RESUMEN

The evolution of bismuth crystal structure upon excitation of its A1g phonon has been intensely studied with short pulse optical lasers. Here we present the first-time observation of a hard x-ray induced ultrafast phase transition in a bismuth single crystal at high intensities (~1014 W/cm2). The lattice evolution was followed using a recently demonstrated x-ray single-shot probing setup. The time evolution of the (111) Bragg peak intensity showed strong dependence on the excitation fluence. After exposure to a sufficiently intense x-ray pulse, the peak intensity dropped to zero within 300 fs, i.e. faster than one oscillation period of the A1g mode at room temperature. Our analysis indicates a nonthermal origin of a lattice disordering process, and excludes interpretations based on electron-ion equilibration process, or on thermodynamic heating process leading to plasma formation.

RESUMEN

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 & inhibidores

RESUMEN

CTLs kill targets by inducing them to die through apoptosis. A number of morphological and biochemical events are now recognized as characteristic features of the apoptotic program. Among these, the disruption of the inner mitochondrial transmembrane potential (Delta Psi m) and the release of cytochrome c into the cytoplasm appear to be early events in many systems, leading to the activation of caspase-3 and, subsequently, nuclear apoptosis. We show here that, in Jurkat targets treated in vitro with purified granzyme B and perforin or granzyme B and adenovirus, Delta Psi m collapse, reactive oxygen species production, and cytochrome c release from mitochondria were observed. Loss of Delta Psi m was also detected in an in vivo system where green fluorescent protein-expressing targets were attacked by a cytotoxic T cell line that kills predominantly through the granzyme pathway. DNA fragmentation, phosphatidylserine externalization, and reactive oxygen species production were inhibited in the presence of the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (zDEVD-fmk) in our in vitro system. Importantly, in either the in vitro or in vivo systems, these inhibitors at concentrations up to 100 microM did not prevent Delta Psi m collapse. In addition, cytochrome c release was observed in the in vitro system in the absence or presence of zVAD-fmk. Thus the granzyme B-dependent killing pathway in Jurkat targets involves mitochondrial alterations that occur independently of caspases.

Asunto(s)

Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Membranas Intracelulares/enzimología , Mitocondrias/enzimología , Serina Endopeptidasas/fisiología , Adenovirus Humanos/inmunología , Animales , Apoptosis/inmunología , Ciclosporina/farmacología , Fragmentación del ADN/inmunología , Activación Enzimática/efectos de los fármacos , Granzimas , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patología , Células Jurkat , Glicoproteínas de Membrana/inmunología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/inmunología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Perforina , Fosfatidilserinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros , Ratas , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T Citotóxicos/enzimología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo

RESUMEN

In TCR-alphabeta transgenic mice, CD4-CD8- TCR-alphabeta+ (alphabeta DN) cells arise in the absence of positively selecting MHC molecules and are resistant to clonal deletion in Ag-expressing mice. In this study the activation requirements and functional properties of alphabeta double-negative (DN) cells were compared with those of positively selected CD8+ cells expressing equivalent levels of the same MHC class I-restricted transgenic TCR. We found that positively selected CD8+ cells required a lower density of the antigenic ligand for optimal proliferative responses compared with alphabeta DN cells derived from nonpositively selecting mice. However, when the CD8 coreceptor on CD8+ cells was blocked with an anti-CD8 mAb, both alphabeta DN and CD8+ cells exhibited the same dose-response curve to the antigenic ligand and the same dependence on CD28/B7 costimulation. Positively selected CD8+ cells also differed from alphabeta DN cells in that they differentiated into more efficient killers and IL-2 producers after Ag stimulation, even after CD8 blockade. However, Ag-activated alphabeta DN and CD8+ cells were equally efficient in producing IFN-gamma, suggesting that this functional property is independent of positive selection. We also found that alphabeta DN cells recovered from the lymph nodes of Ag-expressing mice were functionally anergic. This anergic state was associated with defective proliferation and IL-2 production in response to Ag stimulation. These observations indicate that alphabeta DN cells can be anergized in vivo by physiological levels of the antigenic ligand.

