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CD28-mediated activation of NF-κB in CD45RA+ and CD45RO+ T cells
80
Transcription control in lymphocytes
inhibited by antioxidants and enhanced by ROS-forming agents. ROS induce or augment the phosphorylatfon of the IwB cytopfasmic inhibitors, thus allowing the liberation of NF-KB proteins and their translocation to the nudeus. As it has been observed that chronic oxtdative stress downregulates T cell activation, we have now analyzed the effect of timing and duration of oxidative stress on the activation of NF-KB. Materials and Methods: Jurkat T cells were exposed to hydrogen peroxide either before or simultaneously with the activating signal (TNF or phorbd dibutyrate. PDBu). The transcriptional activity was analyzed by using a transfected NF-KB-luctferase reporter plasmid and the DNA-binding capacity by the electrophoretic mobility shift assay (EMSA). Reeuh In accordance to previous findings we observed that simultaneous oxidattve stress potentiated the NF-KB-dependent transcrfption, but in contrast to this, hydrogen peroxide pre-exposure (3-26 hours previously) had a strong decreasing effect. This took place without a detectable decrease in the nuclear DNA-binding NF-KB and atso the subunit composition of the binding complex rematned unchanged, suggesting that the in vitro DNA-binding NF-cB was defective in its capacity to activate transcription in vivo. Conclusions: These data suggest that oxidattve stress-induced signals downregulate NF-&dependent transcription, if the cells are exposed to it before the activating signal. As this took place without changes in the nuclear translocation of NF-KB proteins, the data suggest that these proteins are funcConally defective in vivo. As it is known that in vitro oxidized NF-KB behaves in this way, it is very probable that exposure of cells to oxidattt stress for a longer time oxidizes the cytoplasmic NF-KB. This could be one of the mechanisms behind the immune deficiency associated to chronic oxidative stress.
P.1 .l 1.18 Differential expression of alternative isoforms of Paxdduring B-ceil development P. Zwollo 1,2,H. Arrieta I, K. Molinder ‘, K. Ede ‘, M.Koshland 2, R. Pollock l. ’ Department of Biology at Occidental College, Los Angeles, California, USA, *Department of Molecular and Cellular Biology at the University of Cahbmia at Berkeley, Berkeley, California, USA The transcription factor Pax-5 encodes the B-cell specific activator protein (BSAP) and is expressed during the early stages of B-cell dierentiaiion. It has been shown that several B-cell specific genes contain BSAP binding sites, including the CD19, CD20, Apes, A5, J-chain, and Mkpmmoters, as well as /9H switch regions and the Ig 3, enhancer, using mobility shii assays. In addition to the existfng isoform (&x5, which we have named Pax&), we have isolated and characterized three new isoforms, Pax-5b. So, and 5e, from mudne spleen and B-lymphoid cell lines, using library screenings, PCR-ampliftcation, RNase protection, Western analysis and mobility shifts. lsoforms Pax-56 and 5e have spliced out their second exon, resulting in proteins with only a partial DNA-binding domain. lsoforms Pax-Sdand 5e have deleted the 3’ region which encodes the transactivating domain, and replaced it with a novel sequence. Cell lines representing earlier stages of development, including pro-, pre-, and mature B cdl lines, generally had high levels of both Pax&s and low or undetectable levels of 5b, in Western analysis. In contrast, low levels of both 5a and 5b were detected in 4 plasmacytomas. Thus, the 5a:5b ratio was significantly lower in plasmacytomas, compared to earlier B-cell lines. Mobility shift assays showed that in vifro translated Pax-5a and 5d interacted equally well with various BSAP-binding sites, including the TSAP binding site, as well as BSAP sites on the CDfSand blkpmmoters. Very low levels of Pax-M were detected in nuclear extracts from 3 of 17 B-cell lines tested, including one pro-B, one mature-B, and one plasma cell line. We conclude that Pax-5 isofonns are diierentiaily expressed during B-cell development. This may provide a mechanism to regulate transcription activation of Pax-5 target genes during B-cell development and/or activation.
