Examination of MCP-1 (CCL2) partitioning and presentation during transendothelial leukocyte migration

Abstract

It is proposed that a chemokine concentration gradient promotes vectorial leukocyte migration across the vascular endothelium during inflammation. In this study, monocyte migration across a model endothelial monolayer was assessed at defined time-points after the addition of MCP-1 (CCL2). At each time-point transendothelial migration was quantified, medium from the apical and basal surface was collected for ELISA and monolayers were stained to detect both heparan sulfate and MCP-1. Statistically significant monocyte migration was observed within 60 min of chemokine addition to the basal surface of the endothelium and an asymmetric distribution of MCP-1 across the monolayer was observed at all time-points. Dual color immunofluorescence analysis demonstrated that MCP-1 was focused into heparan sulfate-containing domains on the apical surface of some of the endothelial cells. Furthermore, no uniform concentration gradient of chemokine was observed within the space between adjacent endothelial cells with apical MCP-1 application resulting in a staining pattern identical to that observed after basal application. The addition of a functional, monomeric form of MCP-1 produced a staining pattern identical to that observed using the wild-type protein, suggesting that localized chemokine oligomerization is not responsible for generating the focal chemokine distribution. Together, these data suggest that apical presentation of concentrated, chemokine-containing domains provides sufficient stimulus to promote transendothelial leukocyte migration in the absence of the formation of a formal haptotactic concentration gradient between endothelial cells.

The CCL2 synthesis inhibitor bindarit targets cells of the neurovascular unit, and suppresses experimental autoimmune encephalomyelitis

“…Furthermore, intravenously administered anti-CCL2 antibody blocked heightened leukocyte adhesion to pial venular endothelium in vivo in mice suffering acute EAE [72], as well as prevented recurring clinical episodes in a chronic relapsing EAE model [73], possibly by antagonizing CCL2 at the luminal endothelial surface. Supporting this possibility, CCL2 harbors in its C-terminal α-helix a binding site for GAGs typically found on the luminal endothelial surface [74], and has been shown to bind to the luminal surface of cultured endothelial cells and then trigger firm adhesion followed by transmigration of mononuclear leukocytes [75,76]. Binding of CCL2 released from BMEC in culture has most recently been shown to switch from the luminal to the abluminal surface following cytokine-induced activation [77], possibly reflecting the changing roles of this chemokine pool from first promoting leukocyte adhesion to later directing extravasation into the parenchyma.…”

Long pentraxin PTX3 deficiency worsens LPS-induced acute lung injury

“…TNF-a is one of the most prominent pro-inflammatory cytokines that can increase pulmonary permeability, induce cell death, and induce the production of multiple cytokines and chemokines [27]. MCP-1 is a CC chemokine for the regulation and recruitment of monocytes and other inflammatory cells to the sites of tissue injury or infection [28,29]. Therefore, the presence of PTX3 may provide a negative feedback signal to dampen inflammatory reactions.…”

Effect of storage conditions on the stability of recombinant human MCP-1/CCL2

“…With the advancements in technology, MCP-1 is now commercially available for laboratory use in the form of recombinant protein. Recombinant MCP-1 is frequently used in many in vitro studies [10–13] including those that focus on the transendothelial migration of monocytes [14, 15]. In our previous study, we used recombinant human MCP-1 to examine the formation of MCP-1 concentration gradients across the collagen matrix of a 3D in vitro vascular tissue model (Manuscript in review).…”