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Die Bedeutung von Zinkionen für mononucleäre Leukocyten
No full textZinc is an essential trace element for the immune system. In this work, important influences of zinc on mononuclear cells were examined.For the first time, a direct stimulation of cells by zinc could be shown, which was dependent on zinc concentration and could not be induced by other cations. Even in physiological doses, zinc interacts significantly with intracellular processes. Cell-lines as well as primary cells express the zinc uptake transporters hZIP1, hZIP2 and hZIP3. Quantitative analyses showed, that hZIP3 is the main zinc transporter in monocytes, T-cells and PBMC, whereas hZIP1 is the highest expressed zinc transporter in B-cells. hZIP2 was only detectable at a very low level in all examined cells. All three transporters were upregulated in response to zinc deprivation. The assessment of transporter expression in cell lines and in primary cells showed a five- to tenfold expression in primary cells (PBMCs) in comparison with cell lines. Also, a decrease in transporter expression was detectable when primary cells were induced to proliferate by the superantigen SPEA or were immortalised by Epstein-Barr-Virus. A similar phenomenon could be observed in vivo, since hZIP transporter expression in PBMC of patients with chronic lymphatic leukaemia was significantly decreased when compared to the PBMC of patients with bronchial carcinoma. Additionally, proliferation assays demonstrated a central role of zinc in apoptosis.An examination of the interaction of zinc with intracellular signal transduction showed that zinc is able to inhibit IL-4 mediated proliferation of the T cell line CTLL-2. Contrary, zinc in combination with IL-4 acted synergistically on the B cell line B9 and induced a significantly higher proliferation rate than IL-4 alone. Both cell lines differ in their CD45 expression. Furthermore, zinc was able to abolish the inhibitory effect of an anti-CD45 antibody on the mixed lymphocyte culture (MLC).The mechanisms of direct and indirect zinc stimulation and the function of zinc uptake transporters that were examined in this work emphasize the importance of zinc for mononuclear cells in vitro and in vivo
Die Rolle des neutrophilen Granulocyten innerhalb des Immunsystems charakterisiert anhand seiner Cytokinproduktion
No full textApproximately 60–70% of the leukocytes in peripheral blood are neutrophils (PMN), which are the first cells to migrate into infected tissue. They provide innate immunity through phagocytosis of invading pathogenes.For a long time, the role of PMN in the immune system has been regarded as mere phagocytes, however recent studies have described cytokine production by PMN with results that are quite controversial. Despite a similar setup of the experiments, the results vary considerably, so that purity of the examined cells can be questioned. Aim of a thesis proceeding this work was therefore to establish an isolation protocol, obtaining PMN of a high purity and with no detectable pre-activation from peripheral blood.In the course of the present thesis it could be shown that these neutrophils of a high purity do not produce the pro-inflammatory cytokines IL-1ß and IL-6. However, differential production of IL-8 and of two isoforms of IL-1RA could be detected on mRNA and on protein-level. While synthesis of IL-8 and of the intracellular IL-1RA variant (icIL-1RA) increases only slightly after stimulation, the secreted isoform sIL-1RA can be strongly induced by LPS and also by GM-CSF. Contrary to sIL-1RA, icIL-1RA of 16 kDa and IL-8 can be found in unstimulated cells as well.Using subcellular fractionation techniques, icIL-1RA could be localized in the cytoplasm, and sIL-1RA and IL-8 could be found in a fraction containing the Golgi apparatus. Presence of the examined cytokines in neutrophilic granules could be excluded. However, preformed IL-8 could be detected in the cells, ready to be released after ligation of CD66b.Further, CD34+ hematopoietic stemcells could be differentiated into granulocytic cells. With these cells, the cytokine profile of mature Neutrophils could be confirmed. The in vitro differentiated granulocytic cells did not show any expression of IL-1ß and IL-6. IL-1RA and IL-8, however, were detected. Monocytic cells, differentiated from stemcells of the same donor, showed expression of the pro-inflammatory IL-1ß and IL-6 in addition to IL-1RA and IL-8. This points to a cell-type specific expression of IL-1ß and IL-6 in monocytic, but not in granulocytic cells.As the first cells to enter the site of an infection, neutrophils dominate the surrounding milieu with their anti-inflammatory cytokine profile. By the release of preformed IL-8 during extravasation, a track may be established for the succeeding cells to follow. As anti-inflammatory cells, neutrophils are essential for the control and the ending of inflammatory reactions.Moreover, a “quiet” infection may be postulated, during which neutrophils may able to eliminate the pathogens in the absence of pro-inflammatory cytokines. In spite of daily contact with pathogens, an inflammatory reaction rarely develops. neutrophils therefore represent an important and unique part in immune regulation
Die Regulation der Zinktransporter und ihr Einfluss auf die intrazelluläre Zinkhomöostase in Leukocyten
