An autoimmunity odyssey: how autoreactive T cells infiltrate into the CNS

Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model of multiple sclerosis (MS), a human autoimmune disease. To explore how EAE and ultimately MS is induced, autoantigen-specific T cells were established, were labeled with fluorescent protein by retroviral gene transfer, and were tracked in vivo after adoptive transfer. Intravital imaging with two-photon microscopy was used to record the entire entry process of autoreactive T cells into the CNS: a small number of T cells first appear in the CNS leptomeninges before onset of EAE, and crawl on the intraluminal surface of blood vessels, which is integrin α4 and αL dependent. After extravasation, the T cells continue into the perivascular space, meeting local antigen-presenting cells (APCs), which present endogenous antigens. This interaction activates the T cells and guides them to penetrate the CNS parenchyma. As the local APCs in the CNS are not saturated with endogenous antigens, exogenous antigens stimulate the autoreactive T cells more strongly and, as a result, exacerbate the clinical outcome. Currently, we are attempting to visualize T-cell activation in vivo in both rat T-cell-mediated EAE and mouse spontaneous EAE models.     

A size selective vascular barrier in the rat area postrema formed by perivascular macrophages and the extracellular matrix

The fenestrated microvasculature of the area postrema shows a less restrictive blood-brain barrier than is found in other areas of the CNS. We have studied the expression and relationship of vascular endothelial tight junctional proteins, astrocytes, macrophages, and the extracellular matrix with the extravasation of fluorescently tagged dextrans and sodium fluorescein in the rat area postrema. Glial fibrillary acidic protein (GFAP) -positive astrocytes were present within the area postrema which was surrounded by a dense zone of highly GFAP-reactive astrocytes. Expression of the tight junction proteins claudin-5, -12 and occludin was absent, although diffuse cytoplasmic claudin-1 immunoreactivity was present. The extracellular matrix of the endothelium showed two non-fused thickened layers of laminin immunoreactivity. CD163 and CD169 immunoreactive perivascular macrophages were located within lacunae between these two laminin layers. Fluorescently tagged dextrans (10-70 kDa) passed from the vasculature but were retained between the inner and outer laminin walls and rapidly sequestered by the perivascular CD163 and CD169 macrophages. Three-kilodalton dextran diffused into the parenchyma, but was retained within the boundary of the area postrema at the interface with the highly reactive GFAP-astrocytes, while sodium fluorescein (0.3 kDa) passed from the area postrema into surrounding CNS areas. Our observations suggest that despite the absence of a tight blood-brain barrier, a size selective barrier restricting the movement of blood solutes into the parenchyma is present in the area postrema. We suggest that the rapid uptake by CD163 and CD169 macrophages together with the non-fused laminin immunoreactive layers of the extracellular matrix plays a size selective role in restricting movement of serum proteins and other blood borne macromolecules over 10 kDa in to the area postrema.     

Disproportionate recruitment of CD8+ T cells into the central nervous system by professional antigen-presenting cells

Inappropriate immune responses, thought to exacerbate or even to initiate several types of central nervous system (CNS) neuropathology, could arise from failures by either the CNS or the immune system. The extent that the inappropriate appearance of antigen-presenting cell (APC) function contributes to CNS inflammation and pathology is still under debate. Therefore, we characterized the response initiated when professional APCs (dendritic cells) presenting non-CNS antigens were injected into the CNS. These dendritic cells expressed numerous T-cell chemokines, but only in the presence of antigen did leukocytes accumulate in the ventricles, meninges, sub-arachnoid spaces, and injection site. Within the CNS parenchyma, the injected dendritic cells migrated preferentially into the white matter tracts, yet only a small percentage of the recruited leukocytes entered the CNS parenchyma, and then only in the white matter tracts. Although T-cell recruitment was antigen specific and thus mediated by CD4+ T cells in the models used here, CD8+ T cells accumulated in numbers equal to or greater than that of CD4+ T cells. Few of the recruited T cells expressed activation markers (CD25 and VLA-4), and those that did were primarily in the meninges, injection site, ventricles, and perivascular spaces but not in the parenchyma. These results indicate that 1) the CNS modulates the cellular composition and activation states of responding T-cell populations and that 2) myelin-restricted inflammation need not be initiated by a myelin-specific antigen.     

