Erin E. McCandless

CXCL12 Limits Inflammation by Localizing Mononuclear Infiltrates to the Perivascular Space during Experimental Autoimmune Encephalomyelitis

The inflammatory response in the CNS begins with the movement of leukocytes across the blood-brain barrier in a multistep process that requires cells to pass through a perivascular space before entering the parenchyma. The molecular mechanisms that orchestrate this movement are not known. The chemokine CXCL12 is highly expressed throughout the CNS by microendothelial cells under normal conditions, suggesting it might play a role maintaining the blood-brain barrier. We tested this hypothesis in the setting of experimental autoimmune encephalomyelitis (EAE) by using AMD3100, a specific antagonist of the CXCL12 receptor CXCR4. We demonstrate that the loss of CXCR4 activation enhances the migration of infiltrating leukocytes into the CNS parenchyma. CXCL12 is expressed at the basolateral surface of CNS endothelial cells in normal spinal cord and at the onset of EAE. This polarity is lost in vessels associated with an extensive parenchymal invasion of mononuclear cells during the peak of disease. Inhibition of CXCR4 activation during the induction of EAE leads to loss of the typical intense perivascular cuffs, which are replaced with widespread white matter infiltration of mononuclear cells, worsening the clinical severity of the disease and increasing inflammation. Taken together, these data suggest a novel anti-inflammatory role for CXCL12 during EAE in that it functions to localize CXCR4-expressing mononuclear cells to the perivascular space, thereby limiting the parenchymal infiltration of autoreactive effector cells.

CXCR4 promotes differentiation of oligodendrocyte progenitors and remyelination.

Multiple sclerosis is a neurodegenerative disease characterized by episodes of autoimmune attack of oligodendrocytes leading to demyelination and progressive functional deficits. Because many patients exhibit functional recovery in between demyelinating episodes, understanding mechanisms responsible for repair of damaged myelin is critical for developing therapies that promote remyelination and prevent disease progression. The chemokine CXCL12 is a developmental molecule known to orchestrate the migration, proliferation, and differentiation of neuronal precursor cells within the developing CNS. Although studies suggest a role for CXCL12 in oligodendroglia ontogeny in vitro, no studies have investigated the role of CXCL12 in remyelination in vivo in the adult CNS. Using an experimental murine model of demyelination mediated by the copper chelator cuprizone, we evaluated the expression of CXCL12 and its receptor, CXCR4, within the demyelinating and remyelinating corpus callosum (CC). CXCL12 was significantly up-regulated within activated astrocytes and microglia in the CC during demyelination, as were numbers of CXCR4+NG2+ oligodendrocyte precursor cells (OPCs). Loss of CXCR4 signaling via either pharmacological blockade or in vivo RNA silencing led to decreased OPCs maturation and failure to remyelinate. These data indicate that CXCR4 activation, by promoting the differentiation of OPCs into oligodendrocytes, is critical for remyelination of the injured adult CNS.

CXCR7 influences leukocyte entry into the CNS parenchyma by controlling abluminal CXCL12 abundance during autoimmunity

During CNS autoimmunity, brain endothelial cell CXCR7 internalizes CXCL12 from the perivascular space, thereby permitting leukocyte migration into the CNS parenchyma.

Pathological Expression of CXCL12 at the Blood-Brain Barrier Correlates with Severity of Multiple Sclerosis

Dysregulation of blood-brain barrier (BBB) function and transendothelial migration of leukocytes are essential components of the development and propagation of active lesions in multiple sclerosis (MS). Animal studies indicate that polarized expression of the chemokine CXCL12 at the BBB prevents leukocyte extravasation into the central nervous system (CNS) and that disruption of CXCL12 polarity promotes entry of autoreactive leukocytes and inflammation. In the present study, we examined expression of CXCL12 and its receptor, CXCR4, within CNS tissues from MS and non-MS patients. Immunohistochemical analysis of CXCL12 expression at the BBB revealed basolateral localization in tissues derived from non-MS patients and at uninvolved sites in tissues from MS patients. In contrast, within active MS lesions, CXCL12 expression was redistributed toward vessel lumena and was associated with CXCR4 activation in infiltrating leukocytes, as revealed by phospho-CXCR4-specific antibodies. Quantitative assessment of CXCL12 expression by the CNS microvasculature established a positive correlation between CXCL12 redistribution, leukocyte infiltration, and severity of histological disease. These results suggest that CXCL12 normally functions to localize infiltrating leukocytes to perivascular spaces, preventing CNS parenchymal infiltration. In the patient cohort studied, altered patterns of CXCL12 expression at the BBB were specifically associated with MS, possibly facilitating trafficking of CXCR4-expressing mononuclear cells into and out of the perivascular space and leading to progression of disease.

CXCR4 antagonism increases T cell trafficking in the central nervous system and improves survival from West Nile virus encephalitis

The migration of lymphocytes into the CNS during viral encephalitis is hindered by the blood–brain barrier (BBB) such that most infiltrating cells remain localized to perivascular spaces. This sequestration of leukocytes away from the parenchyma is believed to protect the CNS from immunopathologic injury. Infections of the CNS with highly cytopathic neurotropic viruses, such as West Nile virus (WNV), however, require the parenchymal penetration of T lymphocytes for virus clearance and survival, suggesting that perivascular localization might hinder antiviral immune responses during WNV encephalitis. Using human and murine brain specimens from individuals with WNV encephalitis, we evaluated the expression of CXCL12 and its receptor, CXCR4, at the BBB and tested the hypothesis that inhibition of CXCR4 would promote T lymphocyte entry into the CNS parenchyma and increase viral clearance. Antagonism of CXCR4 significantly improved survival from lethal infection through enhanced intraparenchymal migration of WNV-specific CD8+ T cells within the brain, leading to reduced viral loads and, surprisingly, decreased immunopathology at this site. The benefits of enhanced CD8+ T cell infiltration suggest that pharmacologic targeting of CXCR4 may have therapeutic utility for the treatment of acute viral infections of the CNS.

CD40-CD40 Ligand Interactions Promote Trafficking of CD8+ T Cells into the Brain and Protection against West Nile Virus Encephalitis

ABSTRACT Recent studies have established a protective role for T cells during primary West Nile virus (WNV) infection. Binding of CD40 by CD40 ligand (CD40L) on activated CD4+ T cells provides an important costimulatory signal for immunoglobulin class switching, antibody affinity maturation, and priming of CD8+ T-cell responses. We examined here the function of CD40-dependent interactions in limiting primary WNV infection. Compared to congenic wild-type mice, CD40−/− mice uniformly succumbed to WNV infection. Although CD40−/− mice produced low levels of WNV-specific immunoglobulin M (IgM) and IgG, viral clearance from the spleen and serum was not altered, and CD8+ T-cell priming in peripheral lymphoid tissues was normal. Unexpectedly, CD8+ T-cell trafficking to the central nervous system (CNS) was markedly impaired in CD40−/− mice, and this correlated with elevated WNV titers in the CNS and death. In the brains of CD40−/− mice, T cells were retained in the perivascular space and did not migrate into the parenchyma, the predominant site of WNV infection. In contrast, in wild-type mice, T cells trafficked to the site of infection in neurons. Beside its role in maturation of antibody responses, our experiments suggest a novel function of CD40-CD40L interactions: to facilitate T-cell migration across the blood-brain barrier to control WNV infection.

