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Francis W. Luscinskas, PhD
Senior Scientist, Brigham and Women's Hospital
Professor of Pathology, Harvard Medical School

Brigham and Women's Hospital
Department of Pathology
75 Francis Street
Boston, MA 02115

Research Location: Harvard New Research Building


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Research Narrative:

Leukocyte transmigration and accompanying increased vascular permeability are cardinal signs of inflammation.  The goal of my lab’s research is to understand the mechanisms of leukocyte recruitment in the context of the innate and adoptive immune systems. My laboratory studies how circulating blood neutrophils and mononuclear leukocytes adhere and transmigrate into tissues during injury, infection or immune responses, focusing on the role of the vascular endothelium as an active participant in this process. We employ an in vitro flow model for direct microscopic examination of leukocyte - endothelial interactions under defined fluid shear stress conditions. As our research on leukocyte-endothelial adhesion under shear flow evolved, we came to realize that both leukocyte and endothelial cells undergo complex morphological and cytoskeletal rearrangements during these interactions. We postulated that understanding the spatial and temporal behavior of individual adhesion molecules, on both cell types, would provide clues to understanding how individual steps (leukocyte adhesion, apical migration and transmigration) were regulated. This, in turn, would enable us to determine which steps were critical and gain mechanistic insights into the process of leukocyte transmigration.
   
We, therefore, have developed a live-cell, time-lapse, fluorescence imaging system and an adenovirus technique to deliver GFP-tagged fusion proteins to endothelium, to facilitate analyses of these dynamic events. This in vitro model system has been replicated and is now in general use within our BWH Center for Excellence in Vascular Biology and by several other laboratories. The first series of studies using this system revealed that leukocytes transmigrate primarily at endothelial cell-cell junctions. Surprisingly, we discovered that some, but not all junctional proteins, transiently move laterally (“the shower curtain effect”), in the endothelial membrane, out of the path of the transmigrating leukocyte (refs 1).  Notably, the endothelial cell VE-cadherin complex, composed of α-catenin, β-catenin, γ-catenin and p120, transiently undergoes a reversible disassociation to facilitate leukocyte transmigration 2. In contrast, other junctional molecules like ICAM-1 surround the transmigrating leukocyte3 and co-localized with their leukocyte ligand, LFA-1 integrin. Our recent work revealed that VE-cadherin tyrosine phosphorylation by Src kinase destabilized the VE-cadherin complex and revealed that VE-cadherin is a physical “gatekeeper” for leukocyte transmigration 4,5. Further studies are underway to define the molecular pathways that regulate VE-cadherin complex disassociation using imaging approaches with biosensors to detect Src kinase activity and define the spatial and temporal dynamics of VE-cadherin and Src kinases in 3 dimensions using spinning disk confocal microscopy approaches.
   
A second focus has been to determine the contributions of endothelial and leukocyte CD47 in T lymphocyte recruitment using in vivo models of inflammation in wt and CD47 null mice and in our in vitro flow model. Endothelial cell CD47 interacts with its leukocyte ligands, Signal Regulatory Molecules (SIRPs), in leukocyte recruitment. Our initial findings were that monoclonal antibodies to leukocyte or endothelial cell CD47 or T cells SIRPγ blocked > 70% of T cells transmigration 6. In agreement with these studies in human cells, we observed that murine T lymphocytes transmigrate poorly across CD47 deficient TNF-α activated murine endothelium, even though murine T cells appear to lack the human ortholog of SIRPγ. This defect in recruitment was recently replicated in two models of inflammation in CD47 deficient mice. The CD47 null mice, suggesting an important role for CD47 in leukocyte function and recruitment. A future goal is to define the murine ligands for CD47 by collaborating with Dr. Gordon Freeman (DFCI).
 