A tumor necrosis factor receptor 1-dependent conversation between central nervous system-specific T cells and the central nervous system is required for inflammatory infiltration of the spinal cord

We examined the role of tumor necrosis factor receptor 1 (TNFR1) in inflammation initiated by the adoptive transfer of central nervous system (CNS)-specific Th1 cells in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. This adoptive transfer paradigm eliminates the confounding effects of bacterial adjuvants in the analysis of inflammation. We found that although T cells could reach the meninges and perivascular space in the absence of TNFR1, recruitment of other inflammatory cells from the blood was dramatically reduced. The reduction in the recruitment of CD11b(hi) cells correlated with a dramatic reduction in the production of the chemokines CCL2 (MCP-1) and CXLC2 (MIP-2) in TNFR1-deficient hosts. Bone marrow chimera experiments demonstrated that TNF can be effectively supplied by either the hematopoietic system or the CNS, but the essential TNFR1-responsive cells reside in the CNS. Previous work has demonstrated that microglia produce CCL2, and here we demonstrate that astrocytes and endothelial cells produced CXCL2 in the early stages of inflammation. Therefore, productive inflammation results from a conversation, or mutually responding signals, between the initiating T cells and cells in the parenchyma of the spinal cord.     

Defective positioning in granulomas but not lung-homing limits CD4 T-cell interactions with Mycobacterium tuberculosis-infected macrophages in rhesus macaques

Protection against Mycobacterium tuberculosis (Mtb) infection requires CD4 T cells to migrate into the lung and interact with infected macrophages. In mice, less-differentiated CXCR3⁺ CD4 T cells migrate into the lung and suppress growth of Mtb, whereas CX3CR1⁺ terminally differentiated Th1 cells accumulate in the blood vasculature and do not control pulmonary infection. Here we examine CD4 T-cell differentiation and lung homing during primary Mtb infection of rhesus macaques. Mtb-specific CD4 T cells simultaneously appeared in the airways and blood ∼21–28 days post exposure, indicating that recently primed effectors are quickly recruited into the lungs after entering circulation. Mtb-specific CD4 T cells in granulomas display a tissue-parenchymal CXCR3⁺CX3CR1⁻PD-1^hiCTLA-4⁺ phenotype. However, most granuloma CD4 T cells are found within the outer lymphocyte cuff and few localize to the myeloid cell core containing the bacilli. Using the intravascular stain approach, we find essentially all Mtb-specific CD4 T cells in granulomas have extravasated across the vascular endothelium into the parenchyma. Therefore, it is unlikely to be that lung-homing defects introduced by terminal differentiation limit the migration of CD4 T cells into granulomas following primary Mtb infection of macaques. However, intralesional positioning defects within the granuloma may pose a major barrier to T-cell-mediated immunity during tuberculosis.     

After injection into the striatum, in vitro-differentiated microglia- and bone marrow-derived dendritic cells can leave the central nervous system via the blood stream

The prototypic migratory trail of tissue-resident dendritic cells (DCs) is via lymphatic drainage. Since the central nervous system (CNS) lacks classical lymphatic vessels, and antigens and cells injected into both the CNS and cerebrospinal fluid have been found in deep cervical lymph nodes, it was thought that CNS-derived DCs exclusively used the cerebrospinal fluid pathway to exit from tissues. It has become evident, however, that DCs found in peripheral organs can also leave tissues via the blood stream. To study whether DCs derived from microglia and bone marrow can also use this route of emigration from the CNS, we performed a series of experiments in which we injected genetically labeled DCs into the striata of rats. We show here that these cells migrated from the injection site to the perivascular space, integrated into the endothelial lining of the CNS vasculature, and were then present in the lumen of CNS blood vessels days after the injection. Moreover, we also found these cells in both mesenteric lymph nodes and spleens. Hence, microglia- and bone marrow-derived DCs can leave the CNS via the blood stream.     

