Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule found on blood–brain barrier endothelial cells (BBB-ECs) that was previously shown to be involved in leukocyte ...transmigration across the endothelium. In the present study, we found that ALCAM knockout (KO) mice developed a more severe myelin oligodendrocyte glycoprotein (MOG)35–55–induced experimental autoimmune encephalomyelitis (EAE). The exacerbated disease was associated with a significant increase in the number of CNS-infiltrating proinflammatory leukocytes compared with WT controls. Passive EAE transfer experiments suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. In addition, phenotypic characterization of unimmunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further in vivo, in vitro, and molecular analysis confirmed that ALCAM is associated with tight junction molecule assembly at the BBB, explaining the increased permeability of CNS blood vessels in ALCAM KO animals. Collectively, our data point to a biologically important function of ALCAM in maintaining BBB integrity.
In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), myelin-specific T cells are activated in the periphery and differentiate in T helper (Th) 1 and Th17 ...effector cells, which cross the blood-brain barrier (BBB) to reach the central nervous system (CNS), where they induce neuroinflammation. Here, we explored the role of intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in the activation of naïve myelin-specific T cells and in the subsequent migration of differentiated encephalitogenic Th1 and Th17 cells across the BBB
and
. While on antigen-presenting cells ICAM-1, but not ICAM-2 was required for the activation of naïve CD4
T cells, endothelial ICAM-1 and ICAM-2 mediated both Th1 and Th17 cell migration across the BBB. ICAM-1/-2-deficient mice developed ameliorated typical and atypical EAE transferred by encephalitogenic Th1 and Th17 cells, respectively. Our study underscores important yet cell-specific contributions for ICAM-1 and ICAM-2 in EAE pathogenesis.
The extravasation of CD4+ effector/memory T cells (TEM cells) across the blood–brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) or multiple ...sclerosis (MS). Endothelial ICAM‐1 and ICAM‐2 are essential for CD4+ TEM cell crawling on the BBB prior to diapedesis. Here, we investigated the influence of cell surface levels of endothelial ICAM‐1 in determining the cellular route of CD4+ TEM‐cell diapedesis across cytokine treated primary mouse BBB endothelial cells under physiological flow. Inflammatory conditions, inducing high levels of endothelial ICAM‐1, promoted rapid initiation of transcellular diapedesis of CD4+ T cells across the BBB, while intermediate levels of endothelial ICAM‐1 favored paracellular CD4+ T‐cell diapedesis. Importantly, the route of T‐cell diapedesis across the BBB was independent of loss of BBB barrier properties. Unexpectedly, a low number of CD4+ TEM cells was found to cross the inflamed BBB in the absence of endothelial ICAM‐1 and ICAM‐2 via an obviously alternatively regulated transcellular pathway. In vivo, this translated to the development of ameliorated EAE in ICAM‐1null//ICAM‐2−/−C57BL/6J mice. Taken together, our study demonstrates that cell surface levels of endothelial ICAM‐1 rather than the inflammatory stimulus or BBB integrity influence the pathway of T‐cell diapedesis across the BBB.
Listeria monocytogenes (Lm) is a bacterial pathogen of major concern for humans and ruminants due to its neuroinvasive potential and its ability to cause deadly encephalitis (neurolisteriosis). On ...one hand, polymorphonuclear neutrophils (PMN) are key players in the defense against Lm, but on the other hand intracerebral infiltration with PMN is associated with significant neural tissue damage. Lm-PMN interactions in neurolisteriosis are poorly investigated, and factors inducing PMN chemotaxis to infectious foci containing Lm in the central nervous system (CNS) remain unidentified.
In this study, we assessed bovine PMN chemotaxis towards Lm and supernatants of infected endogenous brain cell populations in ex vivo chemotaxis assays, to identify chemotactic stimuli for PMN chemotaxis towards Lm in the brain. In addition, microglial secretion of IL-8 was assessed both ex vivo and in situ.
Our data show that neither Lm cell wall components nor intact bacteria elicit chemotaxis of bovine PMN ex vivo. Moreover, astrocytes and neural cells fail to induce bovine PMN chemotaxis upon infection. In contrast, supernatant from Lm infected microglia readily induced chemotaxis of bovine PMN. Microglial expression and secretion of IL-8 was identified during early Lm infection in vitro and in situ, although IL-8 blocking with a specific antibody could not abrogate PMN chemotaxis towards Lm infected microglial supernatant.
These data provide evidence that host-derived rather than bacterial factors trigger PMN chemotaxis to bacterial foci in the CNS, that microglia have a primary role as initiators of bovine PMN chemotaxis into the brain during neurolisteriosis and that blockade of these factors could be a therapeutic target to limit intrathecal PMN chemotaxis and PMN associated damage in neurolisteriosis.
