RESUMEN
In two-dimensionally separated Toxoplasma gondii lysate, mouse Th1 clone 3Tx15 detects two proteins of apparent molecular weight 40000 and pI of 5.8 and 5.9. Microsequencing of peptide fragments from tryptic digestion of one of these proteins yielded partial sequences of T. gondii lactate dehydrogenase (LDH)1. As shown by Western blot, toxoplasmic LDH co-migrates in two-dimensional gel electrophoresis with both T-cell antigenic proteins. With synthetic peptides spanning the complete primary structure of T. gondii LDH1, the T-cell epitope was mapped to a nine amino acid partial sequence which exhibits a motif for binding to I-E(k), the class II restriction element of antigen recognition by clone 3Tx15. From the two known isoforms of T. gondii LDH, clone 3Tx15 specifically recognises tachyzoite LDH1, but not bradyzoite LDH2, as shown with the corresponding epitope peptides and recombinant proteins. Antigen-presenting cells infected with live bradyzoites stimulate 3Tx15 T cells, while killed bradyzoites provide no antigenic stimulus. This finding implies that a transformation into the tachyzoite stage occurs in cells challenged with bradyzoites. Although LDH1 represents one major constituent of the tachyzoite proteome, the protein does not seem to be immunogenic in T. gondii infection of mice. This is evident from the lack of serum anti-LDH immunoreactivity and the failure of adoptively transferred 3Tx15 T cells to protect against lethal challenge. In conclusion, a T-cell-stimulatory Toxoplasma antigen is identified by means of a novel, high-resolution T-cell blot technique, the clones antigenic fine specificity allowing detection of parasite-stage conversion.
Asunto(s)
Antígenos de Protozoos/inmunología , Linfocitos T CD4-Positivos/inmunología , L-Lactato Deshidrogenasa/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/enzimología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/aislamiento & purificación , Modelos Animales de Enfermedad , Isoenzimas/inmunología , Isoenzimas/aislamiento & purificación , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/aislamiento & purificación , Estadios del Ciclo de Vida , Ratones , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Células TH1/inmunología , Toxoplasma/crecimiento & desarrollo , Toxoplasma/inmunología , Toxoplasmosis Animal/prevención & control , Tripsina/farmacologíaRESUMEN
High resolution two-dimensional separation of Toxoplasma gondii tachyzoite lysate revealed up to 224 distinct protein spots in Coomassie-stained gel. Computional matching of 14 digitized gels yielded a standard two-dimensional proteome map. The excretory T. gondii dense granule proteins GRA1-GRA8, S16/acid phosphatase, nucleoside triphosphate hydrolase, and H4 were identified by Western blotting of both total gel and isolated protein spots. In addition, two excretory antigens defined by parasite-specific monoclonal T cells, p36 and p40, were mapped by a novel T-cell blotting technique based on electroeluting single protein spots and testing the eluates for antigenic activity against the T-cell clones. In summary, these results represent a first step in Toxoplasma proteome analysis.
