摘要:

AbstractT cell activation induced via cross‐linking of the T cell receptor (TcR) stimulates hydrolysis of phosphatidylinositol to the second messengers diacylglycerol (DAG) and inositol 1,4,5‐triphosphate (IP3). DAG is necessary for the activation and function of protein kinase C (PKC) which is suggested to play a key role in the cascade of signal transduction when translocated from the cytosol to the cell membrane. In this report, we investigated responses of restingvs. activated Ly‐2+and L3T4+T lymphocytes in the presence of the PKC inhibitor H7 [1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine]. H7 inhibited the induction of primary T cell proliferation, while interleukin 2 (IL 2) production was fully retained. The effect of the PKC inhibitor on primary T cells depended on the type of ligand interacting with the TcR: increasing doses of concanavalin A or of immobilized anti‐CD3 monoclonal antibody (mAb), but not of anti‐Vβ8 or of anti‐TcR α/β mAb, partly overcame the blockade, indicating a differential signaling compared to the former stimuli. The blockade of T cell proliferation by H7 was not due to an inhibition of PKC translocation, but occurred even 4–8 h after T cell induction and correlated with a significant reduction of IL 2 receptor (IL 2R) expression. In contrast, the mRNA levels of IL 2R and the cellular proto‐oncogenes c‐fos and c‐myc were not affected. On activated T cells, H7 neither blocked proliferation nor IL 2R expression. Consequently, H7 dissects the signal resulting in T cell proliferation from those governing the triggering of other T cell functions,i.e.IL 2 production, during primary responses of Ly

ISSN:0014-2980    

DOI:10.1002/eji.1830210702

出版商:WILEY‐VCH Verlag GmbH    

年代:1991

数据来源: WILEY

摘要:

AbstractAn interaction of MRC OX7 monoclonal antibody with Thy‐1.1 antigen of rat peritoneal and pleural mast cells has been previously shown to induce rat mast cell activation (L'. Dráberová,Eur. J. Immunol.1989.19:1715). In the present study we analyzed the expression and function of the Thy‐1 antigen in rat basophilic leukemia cells, clone RBL‐2H3. Two RBL‐2H3‐derived cell lines with stable expression of murine Thy‐1.2 antigen, after transfection of a genomic clone of murine Thy‐1.2 or Thy‐1.2 cDNA under the control of simian virus 40 promoter, were also analyzed. Direct radioantibody binding assays, indirect immuno‐fluorescence studies and flow cytometry analyses revealed that both endogenous Thy‐1.1 and transfected murine Thy‐1.2 gene products were expressed on the surface of the cells under study. Analysis of the distribution of the Thy‐1 antigenin situin cells grown attached to tissue culture vessels located the Thy‐1 predominantly in regions of cell‐cell contacts. Incubation of RBL‐2H3 cells with Thy‐1.1‐specific antibodies, or of the transfected cells with both Thy‐1.1‐ and Thy‐1.2‐specific antibodies, induced a rapid early increase in the concentration of intracellular free calcium ([Ca2+]i) released from internal stores. Sustained increase of [Ca2+]irequired the presence of Ca2+in the extracellular medium. The increase in [Ca2+]iwas followed by histamine release from the target cells. The combined data indicate that RBL‐derived cells can be used as a useful model system for analysis of Thy‐

ISSN:0014-2980    

DOI:10.1002/eji.1830210703

出版商:WILEY‐VCH Verlag GmbH    

年代:1991

数据来源: WILEY

摘要:

AbstractIntravenous injection of nonfractionated BALB/c‐H‐2dm2(dm2) (Ld−) spleen cells into 4‐week‐old, semi‐allogeneic (H‐2d, Ld+) C.B‐17 scid/scid severe combined immunodeficient (scid) mice (2 × 107cells/mouse) reconstituted T lymphopoiesis in thymi and repopulated the lymphoid white pulp in spleens of these immunodeficient recipients. Transplantation of dm2 thymocytes into young scid mice (5 × 107cells/mouse) established a donor‐derived CD3+T cell population in spleens of recipient scid mice, in which CD4+T cells predominated. This was demonstrated by marker analyses of thymocytes and splenocytes, and determinations of serum immunoglobulin levels in transplanted scid mice. Transfer of splenocytes from young primary scid recipients into young secondary or tertiary recipients (3 × 106cells/mouse) engrafted preferentially dm2‐derived CD3+CD4+CD8−T cells in spleens of scid mice despite the strong selective Ld‐associated alloantigenic stimulus for CD8+T cells. Intravenous injections of nonfractionated dm2 spleen cells (2 × 107cells/mouse) or thymocytes (5 × 107cells/mouse) into 10‐ to 12‐month‐old, “leaky” scid mice induced severe clinical signs of graft‐vs.‐host disease (GVHD) in all scid recipients. Lymphoid repopulation of spleen and thymus in old scid recipients was incomplete. This GVHD was not transferrable by injecting 3 × 106spleen cells from old diseased primary scid recipients into secondary or tertiary young scid recipient mice. In these serial transfers, dm2‐derived CD3+CD4+CD8−T cells were again preferentially engrafted in spleens of scid recipients.Transfer of purified CD4+dm2 T cells into young scid mice (2 × 105to 5 × 105cells/mouse) engrafted this T cell subset into the spleen of semi‐allogeneic scid recipients. This was revealed by histological examinations, surface marker analyses,in vitroisolation of donor‐type CD3+CD4+T cell lines from spleens of transplanted scid mice, and serial transfer experiments. These data indicated that the CD4+T cell compartment of scid mice can be selectively repopulated by semi‐allogeneic T cells.Injections of purified CD8+dm2 T‐cells into young scid mice (2 × 105cells/mouse) did not establish a CD8+T cell graft in spleens of recipients. It was necessary to inject transplanted scid mice biweekly with 104units recombinant interleukin 2 to establish and/or maintain transferred dm2 CD8+T cells in spleens of these recipients. dm2 CD8+T cell‐transplanted and interleukin 2‐treated scid mice did not d

ISSN:0014-2980    

DOI:10.1002/eji.1830210704

出版商:WILEY‐VCH Verlag GmbH    

年代:1991

数据来源: WILEY

摘要:

AbstractTransgenic mouse models have demonstrated clonal deletion as well as clonal anergy of monospecific, high‐avidity autoreactive B cell. The function and fate of naturally activated B cells, many of them displaying degenerate specificity including autoreactivity, are still a matter of debate. The question was pursued in Sp6‐transgenic mice. Sp6, a monoclonal anti‐2,4,6‐trinitrophenyl (TNP) IgM has been shown to react with a variety of self antigens. Responsiveness of antibody‐secreting B cells was followed throughout postnatal development of Sp6‐transgenic mice and was related to the availability of antigen‐ and idiotype‐specific help.Thymus as well as spleen cells of transgenic mice contained a significantly higher number of TNP‐specific B cell than non‐transgenic controls. In contrast to control mice, the number of TNP‐specific B cells remained unchanged or decreased in thymus and spleen of transgenic mice after antigenic stimulation with TNP in T‐dependent (TD) and T‐independent (TI) form.Since the relative frequency of transgenic B cells was in particular diminished after repeated stimulation with TD antigen, it was examined whether limited responsiveness was linked to the available repertoire of helper T cells. Early after birth of transgenic individuals, thymic as well as splenic T cells which proliferated in response to TNP and Sp6 and provided help for B cells were found to be significantly augmented. Their number decreased rapidly during postnatal maturation and Thcells did not expand after antigenic stimulation. There was no indication that in the naive host transgenic B cells would suppress proliferation of TNP‐ and Sp6‐specific T cells, but they did so after antigenic stimulation. Furthermore, and in contrast to B cells of non‐transgenic mice, transgenic B cells were unable to present nominal antigen in a stimulatory way.The decrease in the number of B cells after antigenic stimulation indicated that autoreactive transgenic B cells may be subject to (functional) deletion under selected circumstances. In addition, idiotype‐ and antigen‐specific help was impaired in Sp6‐transgenic mice and this clearly was due to interactions with B cells ex

ISSN:0014-2980    

DOI:10.1002/eji.1830210705

出版商:WILEY‐VCH Verlag GmbH    

年代:1991

数据来源: WILEY

摘要:

AbstractHere we present the cloning of three novel mouse mast cell‐specific serine proteases, MMCP‐1, MMCP‐4 and MMCP‐5. A region of approximately 4 kb covering the five exons and 930 bp 5′ and 280 bp 3′ flanking sequences of the gene for MMCP‐1 was characterized by nucleotide sequence analysis. A comparison with the corresponding region of the rat mucosal mast cell‐specific protease RMCP‐II is presented. cDNA clones for the mast cell proteases MMCP‐4 (950 bp) and MMCP‐5 (1098 bp) were isolated from a cDNA library of a connective tissue mast cell‐like mouse mastocytoma cell line. All three proteases were found to belong to the family of chymotrypic serine proteases as deduced from the absence of the Asp 189 which is characteristic for all serine proteases having cleavage specificities similar to pancreatic trypsin. The active polypeptides, excluding possible post‐translational glycosylations, have an Mrof 25–26 kDa. Analysis of the amino acid composition reveals a positive net charge for all three proteases (MMCP‐1 +3, MMCP‐4 +18 and MMCP‐5 +12). Based on their high sequence identity (88%) and high positive net charges (+18 and +18, respectively) we assume that the MMCP‐4 is the mouse homolog to rat RMCP‐I.Probes specific for each of these three highly homologous protease genes have been generated by subcloning of fragments of approximately 100 bp in length, originating from the 3′ ends of the mRNA into plasmid vectors. Northern blot analysis of mRNA from a number of murine cell lines shows gene expression of these proteases to be specific for the differentiation stage of the mast cell. The MMCP‐1 is expressed only at the mucosal mast cell stage and and 5 only in mast cells of the connective tissue mast cell stage. These serine proteases may serve as highly specific markers in the analysis of mast cell he

ISSN:0014-2980    

DOI:10.1002/eji.1830210706

出版商:WILEY‐VCH Verlag GmbH    

年代:1991

数据来源: WILEY

摘要:

AbstractThe fate of the T cell receptor (TcR)/CD3 complex was examined on a cytotoxic T lymphocyte (CTL) clone (KB5.C20) activated either via binding of an anti‐TcR monoclonal antibody (mAb) or by a Ca2+ionophore and phorbol 12‐myristate 13‐acetate (PMA). After binding of the anti‐TcR mAb, electron microscopy revealed internalization through coated vesicles followed by slow degradation of the antibody as shown by use of radiolabeled mAb. The influence of activation on TcR/CD3 internalization was analyzed. The Ca2+ionophore alone had no effect on internalization, whereas PMA induced an accelerated internalization of anti‐TcR mAb. PMA‐induced internalization was dependent on protein kinase C (PKC) as shown by its absence in PKC‐depleted cells or in the presence of the PKC inhibitor staurosporine. Anti‐TcR mAb‐induced internalization was maintained in PKC‐depleted cells, but unexpectedly remained sensitive to inhibition by staurosporine. The monovalent anti‐TcR mAb Fab fragment is non‐stimulatory for the CTL. It was poorly internalized but its internalization was induced by PMA. Surprisingly, on PKC‐depleted cells, the Fab was internalized more readily than in untreated cells and this internalization was sensitive to inhibition by staurosporine. Inhibition of PMA‐induced phosphorylation of γ and ζ subunits of CD3 was demonstrated after depletion of PKC or in the presence of staurosporine, confirming that PKC function was inhibited in those conditions. Cross‐linking of the TcR via plastic‐coated anti‐TcR mAb led to phosphorylation of CD3 γ and ϵ and also of ζ, known to be phosphorylated on tyrosines. All of these phosphorylation events were inhibited by treatment with staurosporine. Our results indicate that staurosporine inhibits the receptor internalization induced by anti‐TcR mAb by means other than inhibition of PKC, suggesting that other kinases may contro

ISSN:0014-2980    

DOI:10.1002/eji.1830210707

出版商:WILEY‐VCH Verlag GmbH    

年代:1991

数据来源: WILEY

摘要:

AbstractTransforming growth factor‐β1 (TGF‐β1) mediates many immunosuppressive effects on immune cells and can inhibit the production of tumor necrosis factor and interleukin 1 (IL 1). However, TGF‐β1 can stimulate the production of IL6 and platelet‐derived growth factor, indicating that TGF‐β1 initiates complex effects on the production of cytokines. In this report we show that treatment of peripheral blood monocytes with TGF‐β1 leads to the induction of a recently described IL1 receptor antagonist protein (IRAP). The effect of TGF‐β was both dose and time dependent. TGF‐β1 inducedde novosynthesis of IRAP, as Northern blotting experiments indicated a rapid and transient induction of the mRNA encoding IRAP. The induction of IRAP suggests a potential mechanism by which some of the inhibitory effects

