Burkhard Becher

PK11195 binding to the peripheral benzodiazepine receptor as a marker of microglia activation in multiple sclerosis and experimental autoimmune encephalomyelitis

Activated glial cells are implicated in regulating and effecting the immune response that occurs within the CNS as part of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). The peripheral benzodiazepine receptor (PBR) is expressed in glial cells. We examined the utility of using in vitro and in vivo ligand binding to the PBR as a measure of lesion activity in autoimmune CNS demyelinating diseases. Applying a combined autoradiography and immunohistochemical approach to spinal cord and brain tissues from mice with EAE, we found a correlation at sites of inflammatory lesions between [3H]‐PK11195 binding and immunoreactivity for the activated microglial/macrophage marker Mac‐1/CD11b. In MS tissues, [3H]‐PK11195 binding correlated with sites of immunoreactivity for the microglial/macrophage marker CD68, at the edges of chronic active plaques. Positron emission tomography (PET) imaging with [11C]‐PK11195 showed ligand uptake only at sites of active MS lesions defined by magnetic resonance imaging criteria. Our results indicate the potential to develop markers suitable for both in vitro and in vivo use, which will serve to help correlate phenotypic and functional properties of cells which participate in disease or injury responses within the CNS. J. Neurosci. Res. 50:345–353, 1997.

Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia

Microglial cells are resident cells of the CNS and are implicated as regulators and effectors of immune responses which occur within this compartment. The precise role of parenchymal microglia remains speculative because distinctions between these cells, perivascular “microglia,” and blood‐derived monocytes/macrophages are not well defined. The current study describes the phenotype and function of microglia immediately upon isolation from the non‐inflamed adult human CNS and the phenotypic changes which occur in these cells when maintained in tissue culture. We find that the characteristic phenotype of immediately ex vivo parenchymal microglia (CD11c‐/CD45low/CD14‐) corresponds to that found in situ in the “normal” human brain. The phenotype differs from that of perivascular “microglia” in situ and PBDM (both CD45hi/CD14++). The immediately ex vivo microglia express B7‐2 and HLA class II molecules and can support alloantigen‐induced proliferation by CD4+ T cells freshly isolated from peripheral blood. Following in vitro culture, the cells are characterized by a bipolar morphology, continued lower levels of CD45 expression compared to PBDM, and slight upregulation of B7‐1 and HLA‐DR antigen expression. CD14 becomes expressed at high levels on the cells, suggesting that CD14 can serve as an apparent marker of microglia activation which is not based on changes in morphology or APC capacity. Further, treatment of the cells with IFN‐γ and LPS causes further upregulation of HLA‐DR and clear expression of B7‐1 molecules on the surface. The capacity to characterize phenotypic and functional properties of microglia before and after activation provides an opportunity to determine means to manipulate the immune regulatory and effector properties of this cell type.

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

IL-10 PRODUCTION BY ADULT HUMAN DERIVED MICROGLIAL CELLS

Microglia, a population of central nervous system (CNS) macrophages, have been demonstrated to support immune accessory and effector functions in the CNS. Numerous studies support the role of microglia in CNS development and pathology, where activation of microglia is consistently noted. The current study investigated microglial immune functions under basal and activation conditions and assessed the ability of interleukin-10 (IL-10), added exogenously or produced by microglia, to down-regulate microglial functions. This report demonstrates that microglia from the adult human brain produce IL-10 following interferon-gamma/lipopolysaccharide activation. Functionally, recombinant human IL-10 down-regulated basal HLA-DR expression by microglia and inhibited, in a dose-dependent response, the ability of microglia to stimulate CD4+ T-cells in antigen presentation assays. These data, together with recent observations of the inhibition of experimental allergic encephalomyelitis (EAE) following IL-10 administration and reduced CNS infection by Listeria monocytogenes after anti-IL-10 treatment, suggest that IL-10 production by microglia may have important immune-regulatory functions in CNS disease and disease models.

Regulation of CD14 expression on human adult central nervous system‐derived microglia