Asunto(s)
Antígenos de Protozoos/análisis , Proteoma , Toxoplasma/química , Animales , Antígenos de Protozoos/inmunología , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Electroforesis en Gel Bidimensional , Activación de Linfocitos , Mapeo Peptídico/métodos , Proteínas Protozoarias/análisis , Toxoplasma/inmunologíaRESUMEN
Determinants of Toxoplasma gondii virulence are still unknown, although genetic markers associated with T. gondii pathogenicity or host susceptibility to infection have been identified. To define indicator proteins of mouse virulence, type I strain parasites were attenuated by continuous passage in fibroblast culture and compared with the parental strain passaged in mice. The loss of acute virulence, evident by a 1000-fold higher pathogen dose causing 100% lethality in mice correlated with a less efficient infection of inflammatory cells at the site of inoculation, while parasite proliferation and invasiveness in vitro proved unimpaired. Infection with the attenuated parasites elicited earlier local interleukin-12 and strong interferon-gamma responses in vivo, although the activity that triggers interleukin-12 secretion in macrophages is reduced in the attenuated compared to the virulent strain variant. The interleukin-12-inducing T. gondii stimulus was identified as a protein(s) present in tachyzoite excretory products. Comparative proteome analysis combined with immunodetection and quantitation of a variety of T. gondii antigens indicated that the steady-state levels of actin, catalase, microneme protein 5, as well as dense granule proteins 1, 2, 3, 4, 5, 7, 8 and nucleoside triphosphate hydrolase 1 are decreased in the attenuated phenotype, whereas the surface antigen 1 and rhoptry protein 1 are produced at a similar level by virulent and attenuated parasites. In conclusion, these findings reveal a correlation between the efficient establishment of T. gondii infection in vivo and parasite synthesis of actin, catalase and several excretory proteins, and thus postulate a role for these molecules in acute virulence.
Asunto(s)
Interleucina-12/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/patogenicidad , Toxoplasmosis Animal/parasitología , Actinas/metabolismo , Animales , Catalasa/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteoma , Proteínas Protozoarias/inmunología , Toxoplasma/crecimiento & desarrollo , VirulenciaRESUMEN
Microglia subpopulations were studied in mouse experimental autoimmune encephalomyelitis and toxoplasmic encephalitis. CNS inflammation was associated with the proliferation of CD11b(+) brain cells that exhibited the dendritic cell (DC) marker CD11c. These cells constituted up to 30% of the total CD11b(+) brain cell population. In both diseases CD11c(+) brain cells displayed the surface phenotype of myeloid DC and resided at perivascular and intraparenchymatic inflammatory sites. By lacking prominent phagocytic organelles, CD11c(+) cells from inflamed brain proved distinct from other microglia, but strikingly resembled bone marrow-derived DC and thus were identified as DC. This brain DC population comprised cells strongly secreting IL-12p70, whereas coisolated CD11c(-) microglia/brain macrophages predominantly produced TNF-alpha, GM-CSF, and NO. In comparison, the DC were more potent stimulators of naive or allogeneic T cell proliferation. Both DC and CD11c(-) microglia/macrophages from inflamed brain primed naive T cells from DO11.10 TCR transgenic mice for production of Th1 cytokines IFN-gamma and IL-2. Resting microglia that had been purified from normal adult brain generated immature DC upon exposure to GM-CSF, while CD40 ligation triggered terminal maturation. Consistently, a functional maturation of brain DC was observed to occur following the onset of encephalitis. In conclusion, these findings indicate that in addition to inflammatory macrophage-like brain cells, intraparenchymatical DC exist in autoimmune and infectious encephalitis. These DC functionally mature upon disease onset and can differentiate from resident microglia. Their emergence, maturation, and prolonged activity within the brain might contribute to the chronicity of intracerebral Th1 responses.
Asunto(s)
Encéfalo/patología , Células Dendríticas/patología , Encefalomielitis Autoinmune Experimental/inmunología , Macrófagos/patología , Microglía/patología , Toxoplasmosis Animal/inmunología , Animales , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/ultraestructura , Ligando de CD40/inmunología , Ligando de CD40/metabolismo , Ligando de CD40/farmacología , Diferenciación Celular/inmunología , Células Cultivadas , Senescencia Celular/inmunología , Técnicas de Cocultivo , Citocinas/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/ultraestructura , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Inmunofenotipificación , Integrina alfaXbeta2/biosíntesis , Interfase/inmunología , Recuento de Leucocitos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos , Antígeno de Macrófago-1/biosíntesis , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/inmunología , Microglía/metabolismo , Microglía/ultraestructura , Óxido Nítrico/biosíntesis , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Células TH1/metabolismo , Toxoplasmosis Animal/metabolismo , Toxoplasmosis Animal/patologíaRESUMEN
During chronic infection of mice with Toxoplasma gondii, gene message for IL-12p40, CD86, and the potassium channel Kv1.3 was detected in brain mononuclear cells, suggesting the presence of dendritic cells (DC) in the CNS. Consistently, cells bearing the DC markers CD11c and 33D1 were localized at inflammatory sites in the infected brain. The number of isolated CD11c+ brain cells increased until peak inflammation. The cells exhibited the surface phenotype of myeloid DC by coexpressing 33D1 and F4/80, little DEC-205, and no CD8alpha. These brain DC were mature, as indicated by high-level expression of MHC class II, CD40, CD54, CD80, and CD86. They triggered Ag-specific and primary allogeneic T cell responses at very low APC/T cell ratios. Among mononuclear cells from encephalitic brain, DC were the main producers of IL-12. Evidence for a parasite-dependent development of DC from CNS progenitors was obtained in vitro: after inoculation of primary brain cell culture with T. gondii, IL-12-secreting dendriform cells emerged, and DC marker genes were expressed. Different stimuli elicited the generation and maturation of brain DC: neutralization of parasite-induced GM-CSF prevented outgrowth of dendriform cells and concomitant release of IL-12. IL-12 production was up-regulated by external IFN-gamma but was stopped by inhibiting parasite replication. Consistently, DC isolated from GM-CSF-treated brain cell culture were activated to secrete IL-12 by exposure to parasite lysate. In sum, these results demonstrate T. gondii-induced expansion and functional maturation of DC in the CNS and, thus, highlight a mechanism that may contribute to the chronicity of the host response.
Asunto(s)
Encéfalo/inmunología , Células Dendríticas/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Presentación de Antígeno , Antígenos de Superficie/análisis , Encéfalo/metabolismo , Encéfalo/parasitología , Encéfalo/patología , Recuento de Células , Ciclo Celular , Diferenciación Celular/inmunología , Enfermedad Crónica , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Células Dendríticas/patología , Encefalitis/inmunología , Encefalitis/metabolismo , Encefalitis/parasitología , Encefalitis/patología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Inmunofenotipificación , Interferón gamma/farmacología , Interleucina-12/biosíntesis , Interleucina-12/metabolismo , Isoantígenos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis Animal/metabolismo , Toxoplasmosis Animal/parasitología , Toxoplasmosis Animal/patologíaRESUMEN
Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-gamma), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-gamma when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-gamma or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L(-/-) mice infected with T. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-gamma but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L(-/-) mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-gamma, to activate macrophages in vitro to control replication of T. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.
Asunto(s)
Antígenos CD40/fisiología , Encefalitis/inmunología , Glicoproteínas de Membrana/fisiología , Toxoplasmosis Cerebral/inmunología , Animales , Ligando de CD40 , Femenino , Interferón gamma/biosíntesis , Interleucina-12/biosíntesis , Macrófagos/parasitología , Macrófagos/fisiología , Ratones , Ratones Endogámicos CBA , Óxido Nítrico/fisiología , Células TH1/inmunologíaRESUMEN
CD28 deficient (CD28-/-) mice were used to study the role of costimulation in the T cell-mediated, IFN-gamma-dependent mechanism of resistance to Toxoplasma gondii. These mice were resistant to infection with the ME49 strain of T. gondii. Analysis of the immune response of acutely infected CD28-/- mice revealed that IL-12 was required for T cell production of IFN-gamma and this was independent of the CD40/CD40 ligand interaction. A similar mechanism of IL-12-dependent, CD28/B7 independent production of IFN-gamma by T cells was also observed in wild-type mice. Interestingly, although chronically infected wild-type mice were resistant to rechallenge with the virulent RH strain of T. gondii, chronically infected CD28-/- mice were susceptible to rechallenge with the RH strain. This deficiency in the protective memory response by CD28-/- mice correlated with a lack of IL-2 and IFN-gamma in recall responses and reduced numbers of CD4+ T cells expressing a memory phenotype. Together, our findings demonstrate that CD28 is not required for the development of a protective T cell response to T. gondii, but CD28 is required for an optimal secondary immune response.
