Rapid Internalization and Nuclear Translocation of CCL5 and CXCL4 in Endothelial Cells

Abstract

The chemokines CCL5 and CXCL4 are deposited by platelets onto endothelial cells, inducing monocyte arrest. Here, the fate of CCL5 and CXCL4 after endothelial deposition was investigated. Human umbilical vein endothelial cells (HUVECs) and EA.hy926 cells were incubated with CCL5 or CXCL4 for up to 120 min, and chemokine uptake was analyzed by microscopy and by ELISA. Intracellular calcium signaling was visualized upon chemokine treatment, and monocyte arrest was evaluated under laminar flow. Whereas CXCL4 remained partly on the cell surface, all of the CCL5 was internalized into endothelial cells. Endocytosis of CCL5 and CXCL4 was shown as a rapid and active process that primarily depended on dynamin, clathrin, and G protein-coupled receptors (GPCRs), but not on surface proteoglycans. Intracellular calcium signals were increased after chemokine treatment. Confocal microscopy and ELISA measurements in cell organelle fractions indicated that both chemokines accumulated in the nucleus. Internalization did not affect leukocyte arrest, as pretreatment of chemokines and subsequent washing did not alter monocyte adhesion to endothelial cells. Endothelial cells rapidly and actively internalize CCL5 and CXCL4 by clathrin and dynamin-dependent endocytosis, where the chemokines appear to be directed to the nucleus. These findings expand our knowledge of how chemokines attract leukocytes to sites of inflammation.

Differential Effects of Platelet Factor 4 (CXCL4) and Its Non-Allelic Variant (CXCL4L1) on Cultured Human Vascular Smooth Muscle Cells

“…Here, we proved that the endocytosis of CXCL4 by VSMCs was clathrin- and dynamin-independent. This contrasts with our previous work showing an involvement of both proteins during endocytosis of CXCL4 by endothelial cells [ 30 ] suggesting distinct uptake mechanisms in different cell types. Alternatively, chemokines were shown to be internalized through clathrin-independent mechanisms involving lipid-rafts and caveolae [ 37 ].…”

“…Although we found the presence of both receptors in cultured VSMCs, their blockade by specific antibodies did not affect the internalization of CXCL4. This is contrary to the aforementioned studies [ 4 , 6 , 33 , 35 ], but in accordance with our previous observation that CXCR3 was not involved in CXCL4 uptake in endothelial cells [ 30 ]. In another report, a CXCR3 blocking antibody did not affect CXCL4-induced cytokine production in the human carotid artery SMCs [ 20 ].…”

“…Previous work has demonstrated rapid internalization of CXCL4 by endothelial cells [ 30 ]. To investigate whether CXCL4 and its variant CXCL4L1 are taken up by VSMCs, cells were treated with CXCL4 or CXCL4L1, stained with fluorescent antibodies and the measured fluorescence was quantified.…”

“…By staining with a specific antibody, we confirmed these observations on the cellular level, showing that non-treated VSMCs have a baseline content of endogenous CXCL4L1 protein. Since CXCL4 is internalized by endothelial cells [ 30 ], we investigated whether CXCL4 is endocytosed by VSMCs or rather binds to the cell surface, resulting in the intracellular signaling without concurrent endocytosis. Our data clearly indicate that unlike its variant, exogenously added, native CXCL4 was internalized by VSMCs and appeared to be enclosed within endosome-like structures.…”

Role of the Skin Immune System in Wound Healing

“…The chemokine CXCL4 promotes neutrophil activation, including adhesion and degranulation, and contributes to the differentiation of monocytes recruited to the site of skin injury into macrophages [35]. Additionally, CXCL4 and CCL5 increase the adhesiveness of circulating monocytes to the endothelium of the dermal microvasculature [36]. CXCL7 is the most abundant platelet-derived chemokine.…”