1 P.l .l1.19 1 Activation of STAT 5 in B-CLL I 1 C. Kneitz, R. Seggewiss, A. Yaman, M. Goller, H.P. Tony. Madizinische Po/i/dinik University of Wuerrburg Germany A typical feature of B-CLL lymphocytes is the abnom7al high expression of CD23 in vivo and the irregular regulation in vitro. Interteukin-4 is the main inducer of CD23 expression in normal B lymphocytes. In B-CLL IL-4 is also able to induce CD23 exoression. however the amount of inducible CD23 is usuallv less compared to normal cells. Remarkably IFN-y increases the IL-4 induced CD23 expression on B-CLL cells while the expression on normal B lymphocytes is reduced. Furthermore phorbolester induce a very strong expression on BCLL cells whereas the effect is minor on normal B lymphocytes. Results obtained from STAT 6 deficient mice point to the central role of STAT 6 for IL4 mediated CD 23 expression. Aim of our studv was to analvse IL-4 inducible STAT 6 activation in B-CLL cells. Furthermorewe addressed the question if the abnormal expression of CD23 on B-CLL cells is a result of STAT 6 overexpression or increased translocation. In western blot experiments we could show that the amount of STAT 6
23 June 1997 - Poster presentations in B-CLLs resembles that in tonsillar B lymphocytes. IL-4 induces translocation of STAT-6 in B CLL cells as well as in normal B lymphocytes. The amount of translocated STAT 8, as shown by means of electronic mobility shift assays (EMSA), is similar in B-CLL and normal B lymphocytes. Therefore we assume that other causes are responsible for the comparable low IL-4 mediated CD23 expression on B-CLLs. Furthermore IL-4 induced translocation of STAT 8 can be increased if phorbolester (PMA) is added. PMA alone has, as well as IFN-y alone, no effect on the translocation of STAT 6, even though in CLL a strong CD23 response is observed. A possible explanation for the irregular hlgh expression of CD23 on B-CLL cells after costimulation with IL-4 and IFN-y could be the observation that stimulation with IFN-y results in binding of an additional protein on the sequence containing the STAT 6 bindlng site, suggesting that IFN-), mediated signals could also be invotved in CD23 regulation. These preliminary results snow the importance of IL-4 mediated signals for the regulation of CD23 expression on B-CLL and give hints for a better understanding of the observed abnormal regulation of CD23 on B-CLL.
I... P 1 11 20 1CD2&medlated
activation of NF-reB In CD45RA+ and CD45RO+ T ceils
Nina Lahdenpohja, Mikko Hurme. University of Tampa Medica/ School. POB 607, Tampere, Finland Introduction: T cell activation requires the engagement of the TCFUCDI as well as costimulatory signals delivered by CD28 receptor. This leads to the activation of complicated signafling network. One of the proposed events on this pathway is the formatton of reactive oxygen intermediates (ROls). NF-KB is a transcription factor which has a central role in the regulation of genes participating in inflammatory responses and T cell proliferation. We have analyzed the effect of complete T cell activation (anti-CD3 plus anUCD26) on the activation of NF-KB in CD45RA+ (naive) and CD46lXh (memory/effecter) T cells, which represent cell populations in different activation and dferentfation stages. MaterlaIr and Methods: T-cells were purified from human peripheral blood using SRBC-rosettfng. CD45RA+ and CD45RO+ cells were further purified by negative immunomagnettc separation. NF-KB DNA-binding was analyzed using electmphoretic mobility shift assay. The subunit composition was analyzed using supershift-assay. c-Rel and IKBU protein levels were analysed by westem blotting. Intracellular reactive oxygen intermediates (ROls) were measured using fluorescent DCHF which could be detected by flow cytometer. Results: Long exposure (24 h) induced stronger NF-KB DNA-binding in CD45RA+ cells than in CD45RO+ cells. Analysis of the composition of the DNA-binding complex revealed that in both cell populations the amount of ~50 and p65 proteins was very similar but the level of c-Rel was higher in CD45RA+ cells. Analysis of the cytoplasmic inhibitor IvBa indicated that antfCD3+ an&CD26 stimulation induced its longlasting degradatlon in CD45RA+ cells but in CD45RO+ cells the degradation process was more rapid. As the CD26 costimulus is known to induce the production of ROls, the intracellular ROI levels in CD45RA+ and CD46RO+ cells were compared by flow cytometry. ROls were produced in both cell types, but more strongly in CD45RA+ cells. Conclusions: Our resufts demonstrate that the T cells in different activation and differentiation stages exhibit different reactivity to CD26 costimulatton. The main differences between CD45RA+ and CD45RO+ cells are in the degradation of IKB~ and the nudear localization of c-Rel. The data presented further emphasize the differences between CD45RA+ and CD45ROt T lymphocytes in ROI-dependent signaling pathways.
