Increased Transendothelial Transport of CCL3 Is Insufficient to Drive Immune Cell Transmigration through the Blood-Brain Barrier under Inflammatory Conditions In Vitro.

Megha Meena,Zwi Berneman,Roeland Buckinx,Maxime De Laere,Carmelita Sousa,Jean-Pierre Timmermans,Nathalie Cools

doi:10.1155/2017/6752756

Abstract

Many neuroinflammatory diseases are characterized by massive immune cell infiltration into the central nervous system. Identifying the underlying mechanisms could aid in the development of therapeutic strategies specifically interfering with inflammatory cell trafficking. To achieve this, we implemented and validated a blood–brain barrier (BBB) model to study chemokine secretion, chemokine transport, and leukocyte trafficking in vitro. In a coculture model consisting of a human cerebral microvascular endothelial cell line and human astrocytes, proinflammatory stimulation downregulated the expression of tight junction proteins, while the expression of adhesion molecules and chemokines was upregulated. Moreover, chemokine transport across BBB cocultures was upregulated, as evidenced by a significantly increased concentration of the inflammatory chemokine CCL3 at the luminal side following proinflammatory stimulation. CCL3 transport occurred independently of the chemokine receptors CCR1 and CCR5, albeit that migrated cells displayed increased expression of CCR1 and CCR5. However, overall leukocyte transmigration was reduced in inflammatory conditions, although higher numbers of leukocytes adhered to activated endothelial cells. Altogether, our findings demonstrate that prominent barrier activation following proinflammatory stimulation is insufficient to drive immune cell recruitment, suggesting that additional traffic cues are crucial to mediate the increased immune cell infiltration seen in vivo during neuroinflammation.

Highlights

Until recently, the central nervous system (CNS) was considered to be an immunologically isolated and inert compartment
The hCMEC/D3 cell line (Tébu-Bio, Le Perray-en-Yvelines, France) was kept in the exponential growth phase in microvascular endothelial cell growth (EGM-2-MV) medium (Lonza, Verviers, Belgium) consisting of endothelial cell basal (EBM-2) medium supplemented with hydrocortisone, ascorbic acid, vascular endothelial growth factor (VEGF), human basic fibroblast growth factor, recombinant human insulin-like growth factor-1 (R3-IGF-1), human epidermal growth factor (EGF), gentamicin, amphotericin-B, and 2.5% fetal calf serum (FCS), as recommended by the manufacturer in flasks coated with type I rat tail collagen (Sigma; Diegem, Belgium)
The increased expression of this tight junction molecule was not mirrored by an increase in Transendothelial electrical resistance (TEER) in blood–brain barrier (BBB) cocultures when compared to hCMEC/D3 monocultures (Supplementary Figure 1C)

Summary

Introduction

The central nervous system (CNS) was considered to be an immunologically isolated and inert compartment. The concept of this so-called immune privilege has been drastically reviewed and we know that the CNS is an immune-competent and immunespecialized compartment. In this context, a better understanding of the mechanisms of leukocyte trafficking into the CNS might yield insights into the regulation of protective or pathologic immune responses in the brain. The general paradigm of leukocyte trafficking applies to immune cell recruitment to the CNS, the specialized structure of the BBB critically modulates this process [3,4,5]. Its distinguished architecture comprising tight junctions and its low basal expression of adhesion molecules ensure a limited but steady entry of immune cells in normal physiological conditions, while its activation and breakdown during

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