Biomedical subjects
Publications and source records attributed to T Leanderson.
The complement system is old, yet it may still have something new to teach us. For many years, research has existed which shows that C3d, in addition to its established role as an adjuvant, could have an immunosuppressive activity. Being true, it suggests that a common mechanism may be used both by organisms and by their pathogens to prevent unwanted immune responses.
We have analysed the mechanism of action for induction of apoptosis by N-substituted benzamides using declopramide as a lead compound. We show here that declopramide at doses above 250 microM in the mouse 70Z/3 pre-B cell line or in the human promyeolocytic cancer cell line HL60 induced cytochrome c release into the cytosol and caspase-9 activation. The broad spectrum caspase inhibitor zVADfmk and caspase-9 inhibitor zLEDHfmk inhibited apoptosis and improved cell viability when administrated to cells 1 h before exposure to declopramide, whereas the caspase-8 inhibitor zIEDHfmk had less effect. Also, the over expression of Bcl-2 by transfection in 70Z/3 cells inhibited declopramide-induced apoptosis. Prior to the induction of apoptosis, a G(2)/M cell cycle block was induced by declopramide. The cell cycle block was also observed in the presence of broad spectrum caspase inhibitor zVADfmk and in a transfectant expressing high levels of Bcl-2. Furthermore, while p53 was induced in 70Z/3 cells by declopramide, neither the apoptotic mechanism nor the G(2)/M cell cycle block were dependent on p53 activation since both effects were also seen in p53 deficient HL60 cells after addition of declopramide.
The diphtheria toxin A chain (DTA) was gene targeted into the Joining chain (J chain) locus to create a mouse strain selecting against J chain-expressing cells, JDTA mice. Serum immunoglobulin (Ig)M and serum IgG were reduced six to eightfold, while serum IgA was elevated 14-fold in these mice. JDTA mice were immune competent although the serum Ig response compared with wild-type mice was reduced sixfold at day 14 but only fourfold at day 45 after immunization. Exchanging the DTA gene with a cDNA for c-myc resulted in mice with a distinct phenotype with increased Ig production and enhanced humoral immune responses. Analysis of single B cells stimulated by lipopolysaccharide in vitro using reverse transcription-polymerase chain reaction showed that J chain-nonexpressing B cells could be detected that had a secretory phenotype as determined by an abundance of transcript for secretory IgM. Finally, limiting dilution analysis of peripheral B cells showed that J chain expression was a clonal property already established in naive, peripheral B lymphocytes.
We have analyzed the murine CD40 ligand promoter with regard to stimulation of transcriptional activity in Jurkat T cells after signaling via the TCR and the costimulatory molecules CD28 and CD2. TCR engagement was necessary for the induction of transcriptional activity from the CD40 ligand promoter, and costimulation through either CD28 or CD2 further increased the activity. Analysis of promoter deletants showed that the DNA elements needed for transcriptional activity induced by costimulatory molecules were located within two regions containing previously identified transcription factor NFAT sites. Further studies of the proximal NFAT site showed that it was not dependent on AP-1 binding for transcriptional activity induced by costimulation through CD28. Instead, a region between the TATA box and the proximal NFAT site was shown to bind proteins of the early growth response family and to contribute to NFAT-mediated transcriptional activation.
Immunoglobulin (Ig)-kappa promoters from humans and mice share conserved sequences. The octamer element is common to all Ig promoters and pivotal for their function. However, other conserved sequence motifs, that differ between Ig variable gene families, are required for normal promoter function. These conserved motifs do not stimulate transcription in the absence of an octamer. One example is an E-box of the E47/E12 type (5'-CAGCTG-3'), which is found in all promoters of the human and murine Ig-kappa gene subgroups/families, with the exception of subgroups II and VI and their related murine families. In the present study we show that the ubiquitously expressed transcription factor AP-4, and not E47, interacts specifically with the kappa promoter E-boxes when tested in electrophoretic mobility-shift assays using nuclear extracts derived from human and murine B-cell lines. Furthermore, AP-4, unlike E47, did not act as a transactivator, which is in agreement with previous studies on intact kappa promoters, showing that transcription is absent when the octamer element has been mutated. Based on these data, and the conservation of the 5'-CAGCTG-3' motif among human and murine kappa promoters, we propose that AP-4 is the major ligand for Ig-kappa promoter E-boxes.