ISSN:0014-2980    

DOI:10.1002/eji.1830210708

出版商:WILEY‐VCH Verlag GmbH    

年代:1991

数据来源: WILEY

摘要:

AbstractWe report the consequences of low expression of the T cell receptor (TcR)/CD3 complex by T lymphocytes from a 4‐year‐old boy with a mild immunodeficiency. TcR/CD3 expression was found to be deficient on both resting and activated T cells, using both anti‐CD3 and anti‐TcR α/β monoclonal antibodies. As shown by immunofluorescence and immunoprecipitation studies, residual expression (corresponding to about 10% of normal) was detectable on resting and activated TcR α/β+T cells. Other T cell membrane receptors were normally expressed. The functional consequences of this TcR/CD3 expression deficiency included an absence of T cell proliferation, interleukin 2 receptor expression and calcium flux following anti‐CD3 and anti‐CD2 antibody‐triggered T cell activation. Antigen (tetanus toxoid,Candidaand allogeneic cell)‐induced proliferation was detectable. In contrast, cytotoxic T cell activity towards allogeneic cells was deficient. These findings shed light on the function of the TcR/CD3 complex and indicate that the expression of a limited number of TcR/CD3 receptors may be sufficient to trigger antigen‐specific T cell activation (and, possibly, differentiation) and that anti‐CD3 antibody‐induced T cell activation differs somewhat from antigen/major histocompatibility complex molecule‐induced activation. These results also confirm that the CD2 pathway of T cell

ISSN:0014-2980    

DOI:10.1002/eji.1830210709

出版商:WILEY‐VCH Verlag GmbH    

年代:1991

数据来源: WILEY

摘要:

AbstractTumor necrosis factor (TNF) mediates its pleiotropic effects via high‐affinity cell surface receptors. In man, molecular cloning has identified two distinct, independent TNF receptors (TNFR) of 55 and 75 kDa. It is unclear, however, whether the multiple effects of TNF are segregated between the receptor types. In the mouse, previous studies had shown functional heterogeneity of TNFR, since the WEHI 164 fibroblast line is sensitive to the cytotoxic effects of both murine and human TNF, whereas the murine T cell line, CT6, proliferates in response to murine but not human TNF. In this study, the cloning of a cDNA encoding the murine homologue of the p55 TNFR is reported. This receptor binds murine and human TNF with equal affinity and is expressed on WEHI 164 and a number of other cell lines, but only low levels of mRNA and no protein is detectable on CT6 cells. CT6 cells, however, express a second TNFR of approximately 75 kDa, identified by cross‐linking analysis, which is also found on WEHI 164 cells, and binds only murine TNF. These studies establish that there are also two TNFR in the mouse, and suggests that there may be segregation of the cytotoxic and proliferative responses between different receptors, at least in these cell li

ISSN:0014-2980    

DOI:10.1002/eji.1830210710

出版商:WILEY‐VCH Verlag GmbH    

年代:1991

数据来源: WILEY

摘要:

AbstractIn order to assess the possible role of lymphoid cells in the development of thymic epithelium, we compared the organization and maturation of thymic epithelium in scid mice lacking T cell receptor (TcR)‐positive cells with that in scid mice containing TcR+cells. Immunohistologic examination revealed that thymi from TcR−scid mice were deficient in thymic medullary epithelial cells recognized by the monoclonal antibody ER‐TR5, and that the few thymic medullary epithelial cells present were not organized into discrete medullary areas. In contrast, thymi from scid mice containing TcR+cells possessed ER‐TR5+thymic medullary epithelial cells and these cells were organized normally into discrete medullary regions. Thus, normal organization and maturation of thymic medullary epithelial cells did not occur in the absence of TcR+cells, but did occur upon introduction of TcR+cells. We conclude that lympho‐stromal cell interactions in the thymus are not unidirectional, and that a symbiotic relationship exists between maturing epithelial cells and developing ly

ISSN:0014-2980    

DOI:10.1002/eji.1830210711

出版商:WILEY‐VCH Verlag GmbH    

年代:1991

数据来源: WILEY