Education:
PhD

Lab Members:
Pilar Alcaide, PhD, Instructor in Pathology
Marcie Williams, PhD , Postdoctoral Fellow
Veronica Azcutia, PhD , Postdoctoral Fellow

Publications (Pulled from Harvard Catalyst Profiles):

1. Jain S, Van Scoyk A, Morgan EA, Matthews A, Stevenson K, Newton G, Powers F, Autio A, Louissaint A, Pontini G, Aster JC, Luscinskas FW, Weinstock DM. Targeted Inhibition of CD47-SIRPa Requires Fc-Fc?R Interactions to Maximize Activity in T-cell Lymphomas. Blood. 2019 Aug 05.

2. Engelbertsen D, Autio A, Verwilligen RAF, Depuydt MAC, Newton G, Rattik S, Levinsohn E, Saggu G, Jarolim P, Wang H, Velazquez F, Lichtman AH, Luscinskas FW. Increased lymphocyte activation and atherosclerosis in CD47-deficient mice. Sci Rep. 2019 Jul 23; 9(1):10608.

3. Folco EJ, Mawson TL, Vromman A, Bernardes-Souza B, Franck G, Persson O, Nakamura M, Newton G, Luscinskas FW, Libby P. Neutrophil Extracellular Traps Induce Endothelial Cell Activation and Tissue Factor Production Through Interleukin-1a and Cathepsin G. Arterioscler Thromb Vasc Biol. 2018 08; 38(8):1901-1912.

4. Miyabe Y, Miyabe C, Murooka TT, Kim EY, Newton GA, Kim ND, Haribabu B, Luscinskas FW, Mempel TR, Luster AD. Complement C5a Receptor is the Key Initiator of Neutrophil Adhesion Igniting Immune Complex-induced Arthritis. Sci Immunol. 2017 Jan; 2(7).

5. Azcutia V, Bassil R, Herter JM, Engelbertsen D, Newton G, Autio A, Mayadas T, Lichtman AH, Khoury SJ, Parkos CA, Elyaman W, Luscinskas FW. Defects in CD4+ T cell LFA-1 integrin-dependent adhesion and proliferation protect Cd47-/- mice from EAE. J Leukoc Biol. 2017 02; 101(2):493-505.

6. Janssen E, Tohme M, Hedayat M, Leick M, Kumari S, Ramesh N, Massaad MJ, Ullas S, Azcutia V, Goodnow CC, Randall KL, Qiao Q, Wu H, Al-Herz W, Cox D, Hartwig J, Irvine DJ, Luscinskas FW, Geha RS. A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton. J Clin Invest. 2016 10 03; 126(10):3837-3851.

7. Zahr A, Alcaide P, Yang J, Jones A, Gregory M, dela Paz NG, Patel-Hett S, Nevers T, Koirala A, Luscinskas FW, Saint-Geniez M, Ksander B, D'Amore PA, Argüeso P. Endomucin prevents leukocyte-endothelial cell adhesion and has a critical role under resting and inflammatory conditions. Nat Commun. 2016 Feb 02; 7:10363.

8. Herter JM, Grabie N, Cullere X, Azcutia V, Rosetti F, Bennett P, Herter-Sprie GS, Elyaman W, Luscinskas FW, Lichtman AH, Mayadas TN. AKAP9 regulates activation-induced retention of T lymphocytes at sites of inflammation. Nat Commun. 2015 Dec 18; 6:10182.

9. Wang G, van Driel BJ, Liao G, O'Keeffe MS, Halibozek PJ, Flipse J, Yigit B, Azcutia V, Luscinskas FW, Wang N, Terhorst C. Migration of myeloid cells during inflammation is differentially regulated by the cell surface receptors Slamf1 and Slamf8. PLoS One. 2015; 10(3):e0121968.

10. Rosetti F, Chen Y, Sen M, Thayer E, Azcutia V, Herter JM, Luscinskas FW, Cullere X, Zhu C, Mayadas TN. A Lupus-Associated Mac-1 Variant Has Defects in Integrin Allostery and Interaction with Ligands under Force. Cell Rep. 2015 Mar 17; 10(10):1655-1664.