Cerebrovascular miRNAs correlate with the clearance of Aβ through perivascular route in younger 3xTg‐AD mice

The “two‐hit vascular hypothesis for Alzheimer's disease (AD)” and amyloid‐β (Aβ) oligomer hypothesis suggest that impaired soluble Aβ oligomers clearance through the cerebral vasculature may be an initial step of the AD process. Soluble Aβ oligomers are driven into perivascular spaces from the brain parenchyma and toward peripheral blood flow. The underlying vascular‐based mechanism, however, has not been defined. Given that microRNAs (miRNAs), emerging as novel modulators, are involved in numerous physiological and pathological processes, we hypothesized that cerebrovascular miRNAs may regulate the activities of brain blood vessels, which further affects the concentration of Aβ in the AD brain. In this study, perivascular Aβ deposits, higher vascular activation, increased pericyte coverage and up‐regulated capillaries miRNAs at 6 months old (6 mo) were found to correlate with the lower Aβ levels of middle AD stage (9 mo) in 3xTg‐AD (3xTg) mice. It is implicated that at the early stage of AD when intracellular Aβ appeared, higher expression of vessel‐specific miRNAs, elevated pericyte coverage, and activated endothelium facilitate Aβ oligomer clearance through the perivascular route, resulting in a transient reduction of Aβ oligomers at 9 mo. Additionally, ghrelin‐induced upregulation of capillary miRNAs and increased pericyte coverage attenuated Aβ burden at 9 mo, in further support of the relationship between vascular miRNAs and Aβ clearance. This work suggests a cerebral microvessel miRNA may boost endothelial highly activated phenotypes to promote elimination of Aβ oligomers through the perivascular drainage pathway and contribute to AD progression. The targeting of brain vessel‐specific miRNAs may provide a new rationale for the development of innovative therapeutic strategies for AD treatment.     

CD62L is required for the priming of encephalitogenic T cells but does not play a major role in the effector phase of experimental autoimmune encephalomyelitis

CD62L (l ‐selectin, mel 14) regulates naïve T cell homing into lymph nodes and the migration of leucocytes to sites of inflammation. The requirement of CD62L in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has been demonstrated previously. However, it remains controversial as to whether CD62L is required for the induction or the effector phase of EAE. It is also unclear whether other non‐T effector cells need CD62L to enter the central nervous system (CNS) parenchyma and exert their damaging effects on myelin. We report that mice with a targeted mutation of CD62L are resistant to Myelin oligodendrocyte glycoprotein peptide‐induced EAE. CD62L‐deficient mice had no peptide‐specific T cell responses in the draining lymph nodes and had lower levels of peptide‐specific T cell responses in spleens at a later time point. Adoptive transfer studies showed that CD62L‐deficient mice were fully susceptible to adoptive transfer EAE induced by either wildtype or CD62L‐deficient T cells. Moreover, CD62L‐deficient, F4/80⁺ macrophages can be efficiently recruited into the CNS parenchyma. These data suggest that CD62L is required for the induction of encephalitogenic T cells during EAE development, but is not required by T and non‐T effector cells to attack the CNS parenchyma.     

Immune control of the brain

Conclusions In contrast to traditional views, cells of the lymphohematopoietic system can and do enter the CNS. Some of them, for example perivascular cells and cells of the microglia, enter the CNS during development. Others, such as T lymphocytes previously engaged in immune responses, gain access to the CNS parenchyma in the process of immune surveillance, or during inflammatory reactions. Elaborate control systems then ensure that the immune reactivity within the CNS system is limited to an absolute minimum.     

A blood-brain barrier to peroxidase in capillaries surrounded by perivascular spaces

Some investigators have hypothesized that the absence of a blood-brain barrier in specialized regions of the mammalian central nervous system is related to the occurrence of a pericapillary connective tissue space that can accommodate more substance than the usual narrow extracellular space in the remainder of the cerebral parenchyma. The capillaries of the urodele brain are well suited to test this hypothesis. All of the cerebral capillaries examined electron-microscopically in Necturus maculosus are surrounded by a collagen-containing space, about 0.5 m? wide, which is delimited by an endothelial and a glial basement membrane. In Ambystoma tigrinum, some capillaries have perivascular spaces whereas others are surrounded by a single basement membrane shared by endothelium and glia and therefore resemble mammalian vessels exhibiting barrier phenomena. In both Necturus and Ambystoma, horseradish peroxidase administered intravenously one-half to two hours before fixation did not cross the brain capillary endothelium. The bulk of the protein remained in the vessel lumen, although some was incorporated by membrane-bound inclusions within the endothelium, and none ever reached the perivascular basement membrane, space or parenchyma. The amphibian cerebral endothelium is, like the mammalian endothelium, the locus for the barrier to the entry of peroxidase from blood to parenchyma. Thus, the mere occurrence of a pericapillary space is not necessarily coincident with the absence of a blood-brain barrier.     

