Molecular mechanisms of sorting and secretion of chemokines in vascular endothelial cells
Sorting and secretion of chemokines in endothelial cells
Cardiovascular disease and ischemic damage remain the chief cause of death in the Western world, and therapeutic efforts to restore blood flow (thrombolysis) have become established protocols. However, such reperfusion can place ischemic organs at risk of further cellular necrosis and limit the recovery of function.
The molecular changes in I/R injury are those of an acute inflammation, with cells of the innate immune system rapidly adhering to activated endothelial cells. A requisite event in this process is the stepwise engagement of juxtaposed neutrophil and endothelial cell adhesion molecules. Chemokines are small, secreted proteins that bind chemokine receptors on leukocytes and mediate firm adhesion to the endothelial surface. The prototypic neutrophil-recruiting chemokine interleukin-8 (IL-8)/CXCL8 can be stored in cytoplasmic Weibel-Palade bodies (WPB) of endothelial and released in response to inflammatory insults. In addition to IL-8, we demonstrated in the course of the current project that a number of other chemokines can be stored in vascular endothelial cells and released upon stimulation of exocytosis: Eotaxin-3/CCL26 is induced by the allergy-associated cytokine IL-4, and like IL-8 it is sorted to WPB. In contrast, the IL-1ß-inducible chemokines growth-related oncogene-a (GRO-a)/CXCL1 and monocyte chemotactic protein-1 (MCP-1)/CCL2 can both be stored in a compartment for regulated chemokine secretion that was recently discovered by our research group and designated type II granules of regulated secretion in endothelial cells. Together these chemokines are able to mediate the tissue recruitment of eosinophils, neutrophils and monocytes in acute inflammation.
We explored the molecular properties of chemokine sorting in endothelial cells and designed chimeric proteins between IL-8 and the constitutively secreted IP-10. This project has been a fruitful collaboration with Bjørn Dalhus and Magnar Bjørås from the Laboratory for molecular biology at the Centre of Excellence in Neurobiology at our hospital. To further highlight individual residues, we also mutated amino acids of interest to alanine, lysine or serine. All constructs including wild-type IL-8 were transfected into human umbilical vein endothelial cells (HUVECs). By evaluating the sorting of mutant chemokines to WPB by immunocytofluorescence, we have revealed the importance of aspartic acid 45 (D45) in loop 2 of IL-8 by observing that mutation of this residue to alanine or lysine abolished sorting to WPB. Furthermore, when we grafted loop 2 (44SDG47) and the C-terminal a-helix of IL-8 into IP-10, the chimeric protein exhibited a sorting efficiency similar to that of WT IL-8. To our knowledge, this was the first time a protein not usually present in WPB has been directed to this organelle by molecular manipulation.
In rodents, the adhesion molecule P-selectin is stored in WPB in a similar manner to man, but endothelial storage of chemokines has hitherto not been demonstrated. Moreover, no IL-8 homologue exists in mice and rats and we therefore investigated related chemokines like KC/CXCL1, MIP-2/CXCL2 or LIX/CXCL6 which are believed to compensate for its role in neutrophil recruitment. However, none of them possess the characteristic loop 2 signature of IL-8 that we have shown to be essential for sorting to WPB in human endothelial cells. Perhaps accordingly, we found no evidence for sorting of these chemokines to WPB in murine vessels. Instead, it looks like they may be sorted to smaller granules similar to the type II compartment we discovered in human vessels. Unfortunately, that compartment is currently defined by function rather than phenotype, and further characterization in the mouse will require studies of murine cell cultures.
In conclusion, this short-term HSØ grant supported the completion of Johanna Hol’s PhD thesis which was submitted in august 2009. It led to the successful mapping of molecular determinants responsible for sorting of chemokines to compartments of regulated secretion in vascular endothelial cells. It also mapped the sorting of chemokines in the mouse to evaluate it as a useful experimental animal model.
Molecular requirements for sorting of the chemokine interleukin-8/CXCL8 to endothelial Weibel-Palade bodies.
J Biol Chem 2009 Aug;284(35):23532-9. Epub 2009 jul 3
The murine IL-8 homologues KC, MIP-2, and LIX are found in endothelial cytoplasmic granules but not in Weibel-Palade bodies.
J Leukoc Biol 2010 Mar;87(3):501-8. Epub 2009 des 9
Are Chemokines Stored for Regulated Secretion in Rodent Endothelial Cells?
2008 (ikke rapportert i fjor)