Molecular mechanisms of immune cell motility and chemotaxis
Final Report Abstract
Macrophages are scattered throughout all tissues in the body and are typically the first effectors of the innate immune system to respond to tissue damage or invading microbes. In this project we studied the roles of purinergic receptors and Rho GTPases in the regulation of key attributes of these cells, in particular, motility and chemotaxis. We had previously shown that autocrine purinergic signaling is involved in complement C5a-mediated chemotaxis, but it was not clear whether purinergic P2Y2 and P2Y12 receptor ligands, such as the nucleotides ATP and ADP, themselves act as chemoattractants. We found that purinergic ligands can induce lamellipodial membrane protrusions, similar to complement C5a, but macrophages failed to migrate along nucleotide gradients. Thus, our data suggest that nucleotides are not long-range chemoattractants. We also found that short (1-4 min) exposure of macrophages to high ATP levels induces delayed (hours) cell death via P2X7 receptor signaling, and we showed that pannexin-1 is not the large pore pathway associated with P2X7 receptor activation. In further work, we found that genetic deletion of either RhoA or RhoB, Rho subfamily GTPases, had mild phenotypes, and mutual compensation was strongly indicated at the protein level. Indeed, deletion of both RhoA and RhoB induced dramatic phenotypes of highly elongated trailing ends and incomplete retraction of membrane protrusions, but, surprisingly, velocity of the cell body was faster and chemotactic navigation was fully intact. In human monocytes, inhibition of Rho signaling produced the same phenotype as Rho-deficient mouse macrophages on a 2D surface, but had little effect on morphology, velocity and chemotaxis in a 3D collagen matrix, unless the matrix density was high. As part of another study, we found that conditional Cdc42 knockout macrophages have a highly rounded up phenoptype, and impaired chemotaxis, suggesting that the Rho GTPase Cdc42, rather than the Rac subfamily of Rho GTPases, drives membrane protrusive activity and front end function in these cells. In further work, we systematically investigated the roles of various heterotrimeric subunits in complement C5a-mediated Ca2+ signaling, membrane protrusions and chemotaxis. To complete this study, we now plan to test macrophages lacking the β-subunit Gβ2, encoded by the gene Gnb2.
Publications
- (2014). Mouse macrophages completely lacking Rho (RhoA, RhoB and RhoC) have severe lamellipodial retraction defects, but robust chemotactic navigation and altered motility. Journal of Biological Chemistry 289: 30772-307784
Königs, V., Jennings, R., Vogl, T., Horsthemke, M., Bachg, A. C., Xu, Y., Grobe, K., Brakenusch, C., Schwab, A., Bähler, M., Knaus, U. G. & Hanley, P. J.
(See online at https://doi.org/10.1074/jbc.m114.563270) - (2016). Real-time two- and threedimensional imaging of monocyte motility and navigation on planar surfaces and in collagen matrices: roles of Rho. Scientific Reports 6:25016
Bzymek, R., Horsthemke, M., Isfort, K., Mohr, S., Tjaden, K., Müller-Tidow, C., Thomann, M., Schwerdtle, T., Bähler, M., Schwab, A. & Hanley, P. J.
(See online at https://doi.org/10.1038/srep25016)