1P.l .11.21 1 Early B-ii factor (EBF) is involved in the activation of SL chain genes C. Persson, A. Martensson, I.-L. Martensson. immunology Unit, University of Lund, Sd/ve@an, Lund, Sweden The pre8 cell-specific expression of the surrogate light (SL) chain genes, A5 and VpreB, is regulated at the level of transcription. Our woddng hypothesis is that expression of the SL genes is controlled by common regulatory elements, since these genes are expressed in the same ceils during ontogeny and B-lymphocyte differentiation. The transcriptional regulation of the SL genes was studied in transient transfectton assays, using reporter gene constructs carrying relevant DNA sequences (and mutated derivatives thereof). Potentially important DNA sites were anaiysed for transcription factor binding in vitroby gel retardation assays. We have found that a region of approximately 706 bp immediately upstream of the respective translation start site contains both promoter and enhancer sequences. We have defined the 1.5 and VpreB enhancer cores, respectively, and shown that they act in a pre-B cell-specific manner. We are currently defining the DNA elements important for enhancer activity. Early B cell factor (EBF) binds a site in each gene, implicating EBF as one of the factors involved in activating the SL chain genes.
Transcription control in lymphocytes
inhibited by antioxidants and enhanced by ROS-forming agents. ROS induce or augment the phosphorylatfon of the IwB cytopfasmic inhibitors, thus allowing the liberation of NF-KB proteins and their translocation to the nudeus. As it has been observed that chronic oxtdative stress downregulates T cell activation, we have now analyzed the effect of timing and duration of oxidative stress on the activation of NF-KB. Materials and Methods: Jurkat T cells were exposed to hydrogen peroxide either before or simultaneously with the activating signal (TNF or phorbd dibutyrate. PDBu). The transcriptional activity was analyzed by using a transfected NF-KB-luctferase reporter plasmid and the DNA-binding capacity by the electrophoretic mobility shift assay (EMSA). Reeuh In accordance to previous findings we observed that simultaneous oxidattve stress potentiated the NF-KB-dependent transcrfption, but in contrast to this, hydrogen peroxide pre-exposure (3-26 hours previously) had a strong decreasing effect. This took place without a detectable decrease in the nuclear DNA-binding NF-KB and atso the subunit composition of the binding complex rematned unchanged, suggesting that the in vitro DNA-binding NF-cB was defective in its capacity to activate transcription in vivo. Conclusions: These data suggest that oxidattve stress-induced signals downregulate NF-&dependent transcription, if the cells are exposed to it before the activating signal. As this took place without changes in the nuclear translocation of NF-KB proteins, the data suggest that these proteins are funcConally defective in vivo. As it is known that in vitro oxidized NF-KB behaves in this way, it is very probable that exposure of cells to oxidattt stress for a longer time oxidizes the cytoplasmic NF-KB. This could be one of the mechanisms behind the immune deficiency associated to chronic oxidative stress.