No full textZinc is an essential trace element for the immune system. During this work, important mechanisms regarding the intracellular zinc homeostasis and its influence on intracellular signal transduction in mononuclear cells could be elucidated. The analysis of the expression of zinc exporters, together with the characterisation of zinc importers, has led to the finding that immortal cells maintain their intracellular zinc homeostasis at a lower level due to an overall lower expression of zinc transporters. Under physiological conditions, hZnT-1 was the main transporter in Raji cells and PBMCs, whereas hZnT-5 and hZnT-6 were most highly expressed in THP-1 and Molt-4 cells as well as in purified primary B and T cells, respectively. Furthermore, the regulation of intracellular zinc homeostasis in response to zinc stimulation during zinc deficiency was established. After initial zinc uptake, the zinc concentration in Molt-4 cells decreases due to the newly-discovered membrane-localised hZnT-4, whereas the zinc concentration in Raji and THP-1 cells increases due to the induced elevated expression of the zinc exporters hZnT-5, hZnT-6 and hZnT-7 under zinc depletion. In addition, the up-regulation of hZnT-4 in response to stimulation with PHA illustrates the important function of this zinc exporter for the regulation of the zinc concentration in leukocytes during an activation process. Moreover, the reduced expression of the zinc exporter hZnT-8 in type 1 and type 2 diabetics demonstrates the tremendous significance of this transporter for both diseases. While hZnT-1, hZnT-4, hZnT-5, hZnT-6 and hZnT-7 are fundamental for maintaining zinc homeostasis and thereby associated with the optimal functionality of leukocytes, hZnT-9 as well as hZnT-3 and hZnT-2 seem to play a minor role in the immune system. The influence of zinc on intracellular processes such as apoptosis and the signal transduction induced by interleukins were also characterised in more detail. On the one hand, it could be shown for the first time that an activation of T cells by IL-1beta under zinc deficiency results in increased activity, which correlates with the intracellular zinc concentration. On the other hand, the molecular mechanism for the influence of zinc on the IL-4 signal transduction in T cells could be elucidated. The mechanisms for regulation of intracellular zinc homeostasis and functionality of zinc in cells of the immune system that were established under in vitro conditions provide new therapeutic options for zinc substitution
Charakterisierung der Eigenschaften des Mycoplasma arthritidis Superantigens
No full textIn the present study the features of the Mycoplasma arthritidis-derived superantigen (MAM) were characterized. The Mycoplasma genome exhibits a characteristically low G + C content. Apart from very few exceptions, the G + C content of Mycoplasma genomes is within the range of 24 to 33 mol%. That' s the reason why the G + C content of MAM was increased from 28% to 47%.First of all the codon usage patterns of Escherichia coli were compared with that of Mycoplasma arthritidis in order to optimize the expression of MAM in E. coli. The codons in question were altered to the optimal codon for E. coli. These steps increased the yield of recombinant MAM.With site-directed mutagenesis (two-step PCR mutagenesis) amino acid residues of potential binding sites of MAM were substituted by alanine or asparagine and glutamine. The functional activity of MAM mutants was examined with HLA-DR transfected L-cells or B-cells and with V beta-transfected T-cells. The results show that histidine residue 14 and aspartic acid 31 are not involved in zinc-dependent binding of MAM to the MHC-class-II-complex. Here it is clearly shown that histidine 58 is an essential residue for the function of MAM. Because substitution of histidine 58 by alanine interrupts presentation of MAM by MHC-class-II. On the other hand glutamic acid residues 28, 30, 39, 40, and 43 are also dispensable, since its substitution don' t interfere with MHC-II- binding. The alanine mutants are still able to interact with MHC-II-complex. However glutamic acid residue 28 plays an important role in interaction with the T-cell receptor. MAM mutant Glu28Ala failed to activate T-cells.Up to now the exact role of zinc ions for MAM dimerization and function need further investigation.As a further interaction partner of MAM CD1-positive cells of the immune system were identified. The results obtained in the present work point out an obviously binding of MAM to CD1a, CD1b, CD1c positive cells. The best interaction of MAM was observed with CD1c.Moreover the data of growth curves presented here indicate a toxic effect of MAM to E. coli. In comparison with Staphylococcal enterotoxin A and Toxic shock syndrome toxin only MAM inhibits growth and viability of E. coli. No protease-activity of MAM could be detected, but the experimental series revealed DNase-activity of MAM. The inhibitory effect of MAM after overexpression on the host bacteria is explained by the fact of DNase-activity displayed by MAM.This outcome suggests the existence of bifunctional proteins with superantigen-activity. In conclusion these proteins are not only potent stimulators of the immune system but also manage enzymatic tasks of the pathogen.In the future the sequence alignment of proteins will promote the discovery of superantigens with interesting features. Once more the unusual characteristics of MAM indicate the existence of proteins with superantigen activity not corresponding to the classical superantigen protein fold
The characterization of major histocompatibility complex class II signal transduction pathways in antigen presenting cells
