Differential roles for endothelial ICAM-1, ICAM-2, and VCAM-1 in shear-resistant T cell arrest, polarization, and directed crawling on blood-brain barrier endothelium

Endothelial ICAM-1 and ICAM-2 were shown to be essential for T cell diapedesis across the blood-brain barrier (BBB) in vitro under static conditions. Crawling of T cells prior to diapedesis was only recently revealed to occur preferentially against the direction of blood flow on the endothelial surface of inflamed brain microvessels in vivo. Using live cell-imaging techniques, we prove that Th1 memory/effector T cells predominantly crawl against the direction of flow on the surface of BBB endothelium in vitro. Analysis of T cell interaction with wild-type, ICAM-1-deficient, ICAM-2-deficient, or ICAM-1 and ICAM-2 double-deficient primary mouse brain microvascular endothelial cells under physiological flow conditions allowed us to dissect the individual contributions of endothelial ICAM-1, ICAM-2, and VCAM-1 to shear-resistant T cell arrest, polarization, and crawling. Although T cell arrest was mediated by endothelial ICAM-1 and VCAM-1, T cell polarization and crawling were mediated by endothelial ICAM-1 and ICAM-2 but not by endothelial VCAM-1. Therefore, our data delineate a sequential involvement of endothelial ICAM-1 and VCAM-1 in mediating shear-resistant T cell arrest, followed by endothelial ICAM-1 and ICAM-2 in mediating T cell crawling to sites permissive for diapedesis across BBB endothelium.

Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood-brain barrier models for the study of T cell extravasation

Important insights into the molecular mechanism of T cell extravasation across the blood-brain barrier (BBB) have already been obtained using immortalized mouse brain endothelioma cell lines (bEnd). However, compared with bEnd, primary brain endothelial cells have been shown to establish better barrier characteristics, including complex tight junctions and low permeability. In this study, we asked whether bEnd5 and primary mouse brain microvascular endothelial cells (pMBMECs) were equally suited as in vitro models with which to study the cellular and molecular mechanisms of T cell extravasation across the BBB. We found that both in vitro BBB models equally supported both T cell adhesion under static and physiologic flow conditions, and T cell crawling on the endothelial surface against the direction of flow. In contrast, distances of T cell crawling on pMBMECs were strikingly longer than on bEnd5, whereas diapedesis of T cells across pMBMECs was dramatically reduced compared with bEnd5. Thus, both in vitro BBB models are suited to study T cell adhesion. However, because pMBMECs better reflect endothelial BBB specialization in vivo, we propose that more reliable information about the cellular and molecular mechanisms of T cell diapedesis across the BBB can be attained using pMBMECs.

Alpha4beta1 integrin mediates the recruitment of immature dendritic cells across the blood-brain barrier during experimental autoimmune encephalomyelitis

Dendritic cells (DCs) within the CNS are recognized to play an important role in the effector phase and propagation of the immune response in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. However, the mechanisms regulating DC trafficking into the CNS still need to be characterized. In this study, we show by performing intravital fluorescence videomicroscopy of the inflamed spinal cord white-matter microvasculature in SJL mice with EAE that immature, and to a lesser extent, LPS-matured, bone marrow-derived DCs efficiently interact with the CNS endothelium by rolling, capturing, and firm adhesion. Immature but not LPS-matured DCs efficiently migrated across the wall of inflamed parenchymal microvessels into the CNS. Blocking alpha4 integrins interfered with the adhesion but not the rolling or capturing of immature and LPS-matured DCs to the CNS microvascular endothelium, inhibiting their migration across the vascular wall. Functional absence of beta1 integrins but not of beta7 integrins or alpha4beta7 integrin similarly reduced the adhesion of immature DCs to the CNS microvascular endothelium, demonstrating that alpha4beta1 but not alpha4beta7 integrin mediates this step of immature DCs interaction with the inflamed blood-brain barrier during EAE. Our study shows that during EAE, especially immature DCs migrate into the CNS, where they may be crucial for the perpetuation of the CNS-targeted autoimmune response. Thus therapeutic targeting of alpha4 integrins affects DC trafficking into the CNS and may therefore lead to the resolution of the CNS autoimmune inflammation by reducing the number of CNS professional APCs.

The pulmonary blood-gas barrier in the avian embryo: inauguration, development and refinement

In vertebrates, efficient gas exchange depends primarily on establishment of a thin blood-gas barrier (BGB). The primordial air conduits of the developing avian lung are lined with a cuboidal epithelium that is ultimately converted to a squamous one that participates in the formation of the BGB. In the early stages, cells form intraluminal protrusions (aposomes) then transcellular double membranes separating the aposome from the basal part of the cell establish, unzip and sever the aposome from the cell. Additionally, better endowed cells squeeze out adjacent cells or such cells constrict spontaneously thus extruding the squeezed out aposome. Formation of vesicles or vacuoles below the aposome and fusion of such cavities with their neighboring cognates results in severing of the aposome. Augmentation of cavities and their subsequent fusion with the apical plasma membranes results in formation of numerous microfolds separating concavities on the apical part of the cell. Abscission of such microfolds results in a smooth squamous epithelium just before hatching.

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

β1-integrin/matrix interactions support blood-brain barrier integrity

Brain microvascular endothelium forms an active permeability barrier, the blood-brain barrier (BBB). In neurologic disorders, barrier properties of the BBB are often lost indicating their dependance on molecular cues of the brain microenvironment. In this issue, Osada et al demonstrate that the endothelial extracellular matrix (ECM) provides one of these cues. Their study shows that β1-integrin-mediated adhesion of brain endothelial cells to the surrounding ECM is critical for stabilizing claudin-5 in BBB tight junctions (TJs) and BBB integrity. These observations point to a novel intracellular signaling pathway from β1-integrin/ECM endothelial adhesions to BBB TJs contributing to BBB integrity.

TET inducible expression of the α4β7-integrin ligand MAdCAM-1 on the blood-brain barrier does not influence the immunopathogenesis of experimental autoimmune encephalomyelitis

Inhibiting the α4 subunit of the integrin heterodimers α4β1 and α4β7 with the mab natalizumab is an effective treatment of multiple sclerosis (MS). Which of the two α4 heterodimers is involved in disease pathogenesis has, however, remained controversial. Whereas the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, is ameliorated in β7-integrin-deficient C57BL/6 mice, neutralizing antibodies against the β7-integrin subunit or the α4β7-integrin heterodimer fail to interfere with EAE pathogenesis in the SJL mouse. To facilitate α4β7-integrin-mediated immune-cell trafficking across the blood-brain barrier (BBB), we established transgenic C57BL/6 mice with endothelial cell-specific, inducible expression of the α4β7-integrin ligand mucosal addressin cell adhesion molecule (MAdCAM)-1 using the tetracycline (TET)-OFF system. Although TET-regulated MAdCAM-1 induced α4β7-integrin mediated interaction of α4β7(+) /α4β1(-) T cells with the BBB in vitro and in vivo, it failed to influence EAE pathogenesis in C57BL/6 mice. TET-regulated MAdCAM-1 on the BBB neither changed the localization of central nervous system (CNS) perivascular inflammatory cuffs nor did it enhance the percentage of α4β7-integrin(+) inflammatory cells within the CNS during EAE. In conclusion, our study demonstrates that ectopic expression of MAdCAM-1 at the BBB does not increase α4β7-integrin-mediated immune cell trafficking into the CNS during MOG(aa35-55)-induced EAE.

Review: leucocyte-endothelial cell crosstalk at the blood-brain barrier: a prerequisite for successful immune cell entry to the brain

Leucocyte migration into the central nervous system is a key stage in the development of multiple sclerosis. While much has been learnt regarding the sequential steps of leucocyte capture, adhesion and migration across the vasculature, the molecular basis of leucocyte extravasation is only just being unravelled. It is now recognized that bidirectional crosstalk between the immune cell and endothelium is an essential element in mediating diapedesis during both normal immune surveillance and under inflammatory conditions. The induction of various signalling networks, through engagement of cell surface molecules such as integrins on the leucocyte and immunoglobulin superfamily cell adhesion molecules on the endothelial cell, play a major role in determining the pattern and route of leucocyte emigration. In this review we discuss the extent of our knowledge regarding leucocyte migration across the blood-brain barrier and in particular the endothelial cell signalling pathways contributing to this process.

