Expressie en mogelijke functie van granulocyt chemotactisch proteïne-2 (GCP-2/CXCL6) in inflammatoïre darmziekten en gastrointenstinale tumoren.

摘要

During evolution, the immune system of humans has adapted itself to confer protection to a wide range of stress conditions. Besides physical barriers, the human body is equipped with an army of immune cells consisting of different subsets, each fulfilling specific tasks. In case of inflammation due to infection, tumor growth or tissue damage, our body relies on the interplay of the innate immune response, acting fast and non-specific, and the adaptive immune response, acting slower but specific. Neutrophils are present in the blood circulation and belong to the immune cells responding first to microbial agents. Upon stimulation, they leavethe blood circulation and enter the inflamed tissue to scavenge harmfulmicroorganisms. When the activation of neutrophils persists or is initiated too early, they can cause severe tissue damage due to the release of toxic metabolites. By attracting and activating immune cells, chemokines play a crucial role in the onset and perpetuation of inflammatory reactions. Chemokines are small proteins that are released by many different cell types upon stimulation with proinflammatory mediators. Besides leukocyte recruitment in response to physiological stress, chemokines are also responsible for basal leukocyte trafficking to secondary lymphoid organs during homeostasis. In many diseases, overexpression of chemokinescauses an uncontrolled immune response that leads to chronic inflammation. Crohn’s disease and ulcerative colitis are inflammatory bowel diseases (IBD) that are characterized by chronic intestinal inflammation and aconstant influx of leukocytes, mediated by proinflammatory cytokines and chemokines. Granulocyte chemotactic protein‑2 (GCP‑2/CXCL6) is an angiogenic chemokine that selectively attracts neutrophilic granulocytes. Functionally, it shows high homology with interleukin‑8 (IL‑8/CXCL8) and both chemokines bind the same G protein-coupled receptors (CXCR1 and CXCR2) to exert their activity. In this thesis, the intestinal expression of the chemokines IL‑8 and GCP‑2 and the participation of immunocompetent cells in IBD were evaluated. IL‑8 production by peripheral blood mononuclear cells (PBMC) from IBD patients, stimulated with bacterial endotoxin, viral double-stranded RNA or plant lectin, was significantly lowered in patients with Crohn’s disease, but not in ulcerative colitis patients or healthy subjects. The reduced chemokine production by PBMC from IBD patients was both IL‑8 and Crohn’s disease specific, but not inducer dependent. In the serum of IBD patients, chemokine levels were not detectable or remained unaltered compared with control subjects. GCP‑2, but not the structurally related chemokine epithelial cell-derived neutrophil attractant‑78 (ENA‑78/CXCL5), nor IL‑8, was expressed by endothelial cells in inflamed intestinal tissue of IBD patients. In contrast, in vitro stimulated macrovascular endothelial cell cultures produced more IL‑8 than GCP‑2. The selective GCP‑2 staining of endothelial cells at sites of epithelial damage suggests that GCP‑2, despite its low production level in vitro, plays a role in IBD, different from that of structurally (ENA‑78) and functionally (IL‑8) related chemokines. Furthermore, we have shown that GCP‑2, IL‑8 and monocyte chemotactic protein‑1 (MCP‑1/CCL2) are co‑induced in microvascular endothelial cells after stimulation with proinflammatory stimuli. To mimic endothelial cell‑derived GCP‑2 in vivo, GCP‑2 was intravenously injected in rabbits and shown to provoke a dose‑dependent systemic response, composed ofan immediate granulopenia, followed by a profound granulocytosis. Moreover, in vitro GCP‑2 synergized with MCP‑1 in neutrophil chemotaxis which may represent a mechanism for tumor developmentand metastasis by providing efficient leukocyte infiltration. Immunohistochemical analysis of inflamed intestinal tissue sections from IBD patients suggested that GCP‑2 is specifically expressed by newlyformed blood vessels.The precise role of chemokines in neovascularization during inflammation or tumor growth is not yet fully understood. By immunohistochemistry, GCP‑2 was further shown to be expressedby endothelial cells from human patients with gastrointestinal malignancies. GCP‑2 staining correlated with leukocyte infiltration into the tumor and with the expression of the matrix metalloproteinase gelatinase B that facilitates angiogenesis by degrading the extracellularmatrix. Together, these data suggest that the expression of GCP‑2 and IL‑8 is differently regulated in vitro and in vivo and that GCP‑2 and IL‑8 have their own role ininflammatory processes although they are structurally and functionally related. Thus, the chemokine network shows complementarity, rather than redundancy.