Asunto(s)

Antígenos/biosíntesis , Antígenos CD4/genética , Antígenos CD8/genética , Linfocitos T CD8-positivos/inmunología , Anergia Clonal/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Subgrupos de Linfocitos T/inmunología , Animales , Antígenos/genética , Antígenos/metabolismo , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Anergia Clonal/inmunología , Citotoxicidad Inmunológica/genética , Femenino , Antígenos H-2/genética , Antígeno H-Y/inmunología , Receptores de Hialuranos/biosíntesis , Antígenos Comunes de Leucocito/biosíntesis , Activación de Linfocitos/genética , Linfocinas/biosíntesis , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Transgénicos , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo

RESUMEN

The issue of whether the signaling process during positive selection can affect the efficiency by which the positively selected T cells respond to antigenic stimulation has not been addressed. We approached this question by determining the consequences of positive selection of a particular transgenic TCR (2C TCR) in the H-2b and the H-2k thymus. The H-2b thymus provides a strong positive-selecting environment for the 2C TCR, whereas the H-2k thymus selects weakly for the 2C TCR. Although the positively selected CD8 thymocytes from the H-2b or H-2k thymus expressed similar levels of the CD8 coreceptor molecule, those for the H-2k thymus expressed a slightly lower level of the 2C TCR. This lower level of 2C TCR expression by H-2k CD8 thymocytes was not a result of coexpression of endogenous TCRs. Interestingly, CD8 thymocytes from H-2k mice were hyporesponsive to Ag stimulation compared with those from the H-2b mice. The functional maturity of positively selected CD8 thymocytes from the H-2b or H-2k thymus was inversely correlated with the level of heat stable Ag expressed by these cells. Furthermore, TCR-derived signals appear to be more efficiently coupled to downstream pathways leading to proliferation and cytokine production in CD8 thymocytes from H-2b 2C mice than those derived from H-2k 2C mice. These results provide the first demonstration that the intensity of the signaling process during positive selection affects the efficiency by which TCR-derived signals in positively selected thymocytes are coupled to downstream effector pathways.

Asunto(s)

Antígenos CD/biosíntesis , Antígenos H-2/metabolismo , Glicoproteínas de Membrana , Receptores de Antígenos de Linfocitos T/biosíntesis , Subgrupos de Linfocitos T/metabolismo , Timo/metabolismo , Animales , Antígenos/inmunología , Antígenos CD/genética , Antígeno CD24 , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Cruzamientos Genéticos , Antígenos H-2/genética , Inmunofenotipificación , Ligandos , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Timo/citología , Timo/inmunología

RESUMEN

The affinity/avidity model for T cell development postulates that ligands with high affinity for the TCR are efficient in negative selection, whereas those with lower affinity/avidity favor positive selection. Using the 2C TCR transgenic model, we evaluated the efficacy of ligands with widely differing affinity for the TCR (3 x 10(3) to 2 x 10(6) M(-1)) in mediating thymocyte deletion. The relative affinities of the 2C TCR for the p2Ca/Ld, dEV-8/Kb, p2Ca-A3/Ld, and p2Ca/Kb ligands are approximately 1000:50:10:1, respectively. Here we show, using an in vitro assay, that the deletion of 2C CD4+ CD8+ thymocytes is mediated not only by p2Ca/Ld, but also by the lower affinity ligands dEV-8/Kb, p2Ca-A3/Ld, and p2Ca/Kb, albeit at relatively higher peptide concentrations. Deletion mediated by low affinity ligands required CD8, whereas high affinity ligand-mediated deletion was CD8 independent. The p2Ca/Kb and dEV-8/Kb ligands are naturally occurring in H-2b mice, and others have shown that p2Ca/Kb can induce the maturation of CD4- CD8+ 2C-TCR(high) thymocytes in fetal thymic organ culture. In this study we showed that in addition to deletion, the p2Ca/Kb and dEV-8/Kb ligands, in the presence of exogenous IL-2, induced mature 2C T cell proliferation, albeit at a lower level than that induced by the high affinity p2Ca/Ld ligand. Thus, the same low affinity ligands that can effect negative selection and, in the case of p2Ca/Kb, the maturation of CD8 single-positive thymocytes can also induce the activation of mature CD8 T cells.

Asunto(s)

Supresión Clonal , Antígenos de Histocompatibilidad Clase I/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Relación Dosis-Respuesta Inmunológica , Antígenos de Histocompatibilidad Clase I/química , Ligandos , Ratones , Ratones Transgénicos , Péptidos/química , Péptidos/inmunología , Unión Proteica , Timo/citología , Microglobulina beta-2/deficiencia

RESUMEN

Interaction of the TCR on immature thymocytes with ligands on antigen presenting cells can lead to different fates including positive and negative selection. The affinity of the selecting ligands plays an important role in determining these outcomes. We used the 2C TCR transgenic model to evaluate the efficacy of ligands with widely differing affinity (3 x 10(3) - 2 x 10(6) M-1) for the 2C TCR in mediating thymic negative and positive selection. Our results support the conclusions that the deletion of immature thymocytes is not only mediated by high-affinity ligands but also by low-affinity/avidity ligands. However, high- and low-affinity ligands differ in their requirements for negative selection. We also present evidence that positive selection is not an all or none process but depending on the strength of interaction between the ligand and the TCR during the positive selection process can result in single positive thymocytes that are at different stages of functional maturity.

Asunto(s)

Células Presentadoras de Antígenos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Ligandos , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Modelos Inmunológicos , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal/inmunología , Linfocitos T/citología , Timo/citología , Timo/inmunología

RESUMEN

Antigen-activated T cells of the CD4(+)CD8(-) phenotype are susceptible to antigen receptor-stimulated cell death. This form of apoptotic cell death has been shown to be dependent on the expression of the Fas (CD95) antigen and can occur via an autocrine mechanism involving the concomitant up-regulation of Fas and its ligand on activated T cells. Mutation in genes encoding Fas (Ipr) and the Fas ligand (gld) contribute to the development of an autoimmune syndrome similar to systemic lupus erythematosus in mice. These observations led to the suggestion that the Fas signaling pathway is an important regulator of immune responses in vivo. Here we evaluated the importance of the Fas pathway in regulating immune responses by male antigen-specific CD4(-)CD8(+) T cells. We found that the in vivo elimination of these activated cells was independent of Fas expression by these cells. However, the elimination of these activated cells was inhibited by the transgenic expression of Bcl-2, a protein that inhibits multiple forms of apoptotic cell death. The transgenic Bcl-2 protein also inhibited the death of male antigen-activated cells following IL-2 deprivation. Cell death resulting from IL-2 deprivation occurred efficiently in male antigen-activated Fas- cells. We propose that the rapid deletion of male antigen-activated Fas- cells in vivo is due to limiting amounts of IL-2 that are available in the microenvironment of the activated cells at the peak of the response.

Asunto(s)

Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno H-Y/inmunología , Receptor fas/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Supresión Clonal , Proteína Ligando Fas , Femenino , Interleucina-2/inmunología , Ligandos , Masculino , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Transgénicos , Proteínas Tirosina Quinasas/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/inmunología

RESUMEN

The ileal Peyer's patch (PP) in sheep plays a central role in the development and production of B cells. Associated with a tremendous amount of B cell proliferation in this site is the extensive diversification of the Ig repertoire by somatic hypermutation. Very few (< 5%) of the B cells produced in the ileal PP differentiate and emigrate; instead, the vast majority of these cells soon die, and we have previously shown that death is associated with apoptosis. When placed in culture, ileal PP B cells die rapidly by apoptosis, such that after 24 h, 60 +/- 1% of DNA is fragmented. Here, we show that the extent of this spontaneous B cell apoptosis in culture, as quantitated by DNA fragmentation, was significantly increased in a dose-dependent manner by the glucocorticoids hydrocortisone or dexamethasone. Furthermore, treatment of lambs with 2-2.5 mg/kg of dexamethasone resulted in a marked increase in the number of apoptotic cells in the ileal PP and an increase in ileal PP B cell DNA fragmentation to 20 +/- 6%, compared with 2.4 +/- 0.1% in untreated lambs. Anti-immunoglobulin (Ig) antibodies also increased the extent of DNA fragmentation in cultured ileal PP B cells. After 24 or 48 h of culture with anti-Ig (PIg47A), DNA fragmentation was 74 +/- 2% and 75 +/- 3%, respectively. Ileal PP B cells are rescued from apoptosis by agents that activate protein kinase C and increase cytosolic Ca2+, and here we show that this treatment also results in apoptotic rescue in the presence of dexamethasone or anti-Ig. We speculate that the apoptosis of ileal PP B cells in situ may be modulated by glucocorticoids and by the cross-linking of surface Ig. Apoptosis, induced by a signal through surface Ig, may be an important mechanism in the deletion of self-reactive B cells during the expansion of the Ig repertoire in the ileal PP.

Asunto(s)

Anticuerpos Antiidiotipos/farmacología , Apoptosis , Linfocitos B/citología , Glucocorticoides/farmacología , Ganglios Linfáticos Agregados/citología , Animales , Calcio/fisiología , Daño del ADN , Femenino , Íleon/citología , Técnicas In Vitro , Masculino , Proteína Quinasa C/fisiología , Ovinos

RESUMEN

The major site of B-cell genesis in the sheep is the ileal Peyer's patch (PP). The B cells in the ileal PP undergo both extensive proliferation and massive death in association with an ongoing diversification of the immunoglobulin repertoire by somatic hypermutation. Most, if not all, the B-cell death in the ileal PP is due to apoptosis. When placed in culture, ileal PP B cells undergo rapid apoptosis. Here, we investigated the expression of the proto-oncogene bcl-2 in ileal PP cells in situ and in culture. Bcl-2 expression has been correlated with the prevention of apoptosis in many cell types. Western blotting, using anti-Bcl-2 monoclonal antibodies, revealed that a Bcl-2-reactive protein of 26,000 MW was expressed in ileal mesenteric lymph node cells, splenocytes and thymocytes from sheep, but was barely detectable in ileal PP B cells in situ or in culture. However, Bcl-2 expression could be markedly induced in ileal PP B cells cultured with phorbol ester and Ca2+ ionophore, a procedure that is known to rescue these cells from apoptosis. We hypothesize that those few B cells that survive a selection event in the ileal PP may begin to express elevated levels of Bcl-2 as they escape from the apoptotic pathway.

