S39 - Macrophages show differential abilities to migrate upstream during inflammatory insult

Introduction: : In chronic inflammatory conditions, aberrant recruitment of pro-inflammatory (M1) macrophages to the inflammatory site perpetuates the pro-inflammatory cascade by prolonging the release of pro-inflammatory cytokines and chemokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). To traffic to these inflammatory sites, circulating monocytes interact with the apical surface of the endothelium through selectin-mediated interactions. This rolling is halted by stronger, anchoring interactions facilitated by macrophage antigen-1 (Mac-1) (CD11b/CD18) and lymphocyte functional antigen 1 (LFA-1) (CD11a/CD18) integrins on the surface of monocytes that bind to intracellular adhesion molecule-1 (ICAM-1) on macrophages. Very late antigen-4 (VLA-4) (CD49d/CD29) has also been shown to promote arrest on the endothelium through interactions with vascular cell adhesion molecule-1 (VCAM-1). Following this arrest, macrophages migrate in the direction of hydrodynamic blood flow, commonly referred to as downstream migration, to transmigrate through the endothelium where they polarize into the M1 and M2 phenotype to perform their end-effector functions. Previous findings from our laboratory have shown that unstimulated M0 macrophages can be induced to migrate against this shear flow on ICAM-1 coated surfaces, referred to as upstream migration, by blocking Mac-1 (Figure 1). This upstream migration phenomenon has been shown in T-cells, which natively migrate upstream, to promote faster transmigration through the endothelium than cells that migrate downstream. However, this phenomenon was not explored in M0 macrophages. Here we demonstrate that M0 macrophages migrate against shear flow on HUVEC monolayers, and this mode of motility facilitates their faster transmigration.

Materials and

Methods: : The migration traces of U-937 derived M0 macrophages were characterized on human umbilical vein endothelial cells (HUVECs). To conduct the flow chamber experiments, HUVECs were seeded in 35 x 10 mm tissue-culture (TC)-treated polystyrene plates and were grown to confluence. Prior to the flow chamber experiments, HUVECs were supplied with fresh EGM-2 media supplemented with 10 ng/mL of Il-1β and were allowed to incubate for 4 h to stimulate robust expression of ICAM-1 and VCAM-1. Flow chamber experiments were conducted by perfusing scraped U-937-derived M0 macrophages at a density of 5 x 105 cells/mL in a vacuum-enclosed circular parallel plate flow chamber (GlycoTech, Gaithersburg, MD) with a rubber gasket to generate a rectangular flow path (h = 127 µm, w = 0.25 cm). M0 macrophages were treated with isotype, anti-CD11a/αL-integrin, anti-CD11b/αM-integrin, anti-CD49d/α4-integrin, anti-CD11a/αL-integrin + anti-CD49d/α4-integrin, or anti-CD11a/αM-integrin + anti-CD49d/α4-integrin and were allowed to attach for 10 minutes. Shear flow rates of 100 s-1 and 800 s-1 were used to induce shear flow mediated migration of macrophages for 30 minutes by perfusing RPMI1640 complete medium. Images were captured with phase contrast microscopy under a 20x objective at 1 minute intervals and cells were tracked using the MTrackJ plugin (https://imagescience.org/meijering/software/mtrackj/). The migration traces were then uploaded into a MATLAB script to characterize migration index (MI) and the time to transmigration. Statistical tests were performed using a paired Student’s t-test.

Results, Conclusions, and Discussions:: At both 100 s⁻¹ and 800 s⁻¹ shear rates, untreated and isotype-treated macrophages predominantly migrated downstream as indicated by a positive mean MI confirming earlier findings on ICAM-1 coated substrates. Blocking CD11b demonstrated upstream migration as indicated by a negative mean MI, whereas blocking CD11a and CD49d yielded downstream migration, also confirming earlier findings on ICAM-1 coated substrates. Blocking both CD11a and CD49d demonstrated more persistent upstream migration as indicated by a negative MI value closer to -1. Additionally, macrophages undergoing upstream migration exhibited a significantly reduced time to transmigration across the endothelium. Therefore, our findings demonstrate that M0 macrophages can be induced to go upstream on endothelial-like surfaces and transmigrate faster through the endothelium. These insights could provide a novel framework for therapeutic strategies aimed at regulating immune cell infiltration in chronic inflammatory diseases.

Acknowledgements and/or References (Optional):: This work was supported by NIH R01GM143357 to ABJ