Asunto(s)
Antígenos CD28/fisiología , Memoria Inmunológica/inmunología , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Animales , Antígeno B7-1/fisiología , Enfermedad Crónica , Femenino , Inmunidad Innata , Inmunización Secundaria , Interleucina-12/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Toxoplasma/patogenicidad , Toxoplasmosis Animal/genética , Toxoplasmosis Animal/parasitología , VirulenciaRESUMEN
Infection of C57BL/6 mice with Toxoplasma gondii leads to chronic encephalitis characterized by infiltration into the brain of T cells that produce IFN-gamma and mediate resistance to the parasite. Our studies revealed that expression of B7.1 and B7.2 was up-regulated in brains of mice with toxoplasmic encephalitis (TE). Because CD28/B7-mediated costimulation is important for T cell activation, we assessed the contribution of this interaction to the production of IFN-gamma by T cells from brains and spleens of mice with TE. Stimulation of splenocytes with Toxoplasma Ag or anti-CD3 mAb resulted in production of IFN-gamma, which was inhibited by 90% in the presence of CTLA4-Ig, an antagonist of B7 stimulation. However, production of IFN-gamma by T cells from the brains of these mice was only slightly reduced (20%) by the addition of CTLA4-Ig. To address the role of the CD28/B7 interaction during TE, we compared the development of disease in C57BL/6 wild-type (wt) and CD28-/- mice. Although the parasite burden was similar in wt and CD28-/- mice, CD28-/- mice developed less severe encephalitis and survived longer than wt mice. Ex vivo recall responses revealed that mononuclear cells isolated from the brains of chronically infected CD28-/- mice produced less IFN-gamma than wt cells, and this correlated with reduced numbers of intracerebral CD4+ T cells in CD28-/- mice compared with wt mice. Taken together, our data show that resistance to T. gondii in the brain is independent of CD28 and suggest a role for CD28 in development of immune-mediated pathology during TE.
Asunto(s)
Antígeno B7-1/fisiología , Antígenos CD28/fisiología , Encefalitis/inmunología , Encefalitis/patología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/patología , Animales , Antígeno B7-1/biosíntesis , Encéfalo/inmunología , Encéfalo/parasitología , Encéfalo/patología , Antígenos CD28/biosíntesis , Antígenos CD28/genética , Enfermedad Crónica , Encefalitis/etiología , Encefalitis/mortalidad , Femenino , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Bazo/inmunología , Bazo/patología , Linfocitos T/inmunología , Toxoplasmosis Animal/etiología , Toxoplasmosis Animal/mortalidadRESUMEN
Monoclonal antibody (mAb) TxE2, reactive with Toxoplasma gondii excretory products, detects an acidic 29 kDa protein (p29) which, in 2D gel electrophoresis, exhibits a migration pattern distinct from those of the toxoplasmic excretory proteins described so far. The sequence of seven peptides from tryptic digestion of isolated p29 allowed the design of primers to obtain the coding DNA sequence. The full-length gene was amplified from genomic DNA of T. gondii strain BK and the sequence was identical with that of the corresponding cDNA, providing evidence for an intron-free gene structure. A single mRNA transcript of 1.3 kb was detected by Northern blot analysis. The deduced 236 amino acid protein contains a putative N-terminal signal peptide, one site of potential N-linked glycosylation, and, close to the C-terminus, a further hydrophobic, putative transmembrane domain. With synthetic peptides spanning the sequence of p29, the epitope for mAb TxE2 was mapped adjacent to the putative signal sequence. The antigen, which represents almost 0.5% of T. gondii protein, is expressed in strains of all three intraspecies subgroups, and is associated with the parasite dense granules as demonstrated by immunoelectron microscopy. In tachyzoite-infected cells, p29 accumulates within the parasitophorous vacuole and co-localizes with its delimiting membrane. In bradyzoite-infected cells, p29 is present within the host cell cytoplasm as detected by immunofluorescence staining, and, furthermore, in the supernatant of cyst-bearing cell culture lacking extracellular parasites as shown by enzyme-linked immunosorbent assay (ELISA). Thus, p29 which is named dense granule protein (GRA)7 may indicate the presence of intracellular toxoplasma.