The interleukin 7 receptor alpha chain (IL-7Rα) is predominately expressed by lymphocytes, and activation by its ligand IL-7 supports the development and maintenance of T cells and boosts T-cell ...mediated immunity. We recently reported that lymphatic endothelial cells (LECs) in dermal lymphatics also express IL-7 and its receptor chains (IL-7Rα and CD132) and that IL-7 supports lymphatic drainage. This suggested that activation of IL-7Rα signaling in lymphatics could exert inflammation-resolving activity, by promoting the clearance of excess tissue fluid. Here we investigated how the potentially opposing effects of IL-7Rα signaling in immune cells and in the lymphatic vasculature would affect the development and progression of psoriasis-like skin inflammation. We found that during acute and chronic skin inflammation mice with an endothelial-specific deletion of IL-7Rα (IL-7Rα
mice) developed more edema compared to control mice, as a consequence of impaired lymphatic drainage. However, systemic treatment of wild-type mice with IL-7 exacerbated edema and immune cell infiltration in spite of increasing lymphatic drainage, whereas treatment with IL-7Rα blocking antibody ameliorated inflammatory symptoms. These data identify IL-7Rα signaling as a new pathway in psoriasis-like skin inflammation and show that its pro-inflammatory effects on the immune compartment override its anti-inflammatory, drainage-enhancing effects on the endothelium.
Intravital multiphoton microscopy has become one of the central tools used in the investigation of dynamic cellular activity and function in living animals under nearly physiological conditions and ...is particularly important for studying the dynamic immune system. During intravital imaging in mice, periodic motion of tissue caused by respiration, induces significant shifts of the imaged region. In slow laser scanning imaging modalities, such as multiphoton microscopy, this movement can lead to considerable distortion and discontinuity in three dimensions of the acquired images. Here, we introduce VivoFollow 2, a toolkit that concurrent with image acquisition performs a precise measurement of the respective image distortion, enabling subsequent automated correction of the imaging data. Recovery of one three-dimensional image stack, corresponding to the tomographic tissue sectioning by the optical plane from each single raw image stack, preserves the time continuity within each image stack. Implementation of VivoFollow 2 thus enables a minimization of motion artifacts in tissues exposed to periodic movements and allows for long-term time-lapse imaging and subsequent precise image analysis of the dynamics of cellular and humoral factors in vivo.
Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults leading to severe disability. Besides genetic traits, environmental factors ...contribute to MS pathogenesis. Cigarette smoking increases the risk of MS in an HLA-dependent fashion, but the underlying mechanisms remain unknown. Here, we explored the effect of cigarette smoke exposure on spontaneous and induced models of experimental autoimmune encephalomyelitis (EAE) by evaluating clinical disease and, when relevant, blood leukocytes and histopathology. In the relapsing-remitting (RR) transgenic model in SJL/J mice, we observed very low incidence in both smoke-exposed and control groups. In the optico-spinal encephalomyelitis (OSE) double transgenic model in C57BL/6 mice, the early onset of EAE prevented a meaningful evaluation of the effects of cigarette smoke. In EAE models induced by immunization, daily exposure to cigarette smoke caused a delayed onset of EAE followed by a protracted disease course in SJL/J mice. In contrast, cigarette smoke exposure ameliorated the EAE clinical score in C57BL/6J mice. Our exploratory studies therefore show that genetic background influences the effects of cigarette smoke on autoimmune neuroinflammation. Importantly, our findings expose the challenge of identifying an animal model for studying the influence of cigarette smoke in MS.
Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism's immune regulation and immune protection. Several ...lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112-CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.
The migration of CD4+ effector/memory T cells across the blood–brain barrier (BBB) is a critical step in MS or its animal model, EAE. T‐cell diapedesis across the BBB can occur paracellular, via the ...complex BBB tight junctions or transcellular via a pore through the brain endothelial cell body. Making use of primary mouse brain microvascular endothelial cells (pMBMECs) as in vitro model of the BBB, we here directly compared the transcriptome profile of pMBMECs favoring transcellular or paracellular T‐cell diapedesis by RNA sequencing (RNA‐seq). We identified the atypical chemokine receptor 1 (Ackr1) as one of the main candidate genes upregulated in pMBMECs favoring transcellular T‐cell diapedesis. We confirmed upregulation of ACKR1 protein in pMBMECs promoting transcellular T‐cell diapedesis and in venular endothelial cells in the CNS during EAE. Lack of endothelial ACKR1 reduced transcellular T‐cell diapedesis across pMBMECs under physiological flow in vitro. Combining our previous observation that endothelial ACKR1 contributes to EAE pathogenesis by shuttling chemokines across the BBB, the present data support that ACKR1 mediated chemokine shuttling enhances transcellular T‐cell diapedesis across the BBB during autoimmune neuroinflammation.
Expression of high levels of the atypical chemokine receptor 1 (ACKR1) promote transcellular T‐cell diapedesis across the blood–brain barrier (BBB) in vitro possibly by shuttling inflammatory chemokines from the abluminal to the luminal side of the BBB.
Considering the 3R rules of animal experimentation, we asked if refined scoring of experimental autoimmune encephalomyelitis (EAE) in mice improves documentation of clinical EAE allowing to perform a ...more powerful statistical analysis. Surprisingly, refined EAE scoring failed to improve statistical outcome comparing the overall disease courses between two groups of mice.
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