Alexandre Prat

Brain-immune connection : Immuno-regulatory properties of CNS-resident cells

Even though the immune privileged status of the central nervous system (CNS) limits access of systemic immune cells through the blood brain barrier (BBB), an immune response can occur in this compartment with or without major breach of the BBB. In this review, we consider properties of resident cells of the CNS, that participate in regulating the neural antigen (Ag)‐directed immune responses implicated in autoimmune diseases such as multiple sclerosis (MS). Under such conditions, the CNS is usually viewed as the target or victim of the immune assault, because such immune responses are thought to be initiated and regulated within the systemic immune compartment. The CNS‐endogenous cells may themselves, however, initiate, regulate and sustain an immune response. We consider the immune regulatory functions within the CNS in terms of events occurring within the CNS parenchyma (microglia, astroglia) and at the vascular interface. These regulatory functions involve antigen presentation to T cells and polarization of the cytokine response of these cells. Such responses may contribute not only to the overall tissue injury in primary immune disorders but also in a wide range of traumatic, ischemic and degenerative processes. GLIA 29:293–304, 2000.

Glial cell influence on the human blood-brain barrier

The blood‐brain barrier (BBB) is a specialized structure of the central nervous system (CNS) that restricts immune cell migration and soluble molecule diffusion from the systemic compartment into the CNS. Astrocytes and microglia are resident cells of the CNS that contribute to the formation of the BBB. In this article, we consider the influence of these glial cells on the immune regulatory functions of the microvascular endothelium, with special emphasis on the human BBB. A series of in vitro studies demonstrate that soluble factors produced by glial cells, under basal culture conditions, help restrict development of inflammation within the CNS. These soluble factor effects include upregulating expression of molecules including HT7, UEA‐1 lectin‐binding sites, and angiotensin receptors that help define the phenotype of endothelial cells. These factors also induce tight junction formation between brain endothelial cells, contributing to the restricted permeability of the BBB. In contrast, these factors have little effect on expression of molecules by ECs that either promote lymphocyte migration, such as chemokines and adhesion molecules or molecules that are required for competent antigen presentation, such as MHC and co‐stimulatory molecules. Glial cells that become activated in response to signals derived from the immune system or generated within the CNS, produce an array of inflammatory molecules that increase permeability and promote lymphocyte trafficking and persistence. These observations emphasize the bidirectional nature of neural‐immune interactions; this dynamic system should be amenable to therapeutic interventions. GLIA 36:145–155, 2001.

Determinants of Human B Cell Migration Across Brain Endothelial Cells

Circulating B cells enter the CNS as part of normal immune surveillance and in pathologic states, including the common and disabling illness multiple sclerosis. However, little is known about the molecular mechanisms that mediate human B cell interaction with the specialized brain endothelial cells comprising the blood-brain barrier (BBB). We studied the molecular mechanisms that regulate the migration of normal human B cells purified ex vivo, across human adult brain-derived endothelial cells (HBECs). We found that B cells migrated across HBECs more efficiently than T cells from the same individuals. B cell migration was significantly inhibited by blocking Abs to the adhesion molecules ICAM-1 and VLA-4, but not VCAM-1, similar to the results previously reported for T cells. Blockade of the chemokines monocyte chemoattractant protein-1 and IL-8, but not RANTES or IFN-γ-inducible protein-10, significantly inhibited B cell migration, and these results were correlated with the chemokine receptor expression of B cells measured by flow cytometry and by RNase protection assay. Tissue inhibitor of metalloproteinase-1, a natural inhibitor of matrix metalloproteinases, significantly decreased B cell migration across the HBECs. A comprehensive RT-PCR comparative analysis of all known matrix metalloproteinases and tissue inhibitors of metalloproteinases in human B and T cells revealed distinct profiles of expression of these molecules in the different cell subsets. Our results provide insights into the molecular mechanisms that underlie human B cell migration across the BBB. Furthermore, they identify potential common, and unique, therapeutic targets for limiting CNS B cell infiltration and predict how therapies currently developed to target T cell migration, such as anti-VLA-4 Abs, may impact on B cell trafficking.