P.1 .l 1.18 Differential expression of alternative isoforms of Paxdduring B-ceil development P. Zwollo 1,2,H. Arrieta I, K. Molinder ‘, K. Ede ‘, M.Koshland 2, R. Pollock l. ’ Department of Biology at Occidental College, Los Angeles, California, USA, *Department of Molecular and Cellular Biology at the University of Cahbmia at Berkeley, Berkeley, California, USA The transcription factor Pax-5 encodes the B-cell specific activator protein (BSAP) and is expressed during the early stages of B-cell dierentiaiion. It has been shown that several B-cell specific genes contain BSAP binding sites, including the CD19, CD20, Apes, A5, J-chain, and Mkpmmoters, as well as /9H switch regions and the Ig 3, enhancer, using mobility shii assays. In addition to the existfng isoform (&x5, which we have named Pax&), we have isolated and characterized three new isoforms, Pax-5b. So, and 5e, from mudne spleen and B-lymphoid cell lines, using library screenings, PCR-ampliftcation, RNase protection, Western analysis and mobility shifts. lsoforms Pax-56 and 5e have spliced out their second exon, resulting in proteins with only a partial DNA-binding domain. lsoforms Pax-Sdand 5e have deleted the 3’ region which encodes the transactivating domain, and replaced it with a novel sequence. Cell lines representing earlier stages of development, including pro-, pre-, and mature B cdl lines, generally had high levels of both Pax&s and low or undetectable levels of 5b, in Western analysis. In contrast, low levels of both 5a and 5b were detected in 4 plasmacytomas. Thus, the 5a:5b ratio was significantly lower in plasmacytomas, compared to earlier B-cell lines. Mobility shift assays showed that in vifro translated Pax-5a and 5d interacted equally well with various BSAP-binding sites, including the TSAP binding site, as well as BSAP sites on the CDfSand blkpmmoters. Very low levels of Pax-M were detected in nuclear extracts from 3 of 17 B-cell lines tested, including one pro-B, one mature-B, and one plasma cell line. We conclude that Pax-5 isofonns are diierentiaily expressed during B-cell development. This may provide a mechanism to regulate transcription activation of Pax-5 target genes during B-cell development and/or activation.
1 P.l .l1.19 1 Activation of STAT 5 in B-CLL I 1 C. Kneitz, R. Seggewiss, A. Yaman, M. Goller, H.P. Tony. Madizinische Po/i/dinik University of Wuerrburg Germany A typical feature of B-CLL lymphocytes is the abnom7al high expression of CD23 in vivo and the irregular regulation in vitro. Interteukin-4 is the main inducer of CD23 expression in normal B lymphocytes. In B-CLL IL-4 is also able to induce CD23 exoression. however the amount of inducible CD23 is usuallv less compared to normal cells. Remarkably IFN-y increases the IL-4 induced CD23 expression on B-CLL cells while the expression on normal B lymphocytes is reduced. Furthermore phorbolester induce a very strong expression on BCLL cells whereas the effect is minor on normal B lymphocytes. Results obtained from STAT 6 deficient mice point to the central role of STAT 6 for IL4 mediated CD 23 expression. Aim of our studv was to analvse IL-4 inducible STAT 6 activation in B-CLL cells. Furthermorewe addressed the question if the abnormal expression of CD23 on B-CLL cells is a result of STAT 6 overexpression or increased translocation. In western blot experiments we could show that the amount of STAT 6
23 June 1997 - Poster presentations in B-CLLs resembles that in tonsillar B lymphocytes. IL-4 induces translocation of STAT-6 in B CLL cells as well as in normal B lymphocytes. The amount of translocated STAT 8, as shown by means of electronic mobility shift assays (EMSA), is similar in B-CLL and normal B lymphocytes. Therefore we assume that other causes are responsible for the comparable low IL-4 mediated CD23 expression on B-CLLs. Furthermore IL-4 induced translocation of STAT 8 can be increased if phorbolester (PMA) is added. PMA alone has, as well as IFN-y alone, no effect on the translocation of STAT 6, even though in CLL a strong CD23 response is observed. A possible explanation for the irregular hlgh expression of CD23 on B-CLL cells after costimulation with IL-4 and IFN-y could be the observation that stimulation with IFN-y results in binding of an additional protein on the sequence containing the STAT 6 bindlng site, suggesting that IFN-), mediated signals could also be invotved in CD23 regulation. These preliminary results snow the importance of IL-4 mediated signals for the regulation of CD23 expression on B-CLL and give hints for a better understanding of the observed abnormal regulation of CD23 on B-CLL.