Somatic hypermutation is initiated as B lymphocytes proliferate in germinal centers. The signals that switch on the mutation process are unknown. We have derived an in vitro system to define signals that will initiate mutation in normal, naive splenic B cells. We find that three signals are required to allow detection of somatic mutation in vitro; these are anti-Ig, anti-CD40, and anti-CD38. If any one of these is omitted, mutation remains off. We show that CD40 is obligatory in vivo, as CD40 knockout mice exhibit no Ag-driven mutation. In contrast, CD38 is not, as CD38 knockout mice mutate normally. We believe that, in vitro, CD38, in combination with other stimuli, drives extensive cell division, allowing the detection of mutated sequences. However, in germinal centers in vivo, proliferative activity is instigated by a different molecule. This is the first demonstration of the initiation of hypermutation in vitro with normal splenic B cells using defined stimuli.
N-substituted benzamides are compounds that have recently been reported to inhibit nuclear factor-kappaB (NF-kappaB) activity and induce apoptosis in a pre-B cell line. In this study, we focused on the effects of N-substituted benzamides on transcriptional regulation in Jurkat T cells. We used a model system where the cells can be stimulated either through TCR/CD28 or by treatment of the cells with PMA and ionomycin to induce transcription factors typical for T lymphocyte activation. Treatment of the Jurkat cells with procainamide did not influence the transcription factor profile of stimulated cells, while treatment with a derivative having an acetyl group in position 4 of the aromatic ring inhibited NF-kappaB and nuclear factor of activated T cells (NFAT) activity. Declopramide, which contains a chloride in position 3 of the aromatic ring, was inactive in this system, whereas also the acetylated derivative of this compound inhibited NF-kappaB and NFAT activity. In contrast, the transcriptional activity and nuclear expression of activator protein 1 induced by TCR/CD28 stimulation or PMA and ionomycin treatment was enhanced by the acetylated variants of the N-substituted benzamides. Finally, we investigated the effect of N-substituted benzamides on intact promoters for two genes central in immune regulation; the CD40 ligand (CD40L) and IL-2 promoters. The transcriptional activity of the CD40L promoter as well as surface expression of the CD40L induced by signaling through TCR/CD28 was inhibited by addition of acetylated N-substituted benzamides, while the transcriptional activity of the IL-2 promoter was enhanced. Taken together, these data indicate that derivatives of N-substituted benzamides are potential drug candidates for quantitative as well as qualitative modulation of immune functions.
A search for an antigen-driven expansion of T lymphocytes in the inflamed joints in rheumatoid arthritis (RA) patients have been going on for decades. We here analyzed the human germinal centre T-cell receptor (TCR) Vbeta gene usage with polymerase chain reaction (PCR) combined with sequence analysis, to address the question of clonality in tonsils and synovial tissue from RA patients. Our data show a large degree of TCR heterogeneity in both these histological structures. Furthermore, clonally related T cells were found within different closely located germinal centres indicating either an active T-cell migration between germinal centres (GC) or that a T-cell clone may seed more than one GC.
We describe a transgenic mouse strain that selectively express a surface marker (huCD25) on transitional B cells, pre-B cells and a lineage unidentified bone marrow (BM) population. We show that a subpopulation of B cells in Peyer's patches, spleen, blood and BM expressed the transgenic huCD25 marker on the cell surface. In the spleen, the huCD25 expression was found on transitional B cells, that had not yet been recruited into the recirculating pool. In the BM a fraction of the B220low surface immunoglobulin (Ig) negative PB493+ pre-B cells were huCD25+. HuCD25 expression was also seen on practically all immature B cells while the mature recirculating B cells did not express huCD25. A huCD25+B220- cell population was also found in the BM that had not rearranged the Ig heavy chain locus and did not express the lineage markers CD3, T-cell receptor (TCR), CD19 and Mac-1. A low expression of CD4 on these cells may indicate that they represent a noncommitted, hematopoetic progenitor cell population.
The GAGA box of the rat serine protease inhibitor 2 (spi 2) genes not only acts as a basal promoter element, but also mediates transcriptional activation by growth hormone and interleukin-6. The GAGA box is separated from the TATA box by only 12 bp, and this close association is required for efficient transcription. Hence, the GAGA box may influence transcription efficiency through interactions between GAGA box binding proteins and some components of the RNA polymerase II complex. Here we report the cloning of two GAGA box-binding proteins termed p38 and p40, that belong to the type A/B heterogeneous nuclear ribonucleoprotein subgroup. GAGA box mutations that diminish the affinity for p38 and p40 decrease basal and GH-induced reporter gene expression. Furthermore, nuclear extracts depleted of p38 and p40 can no longer support GAGA box-dependent in vitro transcription. Therefore, two polypeptides previously assigned to a family of RNA processing proteins also act as DNA-binding, promoter-specific transcription factors.
