(588b) Mac-1 Expression Controls If Immune Cells Can Utilize Upstream Migration

Cells of the adaptive immune system such as CD4⁺ T cells, the innate immune system such as neutrophils, and hematopoietic stem cells (HSPCs), must transit to distal sites such as the lymph nodes, bone marrow, or sites of infection. The recruitment of these immune cells is regulated by a multi-step, leukocyte adhesion cascade of cell rolling, activation, adhesion, and transmigration through the endothelial barrier. After selectin-mediated braking and slow rolling, immune cells migrate along the vascular endothelium through receptor mediated binding to adhesion ligands including Intracellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1). Previous work by ourselves and others has demonstrated an interesting phenomena where cells such as murine and human T lymphocytes (1, 2), hematopoietic stem and progenitor cells (HSPCs)(3), and marginal zone B-cells (4), can migrate efficiently upstream (against the direction of shear-flow) on ICAM-1 surfaces, or surfaces coated with ICAM-1 and VCAM-1, and endothelial monolayers (HUVECs) which express ICAM-1, using the integrin receptor known as Lymphocyte Functional Antigen-1 (LFA-1). Furthermore, cells undergoing upstream migration transmigrate through the endothelium twice as fast as those going downstream (5). Finally, recent work of ours has furthered the phenomenon of upstream migration by demonstrating that innate immune cells such as neutrophils, which express a second ICAM-1 binding integrin named Macrophage-1 antigen (Mac-1), can migrate upstream when Mac-1 is blocked with antibodies (6).

Here, we describe for the first time how Mac-1 integrin expression can be utilized as checkpoint to direct immune cells either with or against the direction of fluid flow on the endothelial surface using genetic engineering. We demonstrate that myeloid cells of the innate immune system, which cannot normally migrate upstream, can be made to swim upstream by removing Mac-1 function (Figure 1). HL-60 cells and primary neutrophils in which Mac-1 is knocked down are able to migrate upstream on ICAM-1, downstream on VCAM-1, upstream on mixed surfaces of ICAM-1 and VCAM-1 (which mimic the vasculature, and upstream on activated HUVEC endothelial monolayers at shear rates seen in the vasculature. On the other hand, lymphoid cells of the adaptive immune system or stem and progenitor cells, which normally can crawl upstream, can be made to crawl with the direction of shear flow (downstream) by expressing Mac-1. We then demonstrate that KG1a cells, primary HSPCs, and CD4+ T cells genetically engineered to express Mac-1 on their surface cannot migrate upstream on ICAM-1 or any endothelial surface. Finally, by transducing Lifeact into these engineered cells, we demonstrate that the F-actin network is fundamentally different between both upstream and downstream migration and that the presence alone of Mac-1 shifts the actin-network between these modes.

In all, while LFA-1 is the critical integrin mediating upstream motility, Mac-1 has the potential to control migration between the upstream and downstream modes. Therefore, through its expression, which is transient in neutrophils, Mac-1 can not only orient immune cells upstream or downstream but also alter the rate of transmigration. This finding has direct implications in understanding how to manipulate both the direction immune cells trafficking as well as their translocation.

REFERENCES:

1. Dominguez GA, Anderson NR, & Hammer DA (2015) The direction of migration of T-lymphocytes under flow depends upon which adhesion receptors are engaged. Integrative biology : quantitative biosciences from nano to macro 7(3):345-355.
2. Valignat M-P, Theodoly O, Gucciardi A, Hogg N, & Lellouch Annemarie C (2013) T Lymphocytes Orient against the Direction of Fluid Flow during LFA-1-Mediated Migration. Biophysical Journal 104(2):322-331.
3. Buffone A, Anderson NR, & Hammer DA (2018) Migration against the direction of flow is LFA-1-dependent in human hematopoietic stem and progenitor cells. Journal of Cell Science 131(1).
4. Tedford K, et al. (2017) The opposing forces of shear flow and sphingosine-1-phosphate control marginal zone B cell shuttling. Nature Communications 8(1):2261.
5. Anderson NR, Buffone A, & Hammer DA (2019) T lymphocytes migrate upstream after completing the leukocyte adhesion cascade. Cell Adhesion & Migration 13(1):163-168.
6. Buffone A, Anderson NR, & Hammer DA (2019) Human Neutrophils Will Crawl Upstream on ICAM-1 If Mac-1 Is Blocked. Biophysical Journal 117(8):1393-1404.