Asunto(s)
Antígenos de Protozoos/química , Proteínas Protozoarias/química , Toxoplasma/química , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Secuencia de Bases , Western Blotting , Células Cultivadas , Citoplasma/química , Gránulos Citoplasmáticos/química , Electroforesis en Gel de Poliacrilamida , Mapeo Epitopo , Técnica del Anticuerpo Fluorescente , Genes Protozoarios , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Alineación de Secuencia , Toxoplasma/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasma/ultraestructura , Vacuolas/químicaRESUMEN
The Toxoplasma gondii-directed CD4+ T cell response in chronically infected mice was studied with respect to both T cell receptor diversity and antigen specificities. T cell receptor chains V beta 4, 6, 8, 10, and 14 were predominantly found on toxoplasma-reactive CD4+ splenocytes. This repertoire was also detected among T. gondii-specific CD4+ T cell clones. Analysis of clonotypic cytokine profiles revealed typical Th1 clones secreting interleukin-2, interferon-gamma and tumour necrosis factor activity and Th2 clones producing interleukin-4 and interleukin-10. Five distinct toxoplasma antigens (p26, p40, p55, p58 and p60) were detected in electrophoretically separated toxoplasma lysate by five individual Th1 clones. Parallel testing of CD4+ T lymphocytes from infected mice confirmed that these specificities constitute the peak immunogenic fractions of toxoplasma lysate. The expression patterns of two clonotypic, T cell-stimulatory parasite antigens were studied in detail. While p55 was expressed by mouse-virulent and avirulent T. gondii isolates and in both the tachyzoite and bradyzoite stages, p58 was detected only in virulent strains from intraspecies subgroup I. Thus, we describe the heterogeneity of toxoplasmic immunodominant T cell antigens including a 58-kDa group I-restricted molecule which may provide a marker for virulent isolates.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Células TH1/inmunología , Toxoplasma/inmunología , Animales , Antígenos de Protozoos/inmunología , Citocinas/biosíntesis , Femenino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BLRESUMEN
To investigate the role of astroglia in intracerebral immune response to Toxoplasma gondii, astrocytes cultured from mouse brain were inoculated with mouse-virulent or -avirulent toxoplasma strains. In comparison to microglia/ brain macrophages, astrocytes as host cells allowed stronger proliferation of avirulent parasites. Toxoplasma infection of astroglia was accompanied by release of interleukin- (IL)1 alpha, IL-6, and granulocyte/macrophage colony-stimulating factor (GM-CSF) activity, whereas alternative challenge by lipopolysaccharide (LPS) evoked no IL-1 response and significantly higher titers of IL-6 and GM-CSF. At the mRNA level, both stimuli induced transcription of all three cytokines in astrocytes. Secretion of IL-1 and IL-6 upon infection was triggered by T. gondii brady- and tachyzoites in a time- and dose-dependent manner. Heat killing of parasites, but not an exposure to polymyxin B, abrogated their cytokine-inducing activity, thus indicating that an LPS-independent stimulus is provided by T. gondii. When administered in combination, LPS synergistically augmented the IL-1-inducing effect of toxoplasma infection. In comparison, T. gondii-induced, but not an LPS-triggered, IL-6 response of astrocytes resisted to antagonization with IL-10. The IL-6 response of parasitized astroglia was up-regulated by external tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta 1, with only TNF-alpha enhancing simultaneous release of IL-1. Substantial secretion of IL-10 and TNF-alpha was detected in T. gondii-infected microglia, but not in astrocyte cultures. A possibly autocrine stimulation of infected astroglia via IL-1 was found to be unlikely, since addition of IL-1 receptor antagonist did not affect the release of IL-6 and GM-CSF while inhibiting these responses in IL-1-treated cells. The findings substantiate a separate, T. gondii-induced pathway of astroglia activation characterized by the release of IL-1 which may drive local inflammatory reaction both at initial infection of the brain and during reactivating toxoplasmosis.