I... P 1 11 20 1CD2&medlated
activation of NF-reB In CD45RA+ and CD45RO+ T ceils
Nina Lahdenpohja, Mikko Hurme. University of Tampa Medica/ School. POB 607, Tampere, Finland Introduction: T cell activation requires the engagement of the TCFUCDI as well as costimulatory signals delivered by CD28 receptor. This leads to the activation of complicated signafling network. One of the proposed events on this pathway is the formatton of reactive oxygen intermediates (ROls). NF-KB is a transcription factor which has a central role in the regulation of genes participating in inflammatory responses and T cell proliferation. We have analyzed the effect of complete T cell activation (anti-CD3 plus anUCD26) on the activation of NF-KB in CD45RA+ (naive) and CD46lXh (memory/effecter) T cells, which represent cell populations in different activation and dferentfation stages. MaterlaIr and Methods: T-cells were purified from human peripheral blood using SRBC-rosettfng. CD45RA+ and CD45RO+ cells were further purified by negative immunomagnettc separation. NF-KB DNA-binding was analyzed using electmphoretic mobility shift assay. The subunit composition was analyzed using supershift-assay. c-Rel and IKBU protein levels were analysed by westem blotting. Intracellular reactive oxygen intermediates (ROls) were measured using fluorescent DCHF which could be detected by flow cytometer. Results: Long exposure (24 h) induced stronger NF-KB DNA-binding in CD45RA+ cells than in CD45RO+ cells. Analysis of the composition of the DNA-binding complex revealed that in both cell populations the amount of ~50 and p65 proteins was very similar but the level of c-Rel was higher in CD45RA+ cells. Analysis of the cytoplasmic inhibitor IvBa indicated that antfCD3+ an&CD26 stimulation induced its longlasting degradatlon in CD45RA+ cells but in CD45RO+ cells the degradation process was more rapid. As the CD26 costimulus is known to induce the production of ROls, the intracellular ROI levels in CD45RA+ and CD46RO+ cells were compared by flow cytometry. ROls were produced in both cell types, but more strongly in CD45RA+ cells. Conclusions: Our resufts demonstrate that the T cells in different activation and differentiation stages exhibit different reactivity to CD26 costimulatton. The main differences between CD45RA+ and CD45RO+ cells are in the degradation of IKB~ and the nudear localization of c-Rel. The data presented further emphasize the differences between CD45RA+ and CD45ROt T lymphocytes in ROI-dependent signaling pathways.
1P.l .11.21 1 Early B-ii factor (EBF) is involved in the activation of SL chain genes C. Persson, A. Martensson, I.-L. Martensson. immunology Unit, University of Lund, Sd/ve@an, Lund, Sweden The pre8 cell-specific expression of the surrogate light (SL) chain genes, A5 and VpreB, is regulated at the level of transcription. Our woddng hypothesis is that expression of the SL genes is controlled by common regulatory elements, since these genes are expressed in the same ceils during ontogeny and B-lymphocyte differentiation. The transcriptional regulation of the SL genes was studied in transient transfectton assays, using reporter gene constructs carrying relevant DNA sequences (and mutated derivatives thereof). Potentially important DNA sites were anaiysed for transcription factor binding in vitroby gel retardation assays. We have found that a region of approximately 706 bp immediately upstream of the respective translation start site contains both promoter and enhancer sequences. We have defined the 1.5 and VpreB enhancer cores, respectively, and shown that they act in a pre-B cell-specific manner. We are currently defining the DNA elements important for enhancer activity. Early B cell factor (EBF) binds a site in each gene, implicating EBF as one of the factors involved in activating the SL chain genes.