Recent publications have provided confusing information on the importance of the J chain for secretion of dimeric IgA at mucosal surfaces. Using J chain-deficient (J chain-/-) mice, we addressed whether a lack of J chain had any functional consequence for the ability to resist challenge with cholera toxin (CT) in intestinal loops. J chain-/- mice had normal levels of IgA plasma cells in the gut mucosa, and the Peyer's patches exhibited normal IgA B cell differentiation and germinal center reactions. The total IgA levels in gut lavage were reduced by roughly 90% as compared with that in wild-type controls, while concomitantly serum IgA levels were significantly increased. Total serum IgM levels were depressed, whereas IgG concentrations were normal. Following oral immunizations with CT, J chain-/- mice developed 10-fold increased serum antitoxin IgA titers, but gut lavage anti-CT IgA levels were substantially reduced. However, anti-CT IgA spot-forming cell frequencies in the gut lamina propria were normal. Anti-CT IgM concentrations were low in serum and gut lavage, whereas anti-CT IgG titers were unaltered. Challenge of small intestinal ligated loops with CT caused dramatic fluid accumulation in immunized J chain-/- mice, and only 20% protection was detected compared with unimmunized controls. In contrast, wild-type mice demonstrated 80% protection against CT challenge. Mice heterozygous for the J chain deletion exhibited intermediate gut lavage anti-CT IgA and intestinal protection levels, arguing for a J chain gene-dosage effect on the transport of secretory IgA. This study unequivocally demonstrates a direct relationship between mucosal transport of secretory SIgA and intestinal immune protection.
A novel Ets protein was isolated by yeast one-hybrid screening of a cDNA library made from lipopolysaccharide-stimulated mouse splenic B cells, using the SP6 kappa promoter kappaY element as a bait. The novel Ets protein was most closely related to PU.1 and Spi-B within the DNA binding Ets domain and was therefore named Spi-C. However, Spi-C may represent a novel subgroup within the Ets protein family, as it differed significantly from Spi-B and PU.1 within helix 1 of the Ets domain. Spi-C was encoded by a single-copy gene that was mapped to chromosome 10, region C. Spi-C interacted with DNA similarly to PU.1 as judged by methylation interference, band-shift and site selection analysis, and activated transcription of a kappaY element reporter gene upon co-transfection of HeLa cells. Spi-C RNA was expressed in mature B lymphocytes and at lower levels in macrophages. Furthermore, pre-B cell and plasma cell lines were Spi-C-negative, suggesting that Spi-C might be a regulatory molecule during a specific phase of B lymphoid development.
Benzamides have been in clinical use for many years in treatment against various disorders. A recent application is that as a sensitizer for radio- or chemotherapies. We have here analysed the mechanism of action of N-substituted benzamides using an in vitro system. We found that while procainamide was biologically inert in our system, the addition of a chloride in the 3' position of the benzamide ring created a compound (declopramide) that induced rapid apoptosis. Furthermore, declopramide also inhibited NFkappaB activation by inhibition of IkappaBbeta breakdown. An acetylated variant of declopramide, N-acetyl declopramide, showed no effect with regard to rapid apoptosis induction but was a potent inhibitor of NFkappaB activation. In fact, the addition of an acetyl group to procainamide in the 4' position was sufficient to convert this biologically inactive substance to a potent inhibitor of NFkappaB activation. These findings suggest two potential mechanisms, induction of early apoptosis and inhibition of NFkappaB mediated salvage from apoptosis, for the biological effect of N-substituted benzamides as radio- and chemo-sensitizers. In addition it suggests that N-substituted benzamides are potential candidates for the development of anti-inflammatory compounds using NFkappaB as a drug target.
