Determinants of CCL5-driven mononuclear cell migration across the blood–brain barrier. Implications for therapeutically modulating neuroinflammation

https://doi.org/10.1016/j.jneuroim.2006.06.004Get rights and content

Abstract

Chemokine receptors and adhesion molecules are used selectively for the transmigration of leukocytes across the blood–brain barrier (BBB) during neuroinflammation. We established an activated in vitro BBB (aIVBBB) using physiological concentrations of cytokines. We studied CCL5-driven migration as a model to determine how chemokine receptors and adhesion molecules regulate T-cell and monocyte migration across the aIVBBB. Increased expression of CCL5 and its receptors, CCR1 and CCR5 have been described in the perivascular space of multiple sclerosis (MS) lesions. Elucidating the determinants of CCL5-mediated mononuclear cell migration may clarify appropriate targets for therapeutic modulation in neuroinflammatory conditions. In response to CCL5, there was a significant increase in total mononuclear cell migration across the aIVBBB. Neutralizing monoclonal antibodies to CCR1 and CCR5 abrogated CCL5-driven transmigration, suggestive of non-redundant receptor usage in mononuclear cell migration to this chemokine in vitro. CCL5-driven transmigration was also dependent on α4β1 integrin/fibronectin connecting segment-1 (FN CS-1) and αLβ2 integrin/intercellular adhesion molecule (ICAM-1) interactions. Monocyte migration to CCL5 was solely dependent on α4β1 integrin/FN CS-1 while T-cell migration required both αLβ2 integrin/ICAM-1 and α4β1 integrin/FN CS-1 interactions. These findings provide plausible molecular targets for the selective inhibition of mononuclear cell trafficking during the acute immune effector phases of MS and other neuroinflammatory diseases.

Introduction

Capillaries of the cerebral vasculature maintain the ionic and biochemical integrity of the central nervous system (CNS) and exclude circulating cells and macromolecules as a consequence of their inter-endothelial tight junctions. Trans-cellular movement of solutes is also precluded, due to the relative lack of endocytic vesicles and fenestrae by these endothelial cells (Pachter et al., 2003, Rubin and Staddon, 1999). The unique properties of the cerebral microvascular endothelium are collectively known as the blood–brain barrier (BBB). Leukocyte migration across the BBB plays an important role in host immune responses to infection and during immunopathologic states (Engelhardt and Ransohoff, 2005, Ransohoff et al., 2003).

Static culture models of the BBB are commonly used to study leukocyte diapedesis in vitro (Callahan and Ransohoff, 2004, Eugenin and Berman, 2003, Persidsky, 1999). Alterations to migrating leukocytes and the endothelial cells are readily studied by such models. It is essential that the in vitro BBB (IVBBB) model possesses the essential characteristics of the BBB, namely inter-endothelial tight junctions and an ability to exclude solutes and macromolecules (Callahan and Ransohoff, 2004). Other techniques employed in vivo include intravital microscopy (IVM) and blockade experiments during experimental autoimmune encephalomyelitis (EAE).

Trafficking of mononuclear cells across the BBB is a sequential and coordinated process that requires selectins, integrins, cell adhesion molecules, chemokines and matrix metalloproteases (Ubogu et al., 2006). There is inconsistent evidence for the role of P-selectin and some preliminary evidence for E-selectin (interacting with a carbohydrate modification of P-selectin glycoprotein-1 ligand and an unknown ligand respectively) in mediating leukocyte rolling and arrest on the endothelium of the BBB based on IVM and EAE blockade experiments (Battistini et al., 2003, Carvalho-Tavares et al., 2000, Engelhardt et al., 2005, Engelhardt et al., 1997, Piccio et al., 2002, Xu et al., 2004). Integrins (such as α4β1 integrin and αLβ2 integrin) expressed on leukocytes and activated by chemokines, interact with cell adhesion molecules of the immunoglobulin (Ig) superfamily to facilitate firm leukocyte adherence and possible transendothelial or inter-endothelial diapedesis, demonstrated by IVM and EAE blockade in vivo and static BBB models in vitro (Baron et al., 1993, Engelhardt and Ransohoff, 2005, Laschinger et al., 2002, Steffen et al., 1994, Ubogu et al., 2006, Vajkoczy et al., 2001, Wong et al., 1999). α4β1 integrin has also been implicated in mediating leukocyte rolling on mouse cerebral vessels and unique “capture” and arrest on the spinal cord and retinal microvessels based on IVM experiments (Engelhardt and Ransohoff, 2005, Vajkoczy et al., 2001).

IVM studies in mice also show that highly activated, neuroantigen-specific encephalitogenic T-cells only interact with superficial cerebral microvessels after endothelial activation with either tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS) (Piccio et al., 2002). Endothelial activation is the process by which endothelial cells respond to proinflammatory cytokines (such as tissue necrosis factor [TNF]-α, interferon [IFN]-γ and interleukin [IL]-1β) or Toll-like receptor ligands, with up-regulation or expression of chemokines and adhesion molecules in vitro or in vivo in a dose- and time-dependent manner (Harkness et al., 2003, Librizzi et al., 2006, Omari et al., 2004, Sobel et al., 1990, Stins et al., 1997, Washington et al., 1994, Wong and Dorovini-Zis, 1992, Wong and Dorovini-Zis, 1995). Endothelial activation in cerebral microvessels can be seen in trauma, infection, ischemia and inflammatory disorders, such as multiple sclerosis (MS) (Sobel et al., 1990, Washington et al., 1994). These observations imply that microvascular endothelial activation by cytokines produced either locally or systemically have roles in CNS leukocyte trafficking.

