Monocyte chemoattractant protein-1 alters expression of tight junction-associated proteins in brain microvascular endothelial cells

Abstract

The chemokine monocyte chemoattractant protein (MCP-1) is recognized to mediate extravasation of mononuclear leukocytes into the brain during a variety of neuroinflammatory conditions. In large part produced by parenchymal neural cells during these disease states, it is unclear how this chemokine can stimulate the migration of circulating leukocytes that lie behind the highly impermeant blood–brain barrier (BBB). Based on the premise that disruption of tight junctions (TJs) could foster leukocyte extravasation, experiments were conducted to test the hypothesis that MCP-1 alters the expression and/or distribution of the TJ-associated proteins zonulae occludens-1 (ZO-1) and occludin in brain microvascular endothelial cells (BMEC) comprising the BBB. Exposure to MCP-1 caused a loss in immunostaining of ZO-1 at inter-endothelial junctional regions in both cultured BMEC and isolated brain microvessels, as well as a similar effect on occludin in cultured BMEC, but did not alter occludin staining in microvessels. In cellular fractionation experiments, ZO-1 associated predominantly with the detergent-resistant cytoskeletal framework (CSK) in both cultured BMEC and brain microvessels, while a slimmer majority of occludin partitioned with the CSK. Following MCP-1 exposure, ZO-1 was reduced in the CSK fraction of cultured BMEC and microvessels, with a shift of ZO-1 to the detergent-soluble fraction in both cases. Occludin exhibited a similar pattern of MCP-1-induced loss and shift from the CSK in cultured BMEC, but remained nearly constant in microvessels. Lastly, expression of caveolin-1, a major structural component of membrane microdomains thought to be functionally complexed with TJs, was additionally altered by MCP-1 treatment of both cultured BMEC and microvessels. These results indicate that, in addition to its chemotactic activity, MCP-1 might alter BBB integrity during CNS inflammation.

Introduction

Monocyte chemoattractant protein (MCP-1, also referred to as CCL2) is a member of the β (CC) class of chemokines (Zlotnick and Yoshio, 2000), and plays a critical role in directing the extravasation of mononuclear cells into the central nervous system (CNS) during a variety of inflammatory, infectious and traumatic conditions Kelder et al., 1998, Adamus et al., 2001, Huang et al., 2001, Rancan et al., 2001, Zink et al., 2001, Hughes et al., 2002, Scarpini et al., 2002. In most of these conditions, astrocytes are the major, if not primary, CNS source of MCP-1 Glabinski et al., 1995, Gourmala et al., 1997, Ransohoff, 1997, Van Der Voorn et al., 1999, Che et al., 2001, Katayama et al., 2002, Strack et al., 2002. However, as astrocytes lie behind the blood–brain barrier (BBB), it is not at all clear how MCP-1 released from these cells stimulates leukocytic infiltration. This confusion stems from the fact that the microvascular endothelial cells of the BBB characteristically express a high level of tight junctions (TJs), which effectively seal-off the paracellular, aqueous pathway connecting the brain interstitial fluid and bloodstream (Wolburg and Lippoldt, 2001). Arguably, this feature would be expected to significantly impede direct contact between blood-born leukocytes and extravascular MCP-1 and, thus, negate any chemotactic effect. That such parenchymally deposited chemokine can stimulate leukocyte migration across the BBB, despite this profound anatomical constraint, has been deftly shown by microinjection of MCP-1, among other chemokines, into the brains of rats (Bell et al., 1996). One possible explanation for this apparent paradox is that, in addition to its recognized chemotactic activity, MCP-1 could potentially disrupt TJ integrity and thereby heighten BBB permeability. In turn, this could facilitate MCP-1 distribution into the vascular compartment.

Such a possible vasoactive action of MCP-1 is not without precedent in the chemokine field. Interleukin-8, a member of the α (CXC) class of chemokines, has been reported to stimulate its cognate receptors, CXCR1 and CXCR2, on microvascular endothelial cells, with activation of CXCR2 specifically leading to Rac-mediated cell retraction and elevated permeability (Schraufstatter et al., 2001). Furthermore, this laboratory recently described MCP-1 binding to the abluminal surface of isolated human and mouse brain microvessels as being mediated by expression of the major, recognized MCP-1 receptor, CCR2, on endothelial cells Andjelkovic et al., 1999, Andjelkovic and Pachter, 2000, Dzenko et al., 2001, and other laboratories have confirmed the expression of this receptor on both cultured human BMEC (Berger et al., 1999) and endothelial cells of rat brain microvessels in situ Banisadr et al., 2002, Jee et al., 2002. Thus, there exists the capacity of MCP-1 to exert TJ disruption through a well-characterized receptor that faces the brain parenchyma. In light of these considerations, we hypothesize that MCP-1 can alter the expression and/or distribution of TJ-associated proteins, an effect that could potentially instigate disruption of BBB integrity and, thereby, foster leukocyte extravasation.

To test this hypothesis, both cultured brain microvascular endothelial cells (BMEC) and freshly isolated brain microvessels were exposed to MCP-1, and the effects on expression and cytoplasmic compartmentation of two TJ-associated proteins, zonulae occludens-1 (ZO-1) and occludin, were assessed. Occludin is a transmembrane protein considered to be responsible for sealing TJs (Lacaz-Viera et al., 1999) and regulating paracellular permeability Balda et al., 2000, Huber et al., 2000. ZO-1 is a submembranous TJ-associated protein that belongs to the membrane-associated guanylate kinase (MAGUK) family. Its characteristic PDZ, SH3 and GK domains enable ZO-1 to link transmembrane proteins, structural elements and signal transduction molecules (Gonzalez-Mariscal et al., 2000). Additionally, we also examined whether MCP-1 altered the expression of caveolin-1, a major structural protein of ‘raft-like’ membrane microdomains, cholesterol-enriched membrane specializations recently evidenced to play an important role in the spatial organization of TJs (Nusrat et al., 2000).

Results indicate that MCP-1 can induce changes in the subcellular expression of both ZO-1 and occludin, as well as alterations in caveolin-1 level, suggesting that another action of this chemokine might be to alter vascular junctional integrity.