Asunto(s)
Astrocitos/inmunología , Citocinas/biosíntesis , Microglía/inmunología , Toxoplasma/fisiología , Animales , Astrocitos/metabolismo , Astrocitos/parasitología , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Femenino , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-1/genética , Interleucina-10/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Microglía/metabolismo , Microglía/parasitología , Toxoplasma/crecimiento & desarrollo , Toxoplasma/patogenicidad , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/parasitología , Transcripción Genética , Factor de Crecimiento Transformador beta/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunología , VirulenciaRESUMEN
Toxoplasma gondii-specific murine CD4+ T cell clone 3Tx9 belongs to the Th1 subtype by virtue of secreting high levels of interleukin(IL)-2, interferon-gamma and tumor necrosis factor without producing IL4 and IL10. To characterize the clonal antigen, Toxoplasma lysate-was separated by SDS-PAGE and probed in T cell blot analysis with 3Tx9 T cells, revealing a fraction of about 60 kD molecular weight. This fraction proved highly stimulatory also for CD4+ splenocytes isolated from infected mice. The expression pattern of the relevant 60 kD antigen was determined by challenge of clone 3Tx9 with T. gondii strains from all three intraspecies subgroups and tachyzoites versus bradyzoites isolated from two strains as a source of antigen. While the T cell clone reacted with tachyzoites of all strains tested, bradyzoites lacked antigenic activity. Parallel T cell blot and ELISA confirmed co-migration of the T cell-stimulatory antigen p60 and rhoptry proteins ROP1, ROP2,3,4, and ROP5 among which ROP1 is a molecule of similar size and has only been shown on tachyzoites. However, a ROP1 knock-out Toxoplasma mutant still had antigen activity for 3Tx9 T cells. Since the two known tachyzoite-specific proteins, surface antigens SAG1/p30 and SAG2/p22, have a much lower molecular weight, we suggest that p60 represents a new T. gondii tachyzoite marker which is defined by clone 3Tx9.
Asunto(s)
Antígenos de Protozoos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Células TH1/inmunología , Toxoplasma/inmunología , Animales , Células Clonales , Citocinas/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BLRESUMEN
In order to identify brain cell types that serve as host cells of Toxoplasma gondii encystation primary cultures from murine brain were infected and stained for neural and parasite stage-specific markers. In mixed culture inoculated with T. gondii tachyzoites, MAP2+ neurons, GFAP+ astrocytes, F4/80+ microglia, and O1+ oligodendrocytes proved to be infected as detected by parallel labeling of SAG1. At 4 days following infection with bradyzoites, cysts developed in neuronal, astroglial, and microglial host cells as clarified using bradyzoite-specific antibody 4F8. Additional staining of SAG1 revealed that astrocytes in bradyzoite-infected brain cell culture can also harbor tachyzoite-containing vacuoles. Stage conversion was observed shortly after inoculation and was accompanied by an increase in] parasite proliferation. However, tachyzoites became rare in prolonged culture. By contrast, the numbers of cysts and of the bradyzoites isolated multiplied during long-term culture. These findings demonstrate that both glial and neuronal host cells allow T. gondii encystation in the absence of T cell-derived cytokines and imply that a brain-internal spreading of bradyzoites may sustain chronic infection.