Estrogen-induced tumors: Changes in the vasculature in two strains of rat

The influence of estrogen on the vasculature of the pars distalis has been studied in two strains of rat that differ in estrogen responsiveness. (Fischer 344 rats are highly estrogen-responsive in comparison to Sprague-Dawley rats.) Ovariectomized adults were implanted with silastic capsules containing 17 β-estradiol benzoate. Control and experimental animals were sacrificed 10 and 20 days after implantation of the silastic capsules. Pituitary weights and plasma prolactin were elevated dramatically in estrogen-treated Fischer rats in comparison to more moderate increases in Sprague-Dawley rats. Although both strains exhibited the hypertrophy of mammotrophs expected after estrogen stimulation, the vasculature in Fischer rats was dramatically altered from normal. The pars distalis of the 20-day, estrogen-treated Fischer rats contained well-formed arteries. In addition, capillaries frequently were disrupted, contributing to the formation of hemorrhagic lakes unlined by an endothelium. Even in intact capillaries, basal laminae delimiting the pericapillary spaces often were disrupted or absent. Perivascular connective tissue cells were prominent within the perivascular spaces and often contained rumerous, large lysosomal dense bodies as well as clusters of small dumbbell-shaped bodies. These granule clusters also were apparent adjacent to the perivascular space within parenchymal cells, most frequently within follicular cells. The vasculature of Sprague-Dawley rats maintained a more normal appearance after estrogen treatment, although perivascular connective tissue cells did appear activated and basal laminae delimiting the pericapillary spaces were disrupted occasionally. However, no capillaries were disrupted, nor were any hemorrhagic lakes evident, and no arteries were present. The results indicate that in some strains of rat, estrogen may influence the integrity of the pars distalis vasculature, including the possibility of stimulating vascular reorganization and arteriogenesis. If stimulation of arteriogenesis decreases normal hypothalamic inhibition of mammotrophs, this could be an important factor underlying the formation of prolactinomas.     

Characterization of CD4−CD8− T cell receptor αβ+ T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis

Inflammation of the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) starts in the subarachnoid space (SAS) and spreads later to the adjacent CNS parenchyma. To characterize the nature of lesion-forming T cells in situ in more detail, T cells were isolated from the SAS and their surface phenotype and the nucleotide sequence of the junctional region of the T cell receptor (TCR) was determined and compared with those of the lymph node (LN) and spinal cord (SC) T cells. Characteristically, more than 70% of SAS TCR αβ⁺ T cells isolated at the early stage of EAE lacked both CD4 and CD8 molecules, whereas those from LN and SC were either CD4⁺ or CD8⁺. Analysis of nucleotide sequences of the junctional region of TCR revealed that T cells bearing a sequence identical to that for encephalitogenic T cell clones were found in both SAS and SC. Furthermore, purified CD4^?CD8^? T cells expressed CD4 molecules after culture. At the same time, these T cells acquired reactivity to myelin basic protein and induced passive EAE in naive animals after adoptive transfer. Our results suggest that CD4^?CD8^? T cells in the SAS are precursors of lesion-forming T cells in the SC and that phenotype switching takes place during the process of T cell infiltration into the CNS parenchyma. The double-negative nature of these T cells may explain an escape of encephalitogenic T cells from negative selection in T cell differentiation.     

Distribution of the thrombomodulin antigen in the rabbit vasculature

The purpose of this study was to determine the distribution of the thrombomodulin (TM) antigen in the rabbit vasculature in vivo. Acetone-fixed, frozen sections of various tissues (brain, heart, aorta, lung, liver, spleen, kidney, small intestine, large intestine, skeletal muscle, bone, and skin) were stained by indirect immunofluorescence with goat affinity-purified antibodies to TM. Both large and small vessels (including capillaries) had demonstrable TM antigen localized to the endothelium while vascular smooth muscle and connective tissue cells were negative. The parenchyma of the organs examined were also negative. Based on antibody dilutions and brightness of staining, there is an indication that lung, followed by heart and intestinal vessels, has the highest concentration of antigen; kidney glomerular capillary loops have the least. These results also indicate that TM is an excellent cell type-specific marker of endothelial cells as the endothelium of all vessels and vascular beds was positive.     

Apoptosis of T lymphocytes in experimental autoimmune encephalomyelitis. Evidence for programmed cell death as a mechanism to control inflammation in the brain.

In experimental autoimmune encephalomyelitis (EAE) myelin-specific T lymphocytes attack the myelinated tissue of the central nervous system (CNS). In the Lewis rat, EAE as a rule has an acute, monophasic course. With spontaneous clinical recovery the inflammatory CNS infiltrates are cleared from the nervous tissue within a few days. This is well in line with the remarkably low incidence of myelin-specific T cells present in EAE infiltrate. Combining immunocytochemical techniques, ultrastructural criteria and in situ nick translation we found up to 49% of T lymphocytes in EAE lesions showing signs of apoptosis at the time of recovery from disease. Our results suggest that apoptosis of T lymphocytes may be one possible mechanism to eliminate T lymphocytes from inflammatory brain lesions.     

Populations of human T lymphocytes that traverse the vascular endothelium stimulated by Borrelia burgdorferi are enriched with cells that secrete gamma interferon

Some diseases are characterized by prevalence in the affected tissues of type 1 T lymphocytes, which secrete gamma interferon (IFN-gamma) and other proinflammatory cytokines. For example, type 1 T cells predominate in the lesions of patients with Lyme disease, which is caused by the bacterium Borrelia burgdorferi. We used an in vitro model of the blood vessel wall to test the premise that the vascular endothelium actively recruits circulating type 1 T cells to such lesions. When T lymphocytes isolated from human peripheral blood were examined, the populations that traversed monolayers of resting human umbilical vein endothelial cells (HUVEC) or HUVEC stimulated by interleukin-1beta or B. burgdorferi were markedly enriched for T cells that produced IFN-gamma compared to the initially added population of T cells. No enrichment was seen for cells that produced interleukin-4, a marker for type 2 T lymphocytes. Very late antigen-4 and CD11/CD18 integrins mediated passage of the T cells across both resting and stimulated HUVEC, and the endothelium-derived chemokine CCL2 (monocyte chemoattractant protein 1) was responsible for the enhanced migration of T cells across stimulated HUVEC. These results suggest that the vascular endothelium may contribute to the selective accumulation of type 1 T cells in certain pathological lesions, including those of Lyme disease.     

Knocking at the brain’s door: intravital two-photon imaging of autoreactive T cell interactions with CNS structures

Since the first applications of two-photon microscopy in immunology 10 years ago, the number of studies using this advanced technology has increased dramatically. The two-photon microscope allows long-term visualization of cell motility in the living tissue with minimal phototoxicity. Using this technique, we examined brain autoantigen-specific T cell behavior in experimental autoimmune encephalitomyelitis, the animal model of human multiple sclerosis. Even before disease symptoms appear, the autoreactive T cells arrive at their target organ. There they crawl along the intraluminal surface of central nervous system (CNS) blood vessels before they extravasate. In the perivascular environment, the T cells meet phagocytes that present autoantigens. This contact activates the T cells to penetrate deep into the CNS parenchyma, where the infiltrated T cells again can find antigen, be further activated, and produce cytokines, resulting in massive immune cell recruitment and clinical disease.     

Shedding of PECAM-1 during HIV infection: a potential role for soluble PECAM-1 in the pathogenesis of NeuroAIDS

Human immunodeficiency virus (HIV) infection is characterized by viral entry into the central nervous system (CNS), which is mediated, in part, by the transmigration of HIV-infected monocytes into the brain. The elaboration of chemokines and other factors by these infected cells contributes to CNS inflammation and cognitive impairment in a significant number of HIV-infected individuals. Recently, we demonstrated that HIV-infected monocyte transmigration into the CNS is enhanced greatly by the chemokine CC chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1. Platelet endothelial cell adhesion molecule-1 (PECAM-1) plays an important role in leukocyte transmigration across the endothelium of the systemic vasculature by mediating homophilic interactions between endothelial cells (EC)-EC and EC-leukocytes, thus preserving vessel integrity. The role of PECAM-1 in HIV-infected leukocyte transmigration across the blood brain barrier (BBB) and NeuroAIDS has not been characterized. We demonstrate that in brain tissue from individuals with HIV encephalitis, there is an accumulation of cleaved, soluble forms of the extracellular region of PECAM-1 (sPECAM-1). In addition, HIV-infected individuals have elevated levels of sPECAM-1 in their sera. Our in vitro data demonstrate that HIV-infected leukocytes, when treated with CCL2, shed sPECAM-1, suggesting a mechanism of extracellular PECAM-1 cleavage and release dependent on HIV infection and CCL2. We hypothesize that sPECAM-1 production by HIV-infected leukocytes, resulting in the accumulation of sPECAM-1 within the CNS vasculature and the generation of truncated, intracellular forms of PECAM-1 within leukocytes, alters PECAM-1 interactions between EC-EC and EC-leukocytes, thus contributing to enhanced transmigration of HIV-infected leukocytes into the CNS and changes in BBB permeability during the pathogenesis of NeuroAIDS.     

The blood-brain-barrier in multiple sclerosis: functional roles and therapeutic targeting

In most regions of the central nervous system (CNS), the composition of the neuronal microenvironment is maintained by virtue of particular blood-brain-barrier (BBB) characteristics, to which vascular endothelial cells (ECs) contribute an important role. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS, characterized at tissue level by multifocal perivascular infiltrates, predominantly of lymphocytes and macrophages. Thus, lymphocyte recruitment into the brain across ECs of the BBB represents a critical event in disease pathogenesis, which is highly restricted and carefully regulated. In recent years, different investigations have identified the crucial components involved in leukocyte migration, providing new insights into mechanisms modulating neuroinflammatory reactions. In this review, several topics relating to these events are discussed, namely: (1) cellular and molecular characteristics of the BBB regulating permeability, as well as signals inducing EC differentiation in the brain and specific cell properties; (2) pathogenic mechanisms guiding the migration of different leukocyte populations through the BBB in MS; and (3) current knowledge on how different MS therapies targeting leukocytes migration across the BBB function. Furthermore, because the BBB has proven to be an important retaining wall preventing drug passage into the CNS, novel strategies directed at successful delivery of large molecules for effective treatment of various inflammatory conditions of the brain, both currently available or still under development, are discussed.     

The blood–brain-barrier in multiple sclerosis: Functional roles and therapeutic targeting

In most regions of the central nervous system (CNS), the composition of the neuronal microenvironment is maintained by virtue of particular blood–brain-barrier (BBB) characteristics, to which vascular endothelial cells (ECs) contribute an important role. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS, characterized at tissue level by multifocal perivascular infiltrates, predominantly of lymphocytes and macrophages. Thus, lymphocyte recruitment into the brain across ECs of the BBB represents a critical event in disease pathogenesis, which is highly restricted and carefully regulated. In recent years, different investigations have identified the crucial components involved in leukocyte migration, providing new insights into mechanisms modulating neuroinflammatory reactions. In this review, several topics relating to these events are discussed, namely: (1) cellular and molecular characteristics of the BBB regulating permeability, as well as signals inducing EC differentiation in the brain and specific cell properties; (2) pathogenic mechanisms guiding the migration of different leukocyte populations through the BBB in MS; and (3) current knowledge on how different MS therapies targeting leukocytes migration across the BBB function. Furthermore, because the BBB has proven to be an important retaining wall preventing drug passage into the CNS, novel strategies directed at successful delivery of large molecules for effective treatment of various inflammatory conditions of the brain, both currently available or still under development, are discussed.