Die Rolle des neutrophilen Granulocyten innerhalb des Immunsystems charakterisiert anhand seiner Cytokinproduktion

Abstract

Approximately 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