Development and remodeling of the vertebrate blood-gas barrier

During vertebrate development, the lung inaugurates as an endodermal bud from the primitive foregut. Dichotomous subdivision of the bud results in arborizing airways that form the prospective gas exchanging chambers, where a thin blood-gas barrier (BGB) is established. In the mammalian lung, this proceeds through conversion of type II cells to type I cells, thinning, and elongation of the cells as well as extrusion of the lamellar bodies. Subsequent diminution of interstitial tissue and apposition of capillaries to the alveolar epithelium establish a thin BGB. In the noncompliant avian lung, attenuation proceeds through cell-cutting processes that result in remarkable thinning of the epithelial layer. A host of morphoregulatory molecules, including transcription factors such as Nkx2.1, GATA, HNF-3, and WNT5a; signaling molecules including FGF, BMP-4, Shh, and TFG- β and extracellular proteins and their receptors have been implicated. During normal physiological function, the BGB may be remodeled in response to alterations in transmural pressures in both blood capillaries and airspaces. Such changes are mitigated through rapid expression of the relevant genes for extracellular matrix proteins and growth factors. While an appreciable amount of information regarding molecular control has been documented in the mammalian lung, very little is available on the avian lung.

Going against the tide--how encephalitogenic T cells breach the blood-brain barrier

During multiple sclerosis or its animal model, experimental autoimmune encephalomyelitis, circulating immune cells enter the central nervous system (CNS) causing neuroinflammation. Extravasation from the blood circulation across the vessel wall occurs through a multistep process regulated by adhesion and signal transducing molecules on the immune cells and on the endothelium. Since the CNS is shielded by the highly specialized blood-brain barrier (BBB), immune cell extravasation into the CNS requires breaching this particularly tight endothelial border. Consequently, travelling into the CNS demands unique adaptations which account for the extreme tightness of the BBB. Modern imaging tools have shown that after arresting on BBB endothelium, in vivo or in vitro encephalitogenic effector/memory T cells crawl for long distances, possibly exceeding 150 µm along the surface of the BBB endothelium before rapidly crossing the BBB. Interestingly, in addition to the distance of crawling, the preferred direction of crawling against the flow is unique for T cell crawling on the luminal surface of CNS microvessels. In this review, we will summarize the cellular and molecular mechanisms involved in the unique T cell behavior that is obviously required for finding a site permissive for diapedesis across the unique vascular bed of the BBB.

Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo

BACKGROUND The central nervous system (CNS) is an immunologically privileged site to which access for circulating immune cells is tightly controlled by the endothelial blood-brain barrier (BBB) located in CNS microvessels. Under physiological conditions immune cell migration across the BBB is low. However, in neuroinflammatory diseases such as multiple sclerosis, many immune cells can cross the BBB and cause neurological symptoms. Extravasation of circulating immune cells is a multi-step process that is regulated by the sequential interaction of different adhesion and signaling molecules on the immune cells and on the endothelium. The specialized barrier characteristics of the BBB, therefore, imply the existence of unique mechanisms for immune cell migration across the BBB.

Immunologic privilege in the central nervous system and the blood-brain barrier

The brain is in many ways an immunologically and pharmacologically privileged site. The blood-brain barrier (BBB) of the cerebrovascular endothelium and its participation in the complex structure of the neurovascular unit (NVU) restrict access of immune cells and immune mediators to the central nervous system (CNS). In pathologic conditions, very well-organized immunologic responses can develop within the CNS, raising important questions about the real nature and the intrinsic and extrinsic regulation of this immune privilege. We assess the interactions of immune cells and immune mediators with the BBB and NVU in neurologic disease, cerebrovascular disease, and intracerebral tumors. The goals of this review are to outline key scientific advances and the status of the science central to both the neuroinflammation and CNS barriers fields, and highlight the opportunities and priorities in advancing brain barriers research in the context of the larger immunology and neuroscience disciplines. This review article was developed from reports presented at the 2011 Annual Blood-Brain Barrier Consortium Meeting.