IL-1R Signaling within the Central Nervous System Regulates CXCL12 Expression at the Blood-Brain Barrier and Disease Severity during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the CNS characterized by disruption of the blood-brain barrier (BBB). This breach in CNS immune privilege allows undeterred trafficking of myelin-specific lymphocytes into the CNS where they induce demyelination. Although the mechanism of BBB compromise is not known, the chemokine CXCL12 has been implicated as a molecular component of the BBB whose pattern of expression is specifically altered during MS and which correlates with disease severity. The inflammatory cytokine IL-1β has recently been shown to contribute not only to BBB permeability but also to the development of IL-17-driven autoimmune responses. Using experimental autoimmune encephalomyelitis, the rodent model of MS, we demonstrate that IL-1β mediates pathologic relocation of CXCL12 during the induction phase of the disease, before the development of BBB disruption. We also show that CD4, CD8, and, surprisingly γδ T cells are all sources of IL-1β. In addition, γδ T cells are also targets of this cytokine, contributing to IL-1β-mediated production of IL-17. Finally, we show that the level of CNS IL-1R determines the clinical severity of experimental autoimmune encephalomyelitis. These data suggest that T cell-derived IL-1β contributes to loss of immune privilege during CNS autoimmunity via pathologic alteration in the expression of CXCL12 at the BBB.

Enhanced sphingosine-1-phosphate receptor 2 expression underlies female CNS autoimmunity susceptibility

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is characterized by BBB dysfunction and has a much higher incidence in females. Compared with other strains of mice, EAE in the SJL mouse strain models multiple features of MS, including an enhanced sensitivity of female mice to disease; however, the molecular mechanisms that underlie the sex- and strain-dependent differences in disease susceptibility have not been described. We identified sphingosine-1-phosphate receptor 2 (S1PR2) as a sex- and strain-specific, disease-modifying molecule that regulates BBB permeability by destabilizing adherens junctions. S1PR2 expression was increased in disease-susceptible regions of the CNS of both female SJL EAE mice and female patients with MS compared with their male counterparts. Pharmacological blockade or lack of S1PR2 signaling decreased EAE disease severity as the result of enhanced endothelial barrier function. Enhanced S1PR2 signaling in an in vitro BBB model altered adherens junction formation via activation of Rho/ROCK, CDC42, and caveolin endocytosis-dependent pathways, resulting in loss of apicobasal polarity and relocation of abluminal CXCL12 to vessel lumina. Furthermore, S1PR2-dependent BBB disruption and CXCL12 relocation were observed in vivo. These results identify a link between S1PR2 signaling and BBB polarity and implicate S1PR2 in sex-specific patterns of disease during CNS autoimmunity.

Region-specific regulation of inflammation and pathogenesis in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis and is characterized by an infiltrate of predominantly T cells and macrophages in the spinal cord and brain. In both the spinal cord and the cerebellum, Th1 cells direct inflammation to antigen-rich white matter tracts, and there is a TNFR1-dependent recruitment of CD11b(hi) cells in both regions. In the spinal cord, parenchymal invasion, demyelination and clinical symptoms are associated with TNFR1-dependant parenchymal induction (especially astrocytes) of VCAM-1 and CXCL2. None of these events occur in the cerebellum despite the fact that an inflammatory infiltrate accumulates in the perivascular space. Therefore regional specificity in astrocyte responses to inflammatory cytokines may regulate regional parenchymal infiltration and pathogenesis.

Molecular targets for disrupting leukocyte trafficking during multiple sclerosis.

Autoimmune diseases of the central nervous system (CNS) involve the migration of abnormal numbers of self-directed leukocytes across the blood-brain barrier that normally separates the CNS from the immune system. The cardinal lesion associated with neuroinflammatory diseases is the perivascular infiltrate, which comprises leukocytes that have traversed the endothelium and have congregated in a subendothelial space between the endothelial-cell basement membrane and the glial limitans. The exit of mononuclear cells from this space can be beneficial, as when virus-specific lymphocytes enter the CNS for pathogen clearance, or might induce CNS damage, such as in the autoimmune disease multiple sclerosis when myelin-specific lymphocytes invade and induce demyelinating lesions. The molecular mechanisms involved in the movement of lymphocytes through these compartments involve multiple signalling pathways between these cells and the microvasculature. In this review, we discuss adhesion, costimulatory, cytokine, chemokine and signalling molecules involved in the dialogue between lymphocytes and endothelial cells that leads to inflammatory infiltrates within the CNS, and the targeting of these molecules as therapies for the treatment of multiple sclerosis.