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.

Immune cell migration across the blood–brain barrier: molecular mechanisms and therapeutic targeting

The endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier protect the CNS from the constantly changing milieu within the bloodstream. The BBB strictly controls immune cell entry into the CNS, which is rare under physiological conditions. During a variety of pathological conditions of the CNS, such as viral or bacterial infections, or during inflammatory diseases, such as multiple sclerosis, immunocompetent cells readily traverse the BBB and subsequently enter the CNS parenchyma. Most of the available information on immune cell entry into the CNS is derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Consequently, our current knowledge on traffic signals mediating immune cell entry across the BBB during immunosurveillance and disease results mainly from experimental data in the EAE model. Therefore, a large part of this review summarizes these findings. Similarly, the potential benefits and risks associated with therapeutic targeting of immune cell trafficking across the BBB will be discussed in the context of multiple sclerosis, since elucidation of the molecular mechanisms relevant to this disease have largely relied on the use of its in vivo model, EAE.

The mucosal immune system at the gastrointestinal barrier

The immune system faces a considerable challenge in its efforts to maintain tissue homeostasis in the intestinal mucosa. It is constantly confronted with a large array of antigens, and has to prevent the dissemination and proliferation of potentially harmful agents while sparing the vital structures of the intestine from immune-mediated destruction. Complex interactions between the highly adapted effector cells and mechanisms of the innate and adaptive immune system generally prevent the luminal microflora from penetrating the intestinal mucosa and from spreading systemically. Non-haematopoietic cells critically contribute to the maintenance of local tissue homeostasis in an antigen-rich environment by producing protective factors (e.g. production of mucus by goblet cells, or secretion of microbicidal defensins by Paneth cells) and also through interactions with the adaptive and innate immune system (such as the production of chemotactic factors that lead to the selective recruitment of immune cell subsets). The complexity of the regulatory mechanisms that control the local immune response to luminal antigens is also reflected in the observation that mutations in immunologically relevant genes often lead to the development of uncontrolled inflammatory reactions in the microbially colonized intestine of experimental animals.

Effect of intramammary administration of prednisolone on the blood-milk barrier during the immune response of the mammary gland to lipopolysaccharide

OBJECTIVE: To investigate effects of intramammary administration of prednisolone on the immune response of mammary glands in cows. ANIMALS: 5 lactating Red Holsteins. PROCEDURES: Cows received a different intramammary infusion in each mammary gland (10 mg of prednisolone, 100 μg of lipopolysaccharide [LPS], 100 μg of LPS and 10 mg of prednisolone, or saline [0.9% NaCl] solution). Milk samples were collected before (time 0) and 3, 6, 9, 12, 24, and 36 hours after treatment. Somatic cell count (SCC), lactate dehydrogenase (LDH) activity, and concentrations of serum albumin (SA) and tumor necrosis factor (TNF)-α in milk and mRNA expression of TNF-α, interleukin (IL)-8, and IL-1β in milk somatic cells were analyzed. RESULTS: Saline solution or prednisolone did not change SCC, LDH activity, and SA and TNF-α concentrations in milk and mRNA expression of TNF-α, IL-1β, and IL-8 in milk somatic cells. The SCC and TNF-α concentration in milk increased similarly in glands infused with LPS, independent of prednisolone administration. However, the increase of LDH activity and SA concentration in milk after LPS infusion was diminished by prednisolone administration. The mRNA expression of TNF-α, IL-8, and IL-1β in milk somatic cells increased after LPS infusion and was unaffected by prednisolone. CONCLUSIONS AND CLINICAL RELEVANCE: Intramammary administration of prednisolone did not induce an immune response and did not change mRNA expression of TNF-α, IL-8, and L-1β during the response to intramammary administration of LPS. However, prednisolone reduced disruption of the blood-milk barrier. This could influence the severity and cure rate of mastitis.

Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

BACKGROUND The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. METHODS We compared cell line models of the mouse BCSFB derived from the Immortomouse(®) and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. RESULTS We found that inducible cell line models established from the Immortomouse(®) or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF. CONCLUSIONS Our study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.

An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

Hypersensitivity and oral tolerance in the absence of a secretory immune system

Mucosal immunity protects the epithelial barrier by immune exclusion of foreign antigens and by anti-inflammatory tolerance mechanisms, but there is a continuing debate about the role of secretory immunoglobulins (SIgs), particularly SIgA, in the protection against allergy and other inflammatory diseases. Lack of secretory antibodies may cause immune dysfunction and affect mucosally induced (oral) tolerance against food antigens.

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.

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.

Fluids and barriers of the CNS establish immune privilege by confining immune surveillance to a two-walled castle moat surrounding the CNS castle

Neuronal activity within the central nervous system (CNS) strictly depends on homeostasis and therefore does not tolerate uncontrolled entry of blood components. It has been generally believed that under normal conditions, the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB) prevent immune cell entry into the CNS. This view has recently changed when it was realized that activated T cells are able to breach the BBB and the BCSFB to perform immune surveillance of the CNS. Here we propose that the immune privilege of the CNS is established by the specific morphological architecture of its borders resembling that of a medieval castle. The BBB and the BCSFB serve as the outer walls of the castle, which can be breached by activated immune cells serving as messengers for outside dangers. Having crossed the BBB or the BCSFB they reach the castle moat, namely the cerebrospinal fluid (CSF)-drained leptomeningeal and perivascular spaces of the CNS. Next to the CNS parenchyma, the castle moat is bordered by a second wall, the glia limitans, composed of astrocytic foot processes and a parenchymal basement membrane. Inside the castle, that is the CNS parenchyma proper, the royal family of sensitive neurons resides with their servants, the glial cells. Within the CSF-drained castle moat, macrophages serve as guards collecting all the information from within the castle, which they can present to the immune-surveying T cells. If in their communication with the castle moat macrophages, T cells recognize their specific antigen and see that the royal family is in danger, they will become activated and by opening doors in the outer wall of the castle allow the entry of additional immune cells into the castle moat. From there, immune cells may breach the inner castle wall with the aim to defend the castle inhabitants by eliminating the invading enemy. If the immune response by unknown mechanisms turns against self, that is the castle inhabitants, this may allow for continuous entry of immune cells into the castle and lead to the death of the castle inhabitants, and finally members of the royal family, the neurons. This review will summarize the molecular traffic signals known to allow immune cells to breach the outer and inner walls of the CNS castle moat and will highlight the importance of the CSF-drained castle moat in maintaining immune surveillance and in mounting immune responses in the CNS.

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.

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.

Regulation of immune cell entry into the central nervous system

The central nervous system (CNS) has long been regarded as an immune privileged organ implying that the immune system avoids the CNS to not disturb its homeostasis, which is critical for proper function of neurons. Meanwhile, it is accepted that immune cells do in fact gain access to the CNS and that immune responses can be mounted within this tissue. However, the unique CNS microenvironment strictly controls these immune reactions starting with tightly controlling immune cell entry into the tissue. The endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid (CSF) barrier, which protect the CNS from the constantly changing milieu within the bloodstream, also strictly control immune cell entry into the CNS. Under physiological conditions, immune cell migration into the CNS is kept at a very low level. In contrast, during a variety of pathological conditions of the CNS such as viral or bacterial infections, or during inflammatory diseases such as multiple sclerosis, immunocompetent cells readily traverse the BBB and likely also the choroid plexus and subsequently enter the CNS parenchyma or CSF spaces. This chapter summarizes our current knowledge of immune cell entry across the blood CNS barriers. A large body of the currently available information on immune cell entry into the CNS has been derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Therefore, most of this chapter discussing immune cell entry during CNS pathogenesis refers to observations in the EAE model, allowing for the possibility that other mechanisms of immune cell entry into the CNS might apply under different pathological conditions such as bacterial meningitis or stroke.