Inside-out signalling of integrins on granulocytes

Eosinophils are associated with allergic diseases such as allergic rhinitis and allergic asthma (Gleich et al. 1986). These cells accumulate in the affected tissues and contribute to the chronic inflammatory reaction which is characteristic of these allergic diseases (Wardlaw et al. 1994). To migrate from the blood to the inflamed tissues, eosinophils use a specific set of adhesion and chemokine receptors (Wardlaw 2001). Rolling, activation, firm adhesion, polarization and extravasation are the sequential steps of leukocytes to migrate out of the blood into the tissue (Figure 1). In general, rolling interactions are mediated by selectins binding to mucin like structures bearing specific carbohydrate moieties. Subsequently, firm adhesion is initiated by activation of the cells through chemoattractants signaling via G protein coupled receptors. These processes result in different states of integrin activation depending on the chemoattractant present (Ulfman et al 2005). Activated integrins bind to vascular adhesion molecules of the Ig Superfamily which eventually results in firm adhesion. Finally, the leukocytes pass the endothelium either para- or transcellular (carman et al 2004).

Figure 1. Chain of events leading to leukocyte emigration and activation: Firm adhesion is mediated by integrins of which α4 integrins on eosinophils are special in the fact that they also contribute to initial attachment

Integrins are heterodimers that consist of an α and a β chain that are non-covalently linked. These integrins are under "inside-out" control: intracellular signals that are induced by inflammatory stimuli (e.g. chemokines) integrate at the intracellular tail of the integrin resulting in an active integrin receptor (Hynes 2002). Subsequently, the functional active integrin is able to bind ligand. Both changes in conformation (affinity) as well as in clustering (valency) have been described to contribute to α4-integrin activity (Chigaev et al. 2003, Grabovsky et al. 2000). An important characteristic of α4-integrins ( α4β1 and α4β7) on lymphocytes, monocytes as well as on eosinophils is that they can pre-exist as adhesion receptors with an intermediate functionality without the apparent need for cellular activation (Chigaev et al. 2001, Johnston et al. 1996, Berlin et al. 1995, Ulfman et al. 1999). Indeed, we have previously shown that α4-integrins on resting, non-activated eosinophils contribute to recruitment to activated endothelial cells (Ulfman et al. 1999). Inhibition of α4-integrins on freshly isolated eosinophils resulted in a 50% decrease in total adhesion. Furthermore, a 200% increase in percentage rolling cells compared to control Moab treated cells was observed, being the consequence of rolling on E-selectin. Overall, α4-integrins are important for the recruitment of leukocytes to inflammatory sites in vivo and, therefore it is important to elucidate the inside-out control mechanisms that control the intermediate and fully active conformations of the α4-containing integrins (Rose et al. 2002).

Current research question:
How is basal α4 integrin function on primary eosinophils regulated?

Approach We addressed this question using several approached: first a pharmacological approach was used since primary cells are hard to transfect with constructs of interest. However, this is possible in CD34+ hematopoietic cells. In collaboration with Miranda Buitenhuis, we combined transduction of cells with a functional adhesion assay. For this CD34+ hematopoietic progenitor cells were transduced with EGFP or constructs of interest (also visible by EGFP) and a fluorescent beads assay was performed. For this, cells were incubated with VCAM-1 coated beads in the absence or presence of an α4 integrin blocking antibody. After 30 minutes of incubation cells were detected by flow cytometer. The EGFP+ cells were gated and the amount of positive events for binding to VCAM-1 coated beads was measured (Figure 2). The advantage of this method is that you do not have to sort the EGFP+ cells before performing a functional assay.

Figure 2. Three day old CD34+ hematopoietic progenitor cells were transduced with EGFP virus particles. In A the size of the cells is visible as determined by the forward/sideward scatter. In B the R2 gate shows all the EGFP positive cells. In C the number of cells (from R1+R2) that bound to fluorescent beads (which are visible in the FL3 channel) are shown.

The data from this research question are currently submitted.

For more information you can contact:

Laurien Ulfman PhD