To evaluate the biological effects of over-expression of interleukin-1beta (IL-1beta) on the immune system we have generated transgenic mice, expressing the IL-1beta gene fused to a heterologous signal sequence under the control of the mouse immunoglobulin enhancer (Emu). A prominent hyperplasia and a disturbed microarchitecture of lymphoid tissues were observed in the transgenic mice. The CD4+ T cells in the hyperplastic lymphoid organs seemed to invade the majority of the lymphoid organs including B-cell restricted areas. Analysis of lymph node cells revealed an increased frequency of CD4+ CD44high CD62L- T cells and local secretion of IL-2 and IL-4, compatible with an elevated number of activated T cells. Furthermore, significant levels of human IL-1beta in sera and high concentrations of serum immunoglobulin G (IgG) were observed in the transgenic mice. The data suggest a role for IL-1beta in controlling lymphoid microarchitecture and, when over-expressed, breaking the threshold in T-helper-B-cell interaction.
We have examined the immunoglobulin variable gene usage and antibody affinities during the memory maintenance phase of the immune response to the hapten phenyl-oxazolone. Hapten-specific hybridomas representing the memory population were generated 4-6 months postimmunization. The V-gene expression of these hybridomas was determined by reverse transcriptase-polymerase chain reaction screening and antibody affinities were estimated by biointeraction analysis (BIA) using the BIAcore biosensor. Our results show that the V-gene repertoire has already been shifted during the memory maintenance phase of the immune response, i.e. prior to a second antigenic challenge, and did not entail any advantages in terms of antigen-binding capacities. Our results concur with the view that antibody affinities are modulated mainly through differences in dissociation rates rather than in association rates, and the implications of this with respect to affinity maturation is discussed.
Early B-cell factor (EBF) is a transcription factor suggested as essential for early B-lymphocyte development by findings in mice where the coding gene has been inactivated by homologous disruption. This makes the identification of genetic targets for this transcription factor pertinent for the understanding of early B-cell development. The lack of B29 transcripts, coding for the beta subunit of the B-cell receptor complex, in pro-B cells from EBF-deficient mice suggested that B29 might be a genetic target for EBF. We here present data suggesting that EBF interacts with three independent sites within the mouse B29 promoter. Furthermore, ectopic expression of EBF in HeLa cells activated a B29 promoter-controlled reporter construct 13-fold and induced a low level of expression from the endogenous B29 gene. Finally, mutations in the EBF binding sites diminished B29 promoter activity in pre-B cells while the same mutations did not have as striking an effect on the promoter function in B-cell lines of later differentiation stages. These data suggest that the B29 gene is a genetic target for EBF in early B-cell development.
A protein interacting with an A-T-rich region that is a positive control element within the SP6 kappa promoter was purified and identified as CArG-box binding factor-A. The purified protein was shown to interact specifically with the coding strand of single-stranded DNA and, with lower affinity, with double-stranded DNA. A mutation that inhibited binding of the protein to the A-T-rich region also aborted the transcriptional stimulatory effect of the region. Two Ets proteins, PU.1 and elf-1, that have previously been shown to bind to an adjacent DNA element were shown to physically interact with CArG-box binding factor-A. An antiserum raised against the protein recognized two different forms indicating either that different splice-forms of CArG-box binding factor-A are expressed, or that the protein is subject to post-translational modification.
By mutational analysis of the octamer-TATA box intervening region in the mouse SP6 kappa promoter, we have mapped two octamer-dependent, costimulatory regions, A and B. The A region was active in late B cells only, while the B region was active throughout B cell differentiation. The B region was TATA proximal and contained a heptamer and an E box of the E2A type that is common in Vkappa promoters. Mutation of the heptamer element did not decrease transcriptional stimulation from this region, but mutations in, or immediately 5' of, the E box core sequence did. A protein binding to this region could be detected in nuclear extracts. The complex could only partially be competed with a muE5 binding site and could not be supershifted with Abs raised to E2A gene products, indicating that it may represent a novel E-box binding complex. The A region was located proximal to the octamer and contained a CCCT element that is conserved both with regard to position and sequence in human VkappaII promoters. By mutational analysis, the transcriptional stimulatory activity was mapped to the CCCT element that also is part of an early B cell factor (EBF) binding site. In late B cells, a novel protein (FA), which did not bind to the EBF binding site in the mb1 promoter, interacted with the A region. This protein was found to be expressed at lower levels in early B cells as well as in HeLa cells. Thus, the octamer-flanking sequence contains positive control elements that may act independently but that differ in the stage of B cell differentiation at which they are active. One of these factors is an example of an ubiquitously expressed transcription factor that participate in differentiation-specific transcriptional activation.