalian aspartic acid-cleaving serine protease. On T cell receptor activation, GrB is released from the CTL cytoplasmic granules by exocytosis, enters the target cells and, in the presence of the granule pore-forming protein perforin, it initiates the processing of caspases and apoptosis. GrB apoptosis is also activated by adenovirus, which can effectively replace perforin. Methods for the purification and quantitation of GrB and perforin, and the preparation and titration of adenovirus, are described. In addition, methods for application of these reagents to the initiation of apoptosis in tumor target cells, with several assays for detecting GrB apoptotic activity, are detailed.
Asunto(s)
Células Asesinas Naturales/enzimología , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Linfocitos T Citotóxicos/enzimología , Animales , Fraccionamiento Celular/métodos , Línea Celular , Centrifugación por Gradiente de Densidad/métodos , Cromatografía de Afinidad , Cromatografía en Gel/métodos , Cromatografía Liquida , Gránulos Citoplasmáticos/enzimología , Gránulos Citoplasmáticos/ultraestructura , Daño del ADN , Fragmentación del ADN , Ensayo de Inmunoadsorción Enzimática/métodos , Granzimas , Hemoglobinas/análisis , Hemólisis , Humanos , Idoxuridina/análisis , Idoxuridina/farmacocinética , Radioisótopos de Yodo , Leucemia Experimental/enzimología , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/metabolismo , Perforina , Proteínas Citotóxicas Formadoras de Poros , Conejos , Ratas , Ratas Endogámicas F344 , Ovinos , Células Tumorales CultivadasRESUMEN
In the very early stages of target cell apoptosis induced by CTL, we found that fluorescence of labeling probes of the target plasma membrane, such as N-(3-triethylammoniumpropyl)-4-(p-dibutylaminostyryl)pyridin ium dibromide (FM1-43), was translocated into intracellular membrane structures including nuclear envelope and mitochondria. This translocation was associated with the execution of CTL-mediated killing, because neither the CTL-target conjugation alone nor the binding of noncytotoxic Th2 clone with target cell was sufficient to provoke the process. Although FM1-43 translocation was observed in perforin-mediated cytotoxicity, examinations with several other dyes failed to detect the evidence for membrane damages that may cause influx of the dye. Moreover, the translocation was also observed in Fas-dependent apoptosis. These data indicate that the translocation precedes the damage of plasma membrane and intracellular organella in the course of apoptotic cell death and may represent the existence of a membrane trafficking that mediates the translocation of plasma membrane components in the early onset of apoptotic cell death.
Asunto(s)
Apoptosis/inmunología , Permeabilidad de la Membrana Celular/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Animales , Apoptosis/genética , Caspasa 3 , Caspasas/deficiencia , Caspasas/genética , Caspasas/metabolismo , Línea Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/genética , Gránulos Citoplasmáticos/inmunología , Gránulos Citoplasmáticos/metabolismo , Citotoxicidad Inmunológica , Colorantes Fluorescentes/metabolismo , Membranas Intracelulares/inmunología , Membranas Intracelulares/metabolismo , Ratones , Mitocondrias/inmunología , Mitocondrias/metabolismo , Permeabilidad , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Linfocitos T Citotóxicos/enzimología , Células Tumorales Cultivadas , Receptor fas/genética , Receptor fas/fisiologíaRESUMEN
beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Cisteína Endopeptidasas/metabolismo , Naftoquinonas/farmacología , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Células Tumorales CultivadasRESUMEN
Granzymes are a family of serine proteinases commonly found in the granules of CD8+ T cells. In HIV infection, CD8+ cells show cytotoxic and noncytotoxic antiviral activities. The latter is mediated, at least in part, by a secreted CD8+ cell antiviral factor, CAF. Because of the antiviral nature of CD8+ cells, we examined the potential anti-HIV activity of free granzymes that can be found in CD8+ cell culture fluids. Pretreatment of CD4+ T cells with granzyme A or granzyme B had no effect on their susceptibility to infection with HIV, nor did incubation of the granzymes with HIV virions alter their infectivity. Continuous culture of acutely infected CD4+ T cells with granzyme A or B showed no effect on cell viability or the replication of HIV. The findings of this study suggest that free granzymes do not control HIV infection and spread in CD4+ T cells.