In several studies, adhesion molecules intercellular adhesion molecule (ICAM)-1 (interacts with αLβ2 integrin) and vascular cell adhesion molecule (VCAM)-1 (interacts with α4β1 integrin) were up-regulated on CNS microvascular endothelial cells during EAE and other experimental systems (Engelhardt and Ransohoff, 2005, Nottet et al., 1996, Sasseville et al., 1992, Steffen et al., 1994). Although ICAM-1 expression is well established in vivo (Bo et al., 1996, Sobel et al., 1990), the expression of VCAM-1 by human cerebral vasculature remains uncertain. Several studies have detected VCAM-1 on human microglia, monocytes or macrophages (Cannella and Raine, 1995, Kivisakk et al., 2003, Peterson et al., 2002) rather than on endothelium. In human atherosclerosis, there was a report of VCAM-1 expression in human coronary artery plaques associated with increased intimal leukocyte accumulation (O'Brien et al., 1993). However, in several other human and animal studies, there was no correlation between VCAM-1 expression on the luminal endothelium and areas corresponding to monocyte entry (Duplaa et al., 1996, Li et al., 1993). These findings raise the question of potential alternative endothelial receptors for α4β1 integrin.

In this regard, an alternatively spliced fibronectin element, termed the type III connecting segment (CS) contains a 25-amino acid, high affinity binding site for α4β1 integrin called CS-1. The LDV peptide within the CS-1 region is critical for α4β1 integrin recognition (Elices et al., 1994, Humphries et al., 1987). The CS-1 binding site on α4β1 integrin is close to, but distinct from the VCAM-1 binding site (Makarem et al., 1994). Almost all cellular fibronectin (in contrast to ∼ 50% of plasma fibronectin) contains the Type III CS region (Elices et al., 1994, Humphries et al., 1987, Shih et al., 1999). Fibronectin CS-1 (FN CS-1) expression on endothelial cells has been demonstrated in vivo (in association with mononuclear cell infiltrate) on rheumatoid arthritis synovial microvasculature (Elices et al., 1994) and human coronary artery lesions (Shih et al., 1999); and on activated human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) in vitro (Boyle et al., 2000, Shih et al., 1999). Accordingly, it is plausible that FN CS-1 could act as an adhesion molecule during leukocyte trafficking in neuroinflammation.

In order to assess chemokine-driven migration in neuroinflammation, we developed a physiological aIVBBB, as local or systemic production of cytokines has been observed during CNS inflammatory processes (Baraczka et al., 2004, Cannella and Raine, 1995, dos Santos et al., 2005, Giovannoni et al., 2001). CCL5 was used for this study as it has been shown to facilitate the chemotaxis of both monocytes and T-cells in vitro via interaction with multiple receptors (Appay and Rowland-Jones, 2001, Schall, 1991) and has been implicated in the pathogenesis of several neuroinflammatory processes, including MS (Appay and Rowland-Jones, 2001, Schall, 1991, Ubogu et al., 2006).

Chemokines (or “chemotactic cytokines”) are small (8–14 kDa), structurally similar proteins that elicit leukocyte migration in a concentration-dependent manner in vitro (Engelhardt and Ransohoff, 2005, Ransohoff et al., 2003, Ubogu et al., 2006). Chemokines govern the migration of hematogenous leukocytes in physiologic and pathologic conditions via interactions with high-affinity G-protein coupled receptors with seven transmembrane spanning regions (Engelhardt and Ransohoff, 2005, Ransohoff et al., 2003, Ubogu et al., 2006). Although chemokines are classically known as regulators of leukocyte migration during inflammation, these molecules play important roles in cellular migration during embryogenesis, including nervous system patterning and development (Ubogu et al., 2006).

Our studies highlighted the differential roles of leukointegrins and adhesion molecules for T-cell and monocyte transmigration across the aIVBBB. We also show that FN CS-1 not VCAM-1 was the relevant α4β1 integrin receptor in this system. Both CCR1 and CCR5 facilitated CCL5-driven PBMC migration, demonstrating a non-redundant role for each of these chemokine receptors in mediating transmigration across the aIVBBB.

Section snippets

Cell culture and cytokine treatment

The endothelial nature of the SV 40 (T antigen) transformed human brain microvascular endothelial cell (THBMEC) line has been previously established and described (Callahan et al., 2004, Stins et al., 1997). Cytokine activation of cultured THBMEC layers was performed by the administration of different concentrations of recombinant human TNF-α and IFN-γ (both from R and D Systems, Minneapolis, MN) 24 h before 100% confluence was expected, as monitored by phase contrast microscopy.

CCL2 ELISA

THBMECs were

Development of a physiological, cytokine-activated in vitro BBB (aIVBBB) model using transformed human brain microvascular endothelial cells (THBMECs)

Results demonstrating the development of the aIVBBB model are available on-line as supplementary data. These experiments showed that THBMEC cytokine activation with 10 U/mL TNF-α and 20 U/mL IFN-γ (10/20 aTHBMECs) resulted in a physiological aIVBBB. Interestingly, these concentrations are within the range reported in the serum of patients with sepsis or systemic inflammatory response syndrome (TNF-α: 4.0–14.6 U/mL, IFN-γ: 0.05–33.5 U/mL) (Brunner et al., 2004, Collighan et al., 2004, Kabir et

Discussion

The initial objective of this study was to develop a physiological aIVBBB to study chemokine-driven PBMC migration during neuroinflammatory processes. Developing a physiological aIVBBB was of importance, as high, non-physiological concentrations of cytokines might compromise THBMEC viability or barrier properties. We were able to define conditions that yielded optimal inflammatory responses (up-regulation of CCL2 and ICAM-1) without affecting BBB properties or endothelial cell viability.

The

Acknowledgments

This research is supported in part by the National Institutes of Health grant P01 NS38667 (to RMR) and a fellowship (to MKC) from the National Multiple Sclerosis Society.

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