Asunto(s)
Encéfalo/parasitología , Toxoplasma/crecimiento & desarrollo , Animales , Células Cultivadas , Quistes/parasitología , Técnica del Anticuerpo Fluorescente Indirecta , Ratones , Ratones Endogámicos BALB C , Neuroglía/parasitología , Neuronas/parasitología , Factores de TiempoRESUMEN
To analyse target molecules of the CD4+ T-cell response to toxoplasma infection, a panel of Toxoplasma gondii-specific murine CD4+ T-cell clones has been established. Clone 3Tx15, belonging to the T helper 1 (Th1) subtype, abolished intracellular parasite growth when co-cultured with macrophages and live toxoplasma at a ratio of 2:2:1. This effect results from macrophage toxoplasmicidal activity induced upon parasite-dependent cellular interaction, an irrelevant Th1 clone failed in this three-party system. Clone 3Tx15 detects its corresponding antigen in the supernatant of infected cells and also reacts with a host cell-free preparation of T. gondii-excreted/secreted antigens. T-cell blot analysis of two-dimensionally separated toxoplasma lysate revealed a molecular weight of about 40,000 for the fractions stimulating clone 3Tx15. As checked in parallel enzyme-linked immunosorbent assay, the 40,000 MW T-cell antigen co-migrates with the excretory protein GRA4, the sole 40,000 MW T. gondii antigen hitherto known to be recognized by T lymphocytes. Nevertheless, neither recombinant GRA4 nor immunoaffinity-purified natural GRA4 was stimulatory for clone 3Tx15. Our findings thus demonstrate that Th1 clone 3Tx15 which induces toxoplasmicidal activity during antigenic interaction with infected macrophages defines a new 40,000 MW excretory T. gondii antigen.
Asunto(s)
Antígenos de Protozoos/análisis , Macrófagos/inmunología , Células TH1/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Animales , Antígenos de Protozoos/química , Encéfalo/inmunología , Técnicas de Cultivo de Célula , Células Clonales/inmunología , Medios de Cultivo Condicionados , Femenino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Peso Molecular , Proteínas Protozoarias/inmunologíaAsunto(s)
Antígenos de Protozoos/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Toxoplasma/genética , Toxoplasma/inmunología , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Protozoario/genética , Epítopos/genética , Genotipo , Humanos , Ratones , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Toxoplasma/clasificación , Toxoplasmosis/parasitología , Virulencia/genéticaRESUMEN
Cloned 10BK.1 T cells with specificity for the ovalbumin peptide OVA257-264 are representative of a novel cell type within the CD8+ subset of T cells. In the presence and in the absence of added antigen presenting cells these T cells react toward antigen (Ag) by proliferation and lymphokine production. These data suggest self-presentation of the Ag by 10BK.1 cells. Here we present evidence that 10BK.1 cells exhibit cytotoxic activity that involves two different cytotoxic effector mechanisms. (i) One mechanism is fast killing activity, apparent within 4 hr. Constitutive mouse T cell-specific proteinase-1 (MTSP-1) activity, constitutive expression of MTSP-1 RNA, increased by Ag challenge, and Ag-inducible perforin RNA expression were observed. Electron microscopic dense granules of the CTL were oriented toward Ag-pulsed target cells. The fast form of cytotoxicity was triggered by Ag recognition and by contact with IL-2. (ii) The other mechanism is slow cytolytic activity, manifested within 2 days. This activity was contained in the supernatant of 10BK.1 cells after Ag activation. It was inhibited by monoclonal anti-TNF antibodies and therefore presumably represents TNF alpha/beta. Cytotoxic T cells capable of antigen self-presentation may be responsible for tissue damage during bacterial and viral infections.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Citotoxicidad Inmunológica/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Células Presentadoras de Antígenos/ultraestructura , Línea Celular , Células Clonales , Gránulos Citoplasmáticos/fisiología , Granzimas , Interleucina-2/fisiología , Activación de Linfocitos/inmunología , Linfotoxina-alfa/fisiología , Glicoproteínas de Membrana/fisiología , Ratones , Ovalbúmina/inmunología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidasas/fisiología , Linfocitos T Citotóxicos/ultraestructura , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