Microglial cells function as regulators of immune reactivity within the CNS and may contribute to tissue injury under inflammatory conditions. Such functions are correlated with their state of activation. In this study, we report the de novo expression of CD14 by adult human CNS‐derived microglia which acquire a bipolar activated morphologic phenotype in dissociated tissue culture. Surface CD14 expression can be down‐regulated by interaction with its ligand lipopolysaccharide (LPS), and by the T‐helper (Th1) cytokine interferon‐γ (IFN‐γ) or the Th2 cytokine interleukin‐4 (IL‐4). Semiquantitative polymerase chain reaction (PCR) analysis of CD14 mRNA expression under each condition suggests a different mechanism accounting for the reduced surface expression. LPS down‐regulates CD14 mRNA, consistent with a feedback signal preventing over‐stimulation. IFN‐γ augments CD14 transcription, suggesting cleavage of surface CD14 consequent to general cell activation. IL‐4 decreases mRNA production likely reflecting a generalized suppressive effect. The effect of LPS, IFN‐γ and IL‐4 on CD14 expression differes from their effect on expression of the immune‐accessory molecules B7‐1 and HLA‐DR, and on production of tumor necrosis factor‐α (TNF‐α), whose secretory pathway is similar to that of CD14. These results indicate the selective effects of molecules, likely to be present in the infected or inflamed CNS, on regulating CD14 expression and that there can be differential regulation of immune response relevant molecules expressed by activated microglia.

CD95–CD95L: can the brain learn from the immune system?

Members of the tumor necrosis factor/nerve growth factor receptor superfamily of cell-surface molecules can play the dual role of mediating either cytotoxicity or cell survival, both in the immune system and in the nervous system. A member of this superfamily, CD95 (also known as ApoI or Fas), was initially identified in the immune system and has been shown to mediate receptor-dependent programmed cell death and to be expressed in the nervous system. In neurodegenerative disorders, CD95-CD95 ligand expression on glial cells might precede receptor-mediated apoptosis by cells of the CNS. It is now being recognized that CD95 signaling by immune cells mediates effects other than apoptosis, such as cell survival and under inflammatory conditions expression of this protein promotes neural-immune interactions. Both neuroscientists and immunologists can contribute to defining the mechanisms underlying these divergent effects and utilize such knowledge to aid understanding of cell death and survival.

Expression of a homologue of rat NG2 on human microglia

The expression of NG2 chondroitin sulfate has been widely associated with oligodendrocyte precursors in rodents. We used a monoclonal antibody (9.2.27) against the human homologue of the rat NG2 to determine whether expression of this molecule was associated with a specific glial cell population present in dissociated cell preparations derived from adult and fetal human brain tissue. Our data, derived using FACS and immunocytochemical analyses of immediately ex vivo or cultured glial cells, indicate that the large majority of NG2 expressing cells belonged to the microglial lineage (CD68, CD11c) rather than to the oligodendrocyte lineage (O4, A2B5, GalC). In situ immunohistochemistry performed on non‐fixed normal spinal cord tissue confirmed the observation that NG2 is expressed by mononuclear phagocytes of the CNS. In contrast, peripheral blood‐derived monocytes were NG2−. Cells from fetal brain tissue showed only small numbers of NG2+ cells, which was consistent with the number of microglial cells in this preparation. In absence of additional markers, we cannot exclude that this anti‐NG2 mAb might also recognize human oligodendrocyte progenitor cells. GLIA 27:259–268, 1999.

Caspase 8 expression and signaling in Fas injury–resistant human fetal astrocytes

Fas (APO‐1/CD95) is a cell surface receptor initially identified in lymphoid cells, but more recently detected in the central nervous system under pathological, usually inflammatory, conditions. In most Fas expressing cells, triggering of Fas by its ligand or by antagonistic antibodies leads to apoptosis. Human fetal astrocytes (HFA) constitutively express Fas yet are resistant to cell death following Fas ligation. In the current study, using dissociated cultures of human fetal central nervous system–derived cells, we attempted to identify a basis for HFA resistance to Fas‐mediated injury. We compared the components of the Fas signaling pathway of HFA to those of two human cell lines susceptible to Fas‐mediated injury, U251 glioma and Jurkat T‐cells. We found that HFA did not express caspase 8 (FLICE), the caspase primarily activated on Fas signaling. Although we could induce caspase 8 in HFA with the inflammatory cytokines IFNγ and TNFα, HFA remained resistant to Fas‐mediated injury. Addition of inflammatory cytokines to the extracellular milieu also increased FLIP mRNA (FLICE inhibitory protein). Furthermore, upon triggering of cytokine‐treated cells with FasL, we observed upregulation of the cleavage product of FLIP (p43‐FLIP) previously shown to associate with the DISC and to block caspase 8 recruitment, thereby inhibiting Fas‐mediated death. Our findings indicate that caspase 8 and its regulators play a central role in determining the response to Fas ligation of HFA and support a role for Fas signaling in the developing central nervous system other than related to cytotoxicity. GLIA 33:217–224, 2001.

Mechanisms of tissue injury in multiple sclerosis: opportunities for neuroprotective therapy

Development of neuroprotective therapies for multiple sclerosis is dependent on defining the precise mechanisms whereby immune effector cells and molecules are able to induce relatively selective injury of oligodendrocytes (OLