Transendothelial migration (TEM) of leukocytes is a critical step in the inflammatory response, since most of the damage due to unwanted inflammation occurs after leukocytes cross blood vessels. TEM is regulated by molecules such as platelet/endothelial cell adhesion molecule-1 (PECAM, CD31) and CD99 expressed on both the leukocytes and at endothelial cell (EC) borders. On EC, these molecules also reside in an interconnected membrane reticulum of membrane called the lateral border recycling compartment (LBRC) that is ?targeted? to the site of TEM. Homophilic interactions between leukocyte PECAM and EC PECAM control the start of diapedesis. Homophilic interactions between leukocyte CD99 and EC CD99 are important for the completion of TEM. In this proposal, we will build on the knowledge gained in the last cycle to test the overarching hypothesis that these leukocyte/endothelial cell interactions recruit the LBRC to promote efficient TEM and that their distribution on the leukocyte is responsible for the sequence in which leukocyte/EC interactions trigger signaling pathways that promote TR to facilitate TEM.
In Aim I we will test the hypothesis that the distribution of PECAM and CD99 on the leukocyte is responsible for the sequential regulation of TEM. Preliminary data show that during TEM, PECAM is concentrated in the front of the leukocyte and CD99 at the rear. We suspect that this concentration is critical to clustering PECAM and CD99 on the endothelial cell. We will test our hypothesis in vitro by switching the cytoplasmic domains of PECAM and CD99 to reverse their distribution on the leukocyte and determine whether this affects the order in which they regulate TEM. We will also test whether they regulate TEM sequentially in vivo using spinning disc confocal intravital microscopy to study the process in real time. CD99L2 (L2) is a molecule we recently discovered to play an important role in TEM.
In Aim II we will identify how L2 functions to regulate TEM. L2 is diffusely distributed on the leukocyte surface. We will test the hypothesis that it regulates a step between those regulated by PECAM and CD99 using the ?sequential block? assay that we have developed and published. We will identify the signaling pathways used by L2 to carry out its function and determine whether it plays a significant role in ischemia/reperfusion (I/R) injury. We will study response to I/R injury by intravital microscopy in the cremaster muscle circulation over hours and in a mouse model of myocardial infarction over days. We recently identified the long-sought mechanism by which endothelial cell CD99 signals in TEM. It resides in a multimolecular complex that activates protein kinase A (PKA).
In Aim III we will identify the CD99 signaling pathways downstream of PKA. Based on preliminary data we hypothesize that Rac1 is a critical downstream effector of PKA to promote targeted recycling. We will test the role of CD99 in I/R injury in the two models as above.

Public Health Relevance

Most diseases are due to inflammation that is uncontrolled, self-directed, or in the wrong place at the wrong time. Most damage in inflammation is done when the white blood cells exit the bloodstream and enter the inflamed tissues. We are studying the molecular mechanisms that control the exit of white blood cells from the bloodstream in order to be able to eventually design better anti-inflammatory therapies.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
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Srinivas, Pothur R
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Northwestern University at Chicago
Schools of Medicine
United States
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Sullivan, David P; Bui, Triet; Muller, William A et al. (2018) In vivo imaging reveals unique neutrophil transendothelial migration patterns in inflamed intestines. Mucosal Immunol 11:1571-1581
Early, Merideth; Schroeder, William G; Unnithan, Ranajana et al. (2017) Differential effect of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) on leukocyte infiltration during contact hypersensitivity responses. PeerJ 5:e3555
Weber, Evan W; Muller, William A (2017) Roles of transient receptor potential channels in regulation of vascular and epithelial barriers. Tissue Barriers 5:e1331722
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Rutledge, Nakisha S; Weber, Evan W; Winger, Ryan et al. (2015) CD99-like 2 (CD99L2)-deficient mice are defective in the acute inflammatory response. Exp Mol Pathol 99:455-9

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