10BK.1 T clone cells of (B10 x B10.BR)F1 genotype specifically react to peptides of OVA, frequently encountered in OVA preparations, and to the synthetic peptide OVA257-264 in an H-2Kb-restricted manner; the T clone cells produce lymphokines and proliferate in the absence of added APC, suggesting self-presentation of the Ag by the T cells. In accordance with their CD8+ CD4- phenotype 10BK.1 cells exhibit cytotoxic capacity. When the target cells were pretreated with OVA257-264 only cells bearing H-2Kb molecules were lysed. However, when the relevant OVA peptide was present during the 4-h 51Cr release assay 10BK.1 cells lysed target cells expressing H-2Kb molecules as well as target cells lacking H-2Kb elements. Likewise, 10BK.1 cells pretreated with OVA257-264 for 1 h and washed extensively to remove residual Ag were able to kill syngeneic and allogeneic target cells. Killing of allogeneic targets in the presence of Ag was inhibited by antibodies directed at H-2Kb. Allogeneic target cells were not killed when 10BK.1 cells were stimulated by peptide-pulsed syngeneic cells. Triggering of the TCR/CD3 complex of 10BK.1 cells was involved during the activation phase but not during the lytic phase. IL-2 did not participate in the MHC-unrestricted killing event. To explain the observed MHC-unrestricted cytotoxicity of 10BK.1 cells, the following model is proposed. In an initial step 10BK.1 cells present the relevant OVA peptide to one another in a H-2Kb-restricted fashion. The cells are thereby triggered to mobilize the lytic machinery. In a second step sensitized 10BK.1 cells lyse allogeneic target cells. Thus, the MHC-unrestricted cytotoxicity can be dissected into an MHC-restricted phase of 10BK.1 cell activation and an MHC-unrestricted lytic phase. Cytotoxic T cells with Ag self-presentation functions may account for tissue damage during bacterial infections.
Asunto(s)
Células Presentadoras de Antígenos/fisiología , Citotoxicidad Inmunológica/genética , Activación de Linfocitos/genética , Complejo Mayor de Histocompatibilidad/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Células Presentadoras de Antígenos/inmunología , Complejo CD3/inmunología , Línea Celular , Células Clonales/inmunología , Células Clonales/metabolismo , Citotoxicidad Inmunológica/inmunología , Antígenos H-2/genética , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Activación de Linfocitos/inmunología , Ratones , Células Tumorales CultivadasAsunto(s)
Diglicéridos/aislamiento & purificación , Glicéridos/aislamiento & purificación , Naftalenos/aislamiento & purificación , Ácidos Fosfatidicos/aislamiento & purificación , Uretano/análogos & derivados , Animales , Bovinos , Cromatografía de Gases , Cromatografía Líquida de Alta Presión , Eritrocitos/análisis , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Uretano/aislamiento & purificaciónRESUMEN
Rat liver mitochondria were incubated with [3H]glycerol 3-phosphate, ATP, CTP and coenzyme A allowing acylatin of glycerophosphate with endogenous fatty acids and the further conversion of labelled phosphatidic acid (PA) to diacylglycerol (DG), CDP-diacylglycerol (CDP-DG) and phosphatidylglycerol (PG). In these glycerolipids, the distribution of label among the individual molecular species was found to be similar, with 16:0-18:1, 16:0-18:2 and 18:0-18:2/16:0-16:0 being the main species. It was concluded that mitochondrial enzymes involved in the de novo synthesis of these glycerolipids exhibited no acyl selectivity for their substrates. The pattern of molecular species of mitochondrial PA, DG and CDP-DG closely approached that of the same glycerolipids synthesized de novo in isolated rat liver microsomes.