Occurrence of Transmembrane Protein 119 in the Retina is Not Restricted to the Microglia: An Immunohistochemical Study.

PURPOSE: Recently, a new marker protein for microglial cells in the brain was postulated, transmembrane protein 119 (TMEM119), raising the hope for a new opportunity to reliably and unambiguously detect microglial cells in histologic sections. It was of interest whether TMEM119 also was a reliable microglial marker in the retina. METHODS: Anti-TMEM119 antibodies of two providers were used to label microglia in the murine retina, and labeling properties were compared to those of antibodies against Iba1 and CD11b. As an example of a pathologic situation, labeling for TMEM119 was also performed in eyes treated by an argon laser as an experimental model for choroidal neovascularization. RESULTS: TMEM119 immunoreactivity (IR) was found on microglial cells in the naïve retina. However, specificity and sensitivity of TMEM119 IR varied clearly depending on the source of the antibody, age of the mouse, and location of retinal microglia. After laser treatment, however, microglial cells lost their IR for TMEM119 at the site of the laser spot. Moreover, other cells became positive for TMEM119; for example, Müller cells. CONCLUSIONS: TMEM119 is a useful marker for the microglia in the brain. However, retinal microglia shows variable IR for TMEM119, and the microglia is not the only cell showing TMEM IR. Therefore, TMEM119 appears not to be applicable as a general marker for the retinal microglia in pathologic situations. TRANSLATIONAL RELEVANCE: Reliable detection and quantification of microglial cells is of high importance to study disease mechanisms and effects of therapeutic approaches in the retina.

EPLIN-α and -ß Isoforms Modulate Endothelial Cell Dynamics through a Spatiotemporally Differentiated Interaction with Actin.

Actin-binding proteins are essential for linear and branched actin filament dynamics that control shape change, cell migration, and cell junction remodeling in vascular endothelium (endothelial cells [ECs]). The epithelial protein lost in neoplasm (EPLIN) is an actin-binding protein, expressed as EPLIN-α and EPLIN-ß by alternative promoters; however, the isoform-specific functions are not yet understood. Aortic compared to cava vein ECs and shear stress-exposed cultured ECs express increased EPLIN-ß levels that stabilize stress fibers. In contrast, EPLIN-α expression is increased in growing and migrating ECs, is targeted to membrane protrusions, and terminates their growth via interaction with the Arp2/3 complex. The data indicate that EPLIN-α controls protrusion dynamics while EPLIN-ß has an actin filament stabilizing role, which is consistent with FRAP analyses demonstrating a lower EPLIN-ß turnover rate compared to EPLIN-α. Together, EPLIN isoforms differentially control actin dynamics in ECs, essential in shear stress responses, cell migration, and barrier function.

Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis.

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

Endothelial CD99 supports arrest of mouse neutrophils in venules and binds to neutrophil PILRs.

CD99 is a crucial regulator of the transmigration (diapedesis) of leukocytes through the blood vessel wall. Here, we report that CD99 acts at 2 different steps in the extravasation process. In agreement with previous antibody-blocking experiments, we found that CD99 gene inactivation caused neutrophil accumulation between venular endothelial cells and the basement membrane in the inflamed cremaster. Unexpectedly, we additionally found that leukocyte attachment to the luminal surface of the venular endothelium was impaired in the absence of CD99. Intravital video microscopy revealed that CD99 supported rapid chemokine-induced leukocyte arrest. Inhibition of leukocyte attachment and extravasation were both solely due to the absence of CD99 on endothelial cells, whereas CD99 on leukocytes was irrelevant. Therefore, we searched for heterophilic ligands of endothelial CD99 on neutrophils. We found that endothelial cells bind to the paired immunoglobulinlike receptors (PILRs) in a strictly CD99-dependent way. In addition, endothelial CD99 was coprecipitated with PILRs from neutrophils that adhered to endothelial cells. Furthermore, soluble CD99 carrying a transferable biotin tag could transfer this tag covalently to PILR when incubated with intact neutrophils. Binding of neutrophils under flow to a surface coated with P-selectin fragment crystallizable (Fc) and intercellular adhesion molecule 1 (ICAM-1) Fc became more shear resistant if CD99 Fc was coimmobilized. This increased shear resistance was lost if neutrophils were preincubated with anti-PILR antibodies. We concluded that endothelial CD99 promotes leukocyte attachment to endothelium in inflamed vessels by a heterophilic ligand. In addition, CD99 binds to PILRs on neutrophils, an interaction that leads to increased shear resistance of the neutrophil attachment to ICAM-1.

Cutting edge: Endothelial-specific gene ablation of CD99L2 impairs leukocyte extravasation in vivo.

CD99-like 2 (CD99L2) is a membrane protein with moderate sequence homology to CD99, which initiates cell aggregation of transfected cells and that is strongly expressed on endothelial cells, neutrophils, and lymphocytes. We showed recently that Abs against CD99L2 inhibit neutrophil, but not T lymphocyte, recruitment into inflamed tissues. In this study, we have generated conditional gene-deficient mice for CD99L2 and show by analyzing them in various inflammation models several results. First, gene ablation of CD99L2 impairs neutrophil recruitment into inflamed cremaster and peritoneum. Second, despite the strong expression of CD99L2 on peripheral neutrophils, only gene ablation on endothelial cells but not on myeloid cells affects neutrophil extravasation. Third, in contrast to our previous Ab-based results, recruitment of activated T cells into inflamed skin was impaired in mice lacking CD99L2 on endothelial cells. We conclude that CD99L2 is an essential endothelial Ag for leukocyte extravasation, which does not require homophilic interactions with CD99L2 on leukocytes.

A CD99-related antigen on endothelial cells mediates neutrophil but not lymphocyte extravasation in vivo.

CD99 is a long-known leukocyte antigen that does not belong to any of the known protein families. It was recently found on endothelial cells, where it mediates transendothelial migration of human monocytes and lymphocyte recruitment into inflamed skin in the mouse. Here, we show that CD99L2, a recently cloned, widely expressed antigen of unknown function with moderate sequence homology to CD99, is expressed on mouse leukocytes and endothelial cells. Using antibodies, we found that CD99L2 and CD99 are involved in transendothelial migration of neutrophils in vitro and in the recruitment of neutrophils into inflamed peritoneum. Intravital and electron microscopy of cremaster venules revealed that blocking CD99L2 inhibited leukocyte transmigration through the vessel wall (diapedesis) at the level of the perivascular basement membrane. We were surprised to find that, in contrast to CD99, CD99L2 was not relevant for the extravasation of lymphocytes into inflamed tissue. Although each protein promoted cell aggregation of transfected cells, endothelial CD99 and CD99L2 participated in neutrophil extravasation independent of these proteins on neutrophils. Our results establish CD99L2 as a new endothelial surface protein involved in neutrophil extravasation. In addition, this is the first evidence for a role of CD99 and CD99L2 in the process of leukocyte diapedesis in vivo.