In the vasculature, inflammation leads not only to the expression of new adhesive ligands on the endothelial cell surface, but also the expression of signaling molecules (chemokines) that attract and activate leukocytes. An important chemokine that acts on neutrophils is IL-8, which is typically found immobilized on carbohydrate structures (the gylcocalyx) on the endothelium. In order to imitate the natural presentation of chemokines to neutrophils, we immobilize IL-8 on a fractalkine stalk on the surface of a bead. By this approach, we can control the timing of the exposure of the cells to stimulus very precisely. We have completed several projects in this area, and continue to pursue others.
Dynamics of adhesion increase after IL-8 stimulus. In this first paper, we used micropipettes to maneuver and control contact between a neutrophil and two types of beads, one coated with the chemikine IL-8 and one coated with the endothelial ligand ICAM-1. The beads were different sizes so we could tell them apart. First we tested adhesion between a neutrophil and the ICAM-1 coated bead to get a baseline for the adhesiveness of the cell for ICAM-1. Then we contacted the cell with an IL-8 bead and monitored the adhesiveness of the cell for ICAM-1 over time by continuously tapping the cell against the ICAM bead and counting the fraction of contacts that resulted in adhesion. The readout is the adhesion probability as a function of time. Essentially, this is a functional test for the activation of beta-2 integrins on the cell surface from their low affinity state to their high affinity state. To our surprise, the first thing ewe observed was that the cell phagocytosed the IL-8 bead! Then after a delay of several minutes, the cell began to adhere to ICAM-1 bead, indicating a long delay before integrin activation.
Blocking calcium release blocks the increase in adhesion
In a second series of experiments we began to explore the signaling pathways connecting the IL-8 stimulus to integrin activation. We used different drugs to block different signaling molecules within the cell. We found that the IL-8 receptor signaled through Phospholipase C to induce calcium release within the cell, and that these steps were critical for the response. Interestingly, the phagocytosis step preceded even when these pathways were inhibited, although in a modified way. Most interestingly, by using molecules to block the different beta-2 ntegrins, we found that the time course of the response was faster for LFA-1 than it was for Mac-1, and furthermore, that Mac-1 was dependent on p38MAP kinase activity, whereas LFA-1 was not. This is the first clear evident that we are aware of that shows differential responses of these two integrins to a common chemokine stimulus.
Signaling pathways leading to integrin activation after IL-8 stimulus
We are continuing these studies by looking at the dynamics of calcium release after the cell contacts an IL-8 coated bead. In this part of the project w are collaborating with the Hammer lab at the University of Pennsylvania to construct an analytical model of the signaling response within the cell.