Asunto(s)

Apoptosis/inmunología , Linfocitos B/inmunología , Ganglios Linfáticos Agregados/inmunología , Proteínas Proto-Oncogénicas/inmunología , Ovinos/inmunología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Western Blotting , Calcimicina/farmacología , Células Cultivadas , Íleon/metabolismo , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos Agregados/metabolismo , Forbol 12,13-Dibutirato/farmacología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2 , Bazo/metabolismo , Timo/metabolismo

RESUMEN

The ileal Peyer's patch (PP) is the major site of B cell production and is a site of immunoglobulin gene diversification in the sheep. Within the ileal PP follicles there is both intense proliferation and death of B cells. We have previously demonstrated that most, if not all of this death can be attributed to apoptosis. Likewise, ileal PP B cells die rapidly by apoptosis in culture--after 6 h many cells appear pyknotic and about 50% of cellular DNA is fragmented. We now show that the DNA fragmentation and cell death of ileal PP B cells can be almost completely abrogated during the first 12 h of culture by the addition of the phorbol esters, phorbol dibutyrate (PBu2) or phorbol myristate acetate. This inhibition of apoptosis could be sustained for greater than 24 h by the concomitant addition of both PBu2 and the Ca2+ ionophore A23187. However, the rescue of B cells from apoptosis by PBu2, with or without Ca2+ ionophore, was prevented by macromolecular synthesis inhibitors or inhibitors of protein kinase C activation. Furthermore, treatment of cultures with PBu2, with or without Ca2+ ionophore, resulted in an activated B cell phenotype and a three- to fourfold increase in cell proliferation. We conclude that protein kinase C activation in conjunction with an increase in intracellular [Ca2+] can provide the signals necessary to rescue ileal PP B cells from apoptosis, and speculate that these ileal PP B cells are destined to die unless they receive a signal that rescues them from the death pathway.

Asunto(s)

Apoptosis , Linfocitos B/enzimología , Proteína Quinasa C/metabolismo , Alcaloides/farmacología , Animales , Antígenos de Superficie/metabolismo , Linfocitos B/citología , Calcimicina/farmacología , Calcio/metabolismo , Tamaño de la Célula/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Íleon , Masculino , Ganglios Linfáticos Agregados/citología , Ovinos , Estaurosporina , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

The ileal Peyer's patch (PP) and the bursa of Fabricius have major roles in populating the B cell system in sheep and chickens, respectively. These tissues contain greater than 90% B cells and possess a massive proliferation index with greater than 5% of B cells entering mitosis per hour. Paradoxically, almost all of the B cells produced in these sites rapidly die in situ. Here we show that the extensive B cell death occurring in the ileal PP and bursa is associated with apoptosis. Gel electrophoresis of ileal PP cell DNA from 7-14 week-old lambs and bursal cell DNA from 4-week-old chickens demonstrated a laddering of DNA in multiples of approximately 200 bp, a pattern indicative of apoptosis. In sheep, the intensity of the laddering pattern seen after agarose gel electrophoresis was always greater with ileal PP cell DNA compared with thymocyte DNA, and usually greater than jejunal PP cell DNA. Likewise, DNA isolated from chicken bursal cells and mouse PP cells always exhibited a more intense laddering pattern than chicken or mouse thymocytes, respectively. When placed in culture ileal PP cells died rapidly less than 40% viable cells were recovered after 24 h. Within 6 h of culture many ileal PP cells exhibited an apoptotic appearance in that they contained condensed chromatin and fragmented nuclei. Moreover, greater than 55% of total cellular DNA was fragmented. Compared with thymocytes, ileal PP cells underwent DNA fragmentation to a much greater extent and with a faster time course in short-term culture. We propose that cell death by apoptosis may make an important contribution to B cell development in the lamb ileal PP and the chicken bursa. Apoptosis may provide a mechanism for the diversification of the B cell immune repertoire and/or the selection of non-self reactive B cells.

Asunto(s)

Linfocitos B/fisiología , Bolsa de Fabricio/citología , Supervivencia Celular , Íleon/inmunología , Ganglios Linfáticos Agregados/citología , Animales , Células Cultivadas , Pollos , ADN/análisis , ADN/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ovinos , Linfocitos T/fisiología