Asunto(s)
Linfocitos T CD4-Positivos/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Serina Endopeptidasas/farmacología , Linfocitos T CD8-positivos/enzimología , Células Cultivadas , Granzimas , Infecciones por VIH/virología , Humanos , Proteínas Recombinantes/farmacología , Virión/efectos de los fármacos , Virión/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Granzyme A (GrA) and B (GrB) together with perforin are the main constituents of cytotoxic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The cytotoxic proteins are released to deliver a lethal hit during contact between the CTL or NK cell and target cell. With the use of an enzyme-linked immunosorbent assay for antigenic levels, we showed in a recent study that plasma of patients with activated CTLs and NK cells contain elevated levels of extracellular GrA. In this study, we determined the form and proteolytic capacity of this extracellular GrA detected in plasma. With the use of various assays, we show that part of the extracellular GrA circulates in the mature conformation and is bound to proteoglycans that protect it against inactivation by protease inhibitors, such as antithrombin III and alpha-2-macroglobulin, whereas another part of GrA circulates as a complex with antithrombin III. Finally, with the use of a novel assay for active GrA, we demonstrate that some plasma samples with high levels of extracellular GrA contain active GrA. These results suggest that various forms of extracellular GrA occur in vivo and that the regulation of GrA activity may be modified by proteoglycans. These data support the notion that granzymes may exert extracellular functions distant from the site of CTL or NK cell interaction with their target cells. (Blood. 2000;95:1465-1472)
Asunto(s)
Células Asesinas Activadas por Linfocinas/enzimología , Inhibidores de Proteasas/farmacología , Proteoglicanos/metabolismo , Serina Endopeptidasas/sangre , Antitrombina III/farmacología , Biotinilación , Células Cultivadas , Cromatografía en Gel , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/inmunología , Gránulos Citoplasmáticos/enzimología , Ensayo de Inmunoadsorción Enzimática , Granzimas , Humanos , Trasplante de Riñón , Cinética , Leucocitos Mononucleares/enzimología , Conformación Proteica , Serina Endopeptidasas/química , Serina Endopeptidasas/efectos de los fármacos , alfa-Macroglobulinas/farmacologíaRESUMEN
Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10% (51)Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal (51)Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin's ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.
Asunto(s)
Apoptosis , Toxinas Bacterianas/metabolismo , Endosomas/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Glicoproteínas de Membrana/fisiología , Serina Endopeptidasas/metabolismo , Estreptolisinas/metabolismo , Animales , Proteínas Bacterianas , Toxinas Bacterianas/farmacología , Núcleo Celular , Citosol , Granzimas , Proteínas de Choque Térmico/farmacología , Proteínas Hemolisinas/farmacología , Humanos , Células Jurkat , Ratones , Perforina , Proteínas Citotóxicas Formadoras de Poros , Estreptolisinas/farmacología , Células Tumorales CultivadasRESUMEN
Cytotoxic cells possess specialized granules which contain perforin and a group of serine proteinases termed granzymes. Granzyme-positive cells have been identified in synovial fluid and tissue of patients with RA, where they may play an important role as mediators of granule-mediated apoptosis, extracellular proteolysis, and cytokine induction. The aim here was to define further the involvement of cytotoxic cells in RA. Plasma and synovial fluid samples from the knee joint were obtained from 31 RA patients. The disease controls included 20 osteoarthritis (OA) patients and 10 reactive arthritis (ReA) patients. A recently developed capture ELISA was used to detect soluble granzymes A and B in all patients. Compared with OA and ReA disease controls, markedly increased levels of soluble granzymes A and B were detected in both plasma and synovial fluid of RA patients (P < 0.00001). When values for soluble granzymes A and B in plasma and synovial fluid were used simultaneously as independent variables, logistic regression analysis indicated that a diagnosis of RA could be predicted correctly in 84% of the RA patients and a diagnosis of non-RA in 90% of the controls. The markedly elevated levels of soluble granzymes A and B in plasma and synovial fluid of RA patients strongly suggest that cytotoxic cells are active participants in the pathogenesis of RA. Moreover, the results suggest that measurement of granzymes may assist the laboratory evaluation of patients with arthritis. Larger studies in patients with early disease may clarify the role of this test system in differential diagnosis.