Engineering therapeutic solutions that involve cell harvesting using microfabricated devices or the targeting of either cells or drug delivery vehicles to specific sites in the vasculature will be facilitated by a detailed understanding of the mechanisms that cells themselves use to target specific sites of injury or infection. For example, strategies for harvesting cells in microdevices may involve the use of natural signals to induce cell adhesion and arrest on natural ligands. Understanding the dynamics of increased cellular adhesion in response to specific signals should facilitate the development of such approaches. In the present report we focus on the response of neutrophils to the immobilized chemokine interleukin-8 (IL-8), identifying critical signaling intermediates in the context of dynamic changes in neutrophil adhesion to the endothelial ligand ICAM-1 (intercellular adhesion molecule-1).
The importance of neutrophil adhesion to endothelium and its regulation is evident in the extensive literature on this topic. It is well known that in the human system, the β2
integrins LFA-1 (αL
, aka CD11a/CD18) and Mac-1 (αM
, aka CD11b/CD18) are critical mediators of neutrophil arrest and migration on inflamed endothelium7, 9, 36
, and furthermore that LFA-1 activity tends to precede Mac-1 mediated interactions12, 28, 29
. However, to our knowledge, differences in the contributions of the different integrins have been observed either in vivo or in vitro in situations where the specific nature and timing of the stimulus, and the subsequent signaling intermediates are not well known. For the integrins to bind their counter-receptors on the endothelium they must be activated, and the mechanisms leading to this activation have also received considerable scrutiny. E-selectin mediated cell rolling has been implicated as an activator of integrins both in vitro35
and in vivo37
, and activation of neutrophil integrins by chemokines, particularly IL-8, is well-documented5
. Two receptors for IL-8 are expressed on neutrophils: CXCR1 and CXCR21
. Both of these are G-protein coupled receptors (GPCR), and their ligation by IL-8 leads to integrin activation23, 32
. Identification of principal pathways that lead from chemokine binding to integrin activation provides critical information in understanding the specific roles that different molecules play in determining neutrophil behavior.
Although chemokines can be released by endothelium into the circulation in soluble form, activation of leukocyte integrins by circulating chemokines may be unfavorable, as it would trigger integrin-mediated arrest remote from the chemokine secretion site. Early work was focused on effects of soluble chemokines5, 31, 32, 42
, but new evidence demonstrates that immobilized chemokines stimulate integrin adhesiveness to endothelial ligands and promote cell motility in a much more successful manner than soluble forms8, 16, 33, 44
. Thus, understanding the dynamics of the neutrophil response to immobilized chemokines is of central physiological relevance, and is also relevant to device design, where it might be advantageous to localize specific cell stimuli to surfaces.
Previously, we have shown that interaction between immobilized IL-8 and human neutrophils results in β2
integrin activation, as assessed by changes in adhesion probability to immobilized ICAM-123
. Integrin activation starts several minutes after the initial contact of the cell with IL-8, and an additional 3 to 5 minutes elapses before the adhesion to ICAM-1 reaches its maximum. This long delay between IL-8 contact and integrin dependent adhesion prompted us to identify signaling pathways induced by immobilized IL-8 in human neutrophils. In these studies we employ a novel micromechanical approach that allows us to control very precisely the time of interaction as well as quantities of interacting molecules. Using different inhibitors to block specific signaling molecules, we were able to identify pathways that are and are not involved in signal transduction from IL-8 binding to its counter receptor on the surface of neutrophils to integrin-mediated adhesion. We show that this process is critically dependent phospholipase C (PLC) and subsequent on inositol 1,4,5-triphosphate (IP3) dependent calcium release, but is independent of phosphoinositide 3 kinase (PI3K) or protein kinase C (PKC) activity. We also present evidence for different temporal responses and different dependence on p38 mitogen activated protein kinase (MAP kinase) activity of LFA-1 and Mac-1 and their contributions to the adhesion.