No full textMajor histocompatibility complex class II (MHC-II) molecules, which can be broken down into three different human isotypes, HLA-DR, HLA-DP, and HLA-DQ, are expressed on a wide range of immune cells including B lymphocytes, monocytes/macrophages and dendritic cells. Although MHC-II molecules are well-known for their ability to present antigens to helper T cells, MHC-II molecules can also contribute to signaling pathways that govern the function and survival of antigen presenting cells. Not all signaling pathways linked to MHC-II molecules have been identified, and therefore the major goal of this work is to further characterize the pathways initiated by MHC-II ligation. MHC-II-induced signaling pathways involve a combination of different kinases and transcription factors that lead to different cellular responses such as proliferation, differentiation, and apoptosis. These signaling components were further characterized and new pathways were identified by activating MHC-II molecules with anti-MHC-II antibodies and superantigens. This dissertation examines MHC-II-mediated calcium mobilization and demonstrates that it is an isotype-independent event, which leads to the dephosphorylation of NFAT. In addition, MHC-II molecules work in conjuction with the B cell receptor to increase NFAT activity. As opposed to calcium mobilization, the classical MAP kinases are activated in an isotype-specific manner where only ligation of HLA-DP and HLA-DQ lead to ERK1/2 and MEK1/2 phosphorylation, which corresponds with an increase in c-Fos protein expression and AP-1 dimer formation. MHC-II most likely requires one signaling partner for the activation of tyrosine kinases leading to calcium mobilization and NFAT dephosphorylation and a different signaling partner for triggering the MAP kinase cascade, whereby the restriction of HLA-DR with the cytoskeleton prevents HLA-DR from associating with the signaling partner. Two important cellular responses were also examined including apoptosis and immunoglobulin synthesis. Apoptosis could only be initiated by crosslinking MHC-II molecules with whole antibodies or Fabs crosslinked with a secondary antibody. MHC-II-mediated cell death was found to occur in an ERK1/2-independent manner. Immunoglobulin synthesis was dramatically reduced after superantigen binding, and this can be explained through the loss of B cells by T cell activation. Anti-MHC-II antibodies can also reduce antibody production, but this cannot be attributed to a decrease in viable B cells. Instead, anti-MHC-II antibodies prevent pokeweed mitogen from carrying out its function in differentiating naïve B cells into plasma antibody-secreting B cells. The novel discovery of the activation of NFAT by MHC-II may also explain the change in antibody production during the differentiation of B lymphocytes. Altogether, this work expands upon known signaling mechansisms as well as illustrates new signaling pathways generated by ligating MHC-II molecules. Extending our understanding on MHC-II-mediated signaling can help provide new therapeutic tools for the fields of cancer, autoimmunity, and transplantation therapy
Die Regulation der Zinktransporter und ihr Einfluss auf die intrazelluläre Zinkhomöostase in Leukocyten
No full textZinc is an essential trace element for the immune system. During this work, important mechanisms regarding the intracellular zinc homeostasis and its influence on intracellular signal transduction in mononuclear cells could be elucidated. The analysis of the expression of zinc exporters, together with the characterisation of zinc importers, has led to the finding that immortal cells maintain their intracellular zinc homeostasis at a lower level due to an overall lower expression of zinc transporters. Under physiological conditions, hZnT-1 was the main transporter in Raji cells and PBMCs, whereas hZnT-5 and hZnT-6 were most highly expressed in THP-1 and Molt-4 cells as well as in purified primary B and T cells, respectively. Furthermore, the regulation of intracellular zinc homeostasis in response to zinc stimulation during zinc deficiency was established. After initial zinc uptake, the zinc concentration in Molt-4 cells decreases due to the newly-discovered membrane-localised hZnT-4, whereas the zinc concentration in Raji and THP-1 cells increases due to the induced elevated expression of the zinc exporters hZnT-5, hZnT-6 and hZnT-7 under zinc depletion. In addition, the up-regulation of hZnT-4 in response to stimulation with PHA illustrates the important function of this zinc exporter for the regulation of the zinc concentration in leukocytes during an activation process. Moreover, the reduced expression of the zinc exporter hZnT-8 in type 1 and type 2 diabetics demonstrates the tremendous significance of this transporter for both diseases. While hZnT-1, hZnT-4, hZnT-5, hZnT-6 and hZnT-7 are fundamental for maintaining zinc homeostasis and thereby associated with the optimal functionality of leukocytes, hZnT-9 as well as hZnT-3 and hZnT-2 seem to play a minor role in the immune system. The influence of zinc on intracellular processes such as apoptosis and the signal transduction induced by interleukins were also characterised in more detail. On the one hand, it could be shown for the first time that an activation of T cells by IL-1beta under zinc deficiency results in increased activity, which correlates with the intracellular zinc concentration. On the other hand, the molecular mechanism for the influence of zinc on the IL-4 signal transduction in T cells could be elucidated. The mechanisms for regulation of intracellular zinc homeostasis and functionality of zinc in cells of the immune system that were established under in vitro conditions provide new therapeutic options for zinc substitution
Entwicklung einer Multiplex-PCR-basierten Methode zur Identifizierung von Toxin-bildenden Streptococcus-pyogenes-Stämmen
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Entwicklung einer Multiplex-PCR-basierten Methode zur Identifizierung von Toxin-bildenden Streptococcus-pyogenes-Stämmen
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