Interferon-γ secretion by peripheral blood T-cell subsets in multiple sclerosis: Correlation with disease phase and interferon-β therapy

Interferon‐γ (IFN‐γ) is implicated as a participant in the immune effector and regulatory mechanisms considered to mediate the pathogenesis of multiple sclerosis (MS). We have used an intracellular cytokine staining technique to demonstrate that the proportion of ex vivo peripheral blood CD4 and CD8 T‐cell subsets expressing IFN‐γ is increased in secondary progressing (SP) MS patients, whereas the values in untreated relapsing‐remitting (RR) MS patients are reduced compared with those of controls. Patients treated with interferon‐β (IFN‐β) have an even more significant reduction in the percentage of IFN‐γ–secreting cells. The finding that the number of IFN‐γ–expressing CD8 cells is increased in SPMS patients, a group with reduced functional suppressor activity, and is most significantly reduced by IFN‐β therapy, which increases suppressor activity, indicates that IFN‐γ secretion by CD8 T cells and functional suppressor defects attributed to this cell subset in MS can be dissociated. Ann Neurol 1999;45:247–250

Characterization of T cell lines derived from glatiramer-acetate-treated multiple sclerosis patients

We analyzed the effects of glatiramer acetate (GA) therapy on in vitro proliferative responses and cytokine production by lymphocytes derived from multiple sclerosis patients receiving this therapy. We confirmed that lymphocytes derived from GA naïve patients show a high frequency of response when initially exposed to GA in vitro; this frequency decreased following GA therapy. The frequency of lymphocytes responding to whole MBP stimulation did not change with GA therapy. GA- and MBP-specific T cell lines generated from these patients by repeated cycles of in vitro stimulation did not cross react. Some (23%) whole MBP-reactive T cell lines did cross react with MBP peptide 83-99. The mean levels of interferon (IFN) gamma secretion and the mean ratio of IFN-gamma/IL-5 were lower for GA-reactive cell lines, derived from patients both prior to and during GA therapy, compared to MBP-reactive T cell lines. The proportion of IFN-gamma(+) cells in unfractionated lymphocyte preparations derived from the GA-treated patients did not differ from that found for healthy controls. Our findings indicate that GA-reactive T cell lines derived from GA-treated MS patients continue to show a relative Th2 cytokine bias consistent with a bystander suppressor function. GA treatment is not associated with a cytokine phenotype shift in the total T cell or MBP-reactive T cell populations.

T lymphocytes conditioned with Interferon β induce membrane and soluble VCAM on human brain endothelial cells

Vascular cell adhesion molecule (VCAM)-1 plays a critical role in mediating inflammatory cell adhesion and migration. Factors regulating the expression of membrane (m)VCAM and its cleaved counterpart soluble (s)VCAM are poorly understood. We previously demonstrated that serum sVCAM levels are increased in multiple sclerosis (MS) patients treated with interferon beta 1b (IFNbeta1b), which correlated with a reduction in gadolinium enhancing lesions on magnetic resonance imaging. However, subsequent studies have shown that IFNbeta does not directly induce VCAM expression on endothelial cells. We demonstrate here that co-culture with IFNbeta-conditioned T cells induces mVCAM on human brain endothelial cells (HBEC). Further, rapid shedding of sVCAM occurs, which mirrors the response after in vivo IFNbeta treatment. The VCAM induction is mediated partially through tumor necrosis factor (TNF)alpha and can be abrogated by sTNF receptor. VCAM could also be induced on astroglioma lines using IFNbeta-conditioned T cells, which suggests the effect is not specific for HBEC. Kinetic studies demonstrated an increase in the sVCAM to mVCAM ratio over time, which may contribute to the ultimate therapeutic effect of IFNbeta in patients. These data have important implications for understanding the events occurring at the blood brain barrier in vivo, and for determining the mechanism of action of IFNbeta in MS.

