Abstract EN:

Type 1 diabetes is a disease that results from a disturbed glucose metabolism due to a deficiency in insulin production. This deficiency is the consequence of immune-mediated damage to the insulin-producing p-cells. The cause of type 1 diabetes is presently unknown and probably multifactorial. The initiation and progression of the inflammatory process that destroys the p-cells involves the interplay of environmental factors with an autoimmune-prone genetic background. Abnormal immune regulation explains the autoimmune phenomena observed in diabetic patients and in spontaneous animal models for the disease to a limited extent only. The precise reason for the immune system to target the pancreatic islets of Langerhans is still unclear. The pathogenic process in the pancreas is characterized by the pathology-related intra-islet infiltration of T and B-lymphocytes that mediate islet destruction. This T and B-celUnfiltration is preceded by an accumulation of macrophages and dendritic cells at the islet periphery. The early peri-islet accumulation of these antigen presenting cells probably reflects the first response of the immune system that is progressively heading for islet destruction. Macrophages are involved in every step of the diabetogenic process. In the non-obese diabetic (NOD) mouse that spontaneously develops diabetes, various macrophage-abnormalities like defective maturation, reduced phagocytosis and increased production of IL-12, have been described previously. Moreover, macrophages are present in higher numbers in the pancreas of the NOD mouse from birth onwards, randomly distributed in the connective tissue and exocrine parenchyma. In this thesis we present the results of our studies on the murine pancreatic macrophage compartment. We have questioned in particular the possible underlying causes for the abnormal early peri-islet accumulation of macrophages. Therefore, the studies were performed with an emphasis on the interactions of macrophages with the extracellular matrix of the pancreatic connective tissue. In our first study we show that the depletion of macrophages and dendritic cells from the endocrine pancreas was accompanied by a total disappearance of lymphocytes from the pancreas. Hence, pancreatic macrophages and dendritic cells are critically involved in the local progression of islet inflammation in NOD mice by mediating the retention and possibly the recruitment of lymphocytes to the pancreas. Importantly, this depletion significantly postponed the onset of diabetes, leading to a strongly decreased incidence by 35 weeks of age. In the second study described, we have phenotypically characterized the pancreatic macrophages. The majority of macrophages were characterized by the expression of MMGL and sialoadhesin. Only a minority of these pancreatic macrophages expressed the macrophage marker F4/80 under non-inflammatory conditions. By contrast, macrophages expressing F4/80 were observed massively before the onset of destructive insulitis in the NOD pancreas, and their presence is particularly associated with islet destruction. This suggests that F4/80 identifies a particular subset of inflammatory macrophages in the pancreas that are virtually absent under non- inflammatory conditions. Interestingly, in the fetal pancreas mature macrophages were exclusively identified by their expression of F4/80 and these macrophages lacked the expression of MMGL and sialoadhesin. In addition, we present data that support the conclusion that macrophages develop from pre-existing precursors that are present in the fetal pancreas at E12. 5. Using an in vitro approach, we demonstrated that their numbers significantly increased in fetal pancreas explants cultured with M-CSF. This increase of F4/80-positive cells was paralleled by an increase in the number of insulin-producing cells, suggesting that macrophages support insulin-cell growth in vitro. These results are in line with the results of the third study that is presented in this thesis. Early postnatal pancreases from NOD mice are characterized by an increase in the percentage of endocrine tissue and by enlarged and irregularly-shaped islets, concomitantly with the presence of increased numbers of macrophages. Importantly, the levels of the extracellular matrix protein fibronectin are significantly increased during this pre-weaning period as well. Fibronectin labeling was mostly localized at the islet-ductal pole, islet periphery and in intralobular septa. Interestingly, pancreatic macrophages mainly reside at sites with intense fibronectin-labeling. In a fourth study we demonstrate that, paradoxically, NOD macrophages exhibit impaired fibronectin-mediated adhesion and migration due to the defective expression of the integrin-type fibronectin receptor a-chain, CD49d. In addition, we show that extracellular-regulated kinase-1/2 (ERK-1/2) is a negative regulator of CD49d expression in macrophages. NOD macrophages were characterized by increased levels of the activated form of this kinase when stimulated with the toll-like receptor-4 (TLR-4) ligand, lipopolysaccharide (LPS). LPS-stimulation resulted in the impaired upregulation of CD49d levels in NOD macrophages as compared to macrophages of other mouse strains. We believe that this specific defect in the macrophage compartment of NOD mice might play a role in the observed peri-islet accumulation. At the end of this thesis, we have postulated the working hypothesis that the observed presence of higher levels of the endogenous TLR-4 ligand fibronectin in the NOD pancreas in combination with the inappropriate TLR-4-responsiveness of NOD macrophages contributes to their local retention and activation. This unfortunate activation may damage the neighboring islets, unintentionally provoking an immune response eventually leading to the development of autoimmunity. We have uncovered several abnormalities that may trigger a mechanism that possibly contributes to the development of spontaneous autoimmune diabetes in the NOD mouse. Whether similar abnormalities are present in type 1 diabetic patients remains to be established.