Intravascular Crawling of Patrolling Monocytes: A Lèvy-Like Motility for Unique Search Functions?

Patrolling monocytes (PMo) are the organism’s preeminent intravascular guardians by their continuous search of damaged endothelial cells and harmful microparticles for their removal and to restore homeostasis. This surveillance is accomplished by PMo crawling on the apical side of the endothelium through regulated interactions of integrins and chemokine receptors with their endothelial ligands. We propose that the search mode governs the intravascular motility of PMo in vivo in a similar way to T cells looking for antigen in tissues. Signs of damage to the luminal side of the endothelium (local death, oxidized LDL, amyloid deposits, tumor cells, pathogens, abnormal red cells, etc.) will change the diffusive random towards a Lèvy-like crawling enhancing their recognition and clearance by PMo damage receptors as the integrin αMβ2 and CD36. This new perspective can help identify new actors to promote unique PMo intravascular actions aimed at maintaining endothelial fitness and combating harmful microparticles involved in diseases as lung metastasis, Alzheimer’s angiopathy, vaso-occlusive disorders, and sepsis.

Abstract 12659: Hepatoma-Derived Growth Factor Related Protein-3 as a Novel Endothelial Ligand

Background: Endothelial ligands extrinsically regulate a broad spectrum of vascular functions with therapeutic potentials, but are traditionally identified on a case-by-case basis with technical challenges. We recently developed open reading frame phage display (OPD) for unbiased identification of phagocytosis ligands. In this study, we identified hepatoma-derived growth factor related protein-3 (HRP-3) as a putative endothelial ligand by OPD. We hypothesized that HRP-3 is a novel endothelial growth factor, capable of promoting endothelial cell (EC) growth and migration. Methods and Results: We performed 3 rounds of in vivo phage binding selection in mice with an OPD library, screened enriched phage clones by next generation DNA sequencing, and identified HRP-3 as one of the putative endothelial ligands. To confirm the finding, clonal phages displaying HRP-3, VEGF and GFP were generated and analyzed for their binding to human umbilical vein endothelial cells (HUVECs). The results show that HRP-3-Phage and VEGF-Phage had significantly higher binding to HUVECs than GFP-Phage. Functional analysis showed that purified recombinant HRP-3 significantly increased the proliferation of HUVECs at 24 and 48 h, whereas VEGF induced significant growth only at 48 h. Consistent with these findings, HRP-3 significantly stimulated cell proliferation by MTT assay. In vitro wound-healing assay indicated that both HRP-3 (500 ng/ml) and VEGF (50 ng/ml) significantly promoted the migration of HUVECs into the denuded area. To dissect the downstream signaling pathway, we demonstrated that HRP-3 significantly induced ERK1/2 phosphorylation in HUVECs after 10 min treatment. Similar effects of HRP-3 and VEGF on EC growth, migration, and ERK activation were also verified using human aorta endothelial cells. Conclusions: Our findings demonstrate that HRP-3 is a novel ligand, capable of promoting proliferation and migration of ECs. The pro-growth effect of HRP-3 is at least partially mediated through ERK pathway activation. These results in turn support the broad applicability of OPD for the systematic discovery of endothelial ligands.

α-Chain phosphorylation of the human leukocyte CD11b/CD18 (Mac-1) integrin is pivotal for integrin activation to bind ICAMs and leukocyte extravasation

The promiscuous CD11b/CD18 (Mac-1) integrin has important roles in regulating many immunologic functions such as leukocyte adhesion and emigration from the bloodstream via interactions with the endothelial ligands ICAM-1 and ICAM-2, iC3b-mediated phagocytosis, and apoptosis. However, the mechanisms for Mac-1 inside-out activation have remained poorly understood. Phosphorylation of integrin cytoplasmic domains is emerging as an important mechanism of regulating integrin functions. Here, we have studied phosphorylation of human CD11b, which takes place on the cytoplasmic Ser1126 in neutrophils. We show that mutation of the serine phosphorylation site leads to inability of Mac-1 to become activated to bind the cellular ligands ICAM-1 and ICAM-2. However, CD11b-mutant cells are fully capable of binding other studied CD11b ligands (ie, iC3b and denatured BSA). Activation epitopes expressed in the extracellular domain of the integrin and affinity for soluble ICAM ligands were decreased for the mutated integrin. Additionally, the mutation resulted in inhibition of chemokine-induced migration in a transendothelial assay in vitro and significantly reduced the accumulation of intravenously administered cells in the spleen and lungs of Balb/c mice. These results characterize a novel selective mechanism of Mac-1–integrin activation, which mediates leukocyte emigration from the bloodstream to the tissues.

Sulphated endothelial ligands for L-selectin in lymphocyte homing and inflammation

Lymphocytes from the blood home to secondary lymphoid tissues through a process of tethering, rolling, firm adhesion and transmigration. Tethering and rolling of lymphocytes is mediated by the interaction of L-selectin on lymphocytes with sulphated ligands expressed by the specialized endothelial cells of high endothelial venules (HEVs). The sulphate-dependent monoclonal antibody MECA79 stains HEVs in peripheral lymph nodes and recognizes the complex of HEV ligands for L-selectin termed peripheral node addressin. High endothelial cell GlcNAc-6-sulphotransferase/L-selectin ligand sulphotransferase is a HEV-expressed sulphotransferase that contributes to the formation of the MECA79 epitope and L-selectin ligands on lymph node HEVs. MECA79-reactive vessels are also common at sites of chronic inflammation, suggesting mechanistic parallels between lymphocyte homing and inflammatory trafficking.

Numerical Analysis of the Deformation of an Adherent Drop Under Shear Flow

The adhesion of leukocytes to substrates is an important biomedical problem and has drawn extensive research. In this study, employing both single and compound drop models, we investigate how hydrodynamics interacts with an adherent liquid drop in a shear flow. These liquid drop models have recently been used to describe the rheological behavior of leukocytes. Numerical simulation confirms that the drop becomes more elongated when either capillary number or initial contact angle increases. Our results show that there exists a thin region between the drop and the wall as the drop undergoes large stretching, which allows high pressure to build up and provides a lift force. In the literature, existing models regard the leukocyte as a rigid body to calculate the force and torque acting on the drop in order to characterize the binding between cell receptors and endothelial ligands. The present study indicates that such a rigid body model is inadequate and the force magnitude obtained from it is less than half of that obtained using the deformable drop models. Furthermore, because of its much higher viscosity, the cell nucleus introduces a hydrodynamic time scale orders of magnitude slower than the cytoplasm. Hence the single and compound drops experience different dynamics during stretching, but exhibit very comparable steady-state shapes. The present work offers a framework to facilitate the development of a comprehensive dynamic model for blood cells.