Differential effects of Th1 and Th2 lymphocyte supernatants on human microglia

We assessed the effects of soluble molecules (supernatants) produced by pro‐ (Th1) and anti‐ (Th2) inflammatory T‐cell lines on the capacity of adult human CNS‐derived microglia to express or produce selected cell surface and soluble molecules that regulate immune reactivity or impact on tissue protection/repair within the CNS. Treatment of microglia with supernatants from allo‐antigen and myelin basic protein‐specific Th1 cell lines augmented expression of cell surface molecules MHC class II, CD80, CD86, CD40, and CD54, enhanced the functional antigen‐presenting cell capacity of microglia in a mixed lymphocyte reaction, and increased cytokine/chemokine secretion (TNFα, IL‐6, and CXCL10/IP‐10). These Th1‐induced effects were not reproduced by interferon‐γ (IFNγ) alone and were only incompletely blocked by anti‐IFNγ antibody. Th2 cell supernatant treatments did not alter costimulatory/adhesion molecule expression or induce cytokine/chemokine production by microglia. Th2 treatment, furthermore, failed to reduce the induction observed in response to Th1 supernatants. Neither Th1 nor Th2 supernatants induced production of the neurotrophin molecules, nerve growth factor, or brain‐derived neurotrophic factor. Our results suggest that soluble molecules released by Th1 and not Th2 cells that infiltrate the CNS can stimulate resident microglia to acquire enhanced effector and accessory cell functions; the Th1‐induced effects were not downregulated by Th2 supernatant‐mediated bystander suppression. GLIA 42:36–45, 2003.

NG2 immunoreactivity on human brain endothelial cells

Abstract. In this study, we evaluated the expression of NG2 on human brain endothelial cells derived from temporal lobe tissue resected as a treatment for intractable epilepsy. Using dissociated cell cultures, we found expression of NG2 on both proliferating and non-proliferating cells, at the mRNA level by reverse transcription-polymerase chain reaction analyses, and at the protein level by immunocytochemistry and immunoblotting. We further observed that human cerebral microvessels in non-malignant CNS tissues immunoreacted with NG2. NG2 protein was detected using both a rabbit antibody raised against the rodent NG2 and a monoclonal antibody raised against the human NG2 (9.2.27). Our findings further define the range of resident cells of the CNS that can express NG2 and indicate that expression of NG2 by endothelial cells is not restricted to proliferating CNS endothelial cells or to endothelial cells found in brain tumors.

Human brain endothelial cells supply support for monocyte immunoregulatory functions

Blood-derived monocytic cells comprise a significant component of most inflammatory responses that occur in the CNS. We utilized human brain-derived endothelial cells (HBECs) coated membranes in Boyden chambers to assess immune function related properties of human blood-derived monocytes following interaction with HBECs. Monocytes in contact with HBECs maintained functional antigen-presenting capacity and chemokine/cytokine production in contrast to monocytes that migrated through the HBEC barrier. These results indicate that HBECs, although themselves incapable of serving as competent antigen-presenting cells during the course of inflammatory CNS disorders, supply support needed for infiltrating perivascular monocytes to maintain their functions. Monocyte migration across HBECs was inhibited by interferon-beta.

Bradykinin B1 receptor regulates the migration of lymphocytes in multiple sclerosis

1 1 3 C h e m o k i n e Binding Sites on H u m a n Bra in M i c r o v e s s d s A. Andjelkovic, J.S. Pachter, University of Connect~cut Health Center, USA 1 1 6 Microgl ia P roduce C h e m o a t t r a e t a n t s in f luenc ing Monocyte Mig ra t ion th rough the Blood-bra in B a r r i e r Y. Persidsky, J. Limoges, J. Rasmussen, UniversityofNebraskaMedicalCenter, USA, M. Stins, Childrens HospitaI of Los Angeles, USA, M. Fiala, University of California at Los AngeIes, USA, K.S. Kim, Childrens Hospital of Los Angeles, USA, H.E. Gendelm an, University of Nebraska Medical Center, USA