Abstract
BackgroundDuring neuroinflammation many chemokines alter the function of the blood-brain barrier (BBB) that regulates the entry of macromolecules and immune cells into the brain. As the milieu of the brain is altered, biochemical and structural changes contribute to the pathogenesis of neuroinflammation and may impact on neurogenesis. The chemokine CCL4, previously known as MIP-1β, is upregulated in a wide variety of central nervous system disorders, including multiple sclerosis, where it is thought to play a key role in the neuroinflammatory process. However, the effect of CCL4 on BBB endothelial cells (ECs) is unknown. Materials and methodsExpression and distribution of CCR5, phosphorylated p38, F-actin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) were analysed in the human BBB EC line hCMEC/D3 by Western blot and/or immunofluorescence in the presence and absence of CCL4. Barrier modulation in response to CCL4 using hCMEC/D3 monolayers was assessed by measuring molecular flux of 70 kDa RITC-dextran and transendothelial lymphocyte migration. Permeability changes in response to CCL4 in vivo were measured by an occlusion technique in pial microvessels of Wistar rats and by fluorescein angiography in mouse retinae. ResultsCCR5, the receptor for CCL4, was expressed in hCMEC/D3 cells. CCL4 stimulation led to phosphorylation of p38 and the formation of actin stress fibres, both indicative of intracellular chemokine signalling. The distribution of junctional proteins was also altered in response to CCL4: junctional ZO-1 was reduced by circa 60% within 60 min. In addition, surface VE-cadherin was redistributed through internalisation. Consistent with these changes, CCL4 induced hyperpermeability in vitro and in vivo and increased transmigration of lymphocytes across monolayers of hCMEC/D3 cells. ConclusionThese results show that CCL4 can modify BBB function and may contribute to disease pathogenesis.
Highlights
Neuroinflammation is the inflammatory response to injury or infection in the brain or spinal cord (Fuster-Matanzo et al, 2013) and can lead to either collateral damage or resolution and repair depending on disease setting
Chemokines play an integral part in this regulation through sequestrating to the endothelial cell glycocalyx where they are Abbreviations: BBB, blood-brain barrier; CNS, central nervous system; EC, endothelial cell; F-actin, filamentous actin; hCMEC/D3, immortalized human cerebral microvascular endothelial cell line; MAPK, mitogen-activated protein kinase; SDS-PAGE, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis; VE-cadherin, vascular endothelial cadherin; ZO-1, zonula occludens-1
When used in indirect immunofluorescence the CCR5 antibody revealed strong staining in hCMEC/D3, which was concentrated in the cell periphery (Fig. 1B)
Summary
Neuroinflammation is the inflammatory response to injury or infection in the brain or spinal cord (Fuster-Matanzo et al, 2013) and can lead to either collateral damage or resolution and repair depending on disease setting. Engagement of chemokines with their G-protein coupled receptors results in a change in cell behaviour, including migration, proliferation, and activation of inflammatory responses. Chemokines play an integral part in this regulation through sequestrating to the endothelial cell glycocalyx where they are Abbreviations: BBB, blood-brain barrier; CNS, central nervous system; EC, endothelial cell; F-actin, filamentous actin; hCMEC/D3, immortalized human cerebral microvascular endothelial cell line; MAPK, mitogen-activated protein kinase; SDS-PAGE, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis; VE-cadherin, vascular endothelial cadherin; ZO-1, zonula occludens-1. Materials and methods: Expression and distribution of CCR5, phosphorylated p38, F-actin, zonula occludens-1 (ZO1) and vascular endothelial cadherin (VE-cadherin) were analysed in the human BBB EC line hCMEC/D3 by Western blot and/or immunofluorescence in the presence and absence of CCL4. Consistent with these changes, CCL4 induced hyperpermeability in vitro and in vivo and increased transmigration of lymphocytes across monolayers of hCMEC/D3 cells. Conclusion: These results show that CCL4 can modify BBB function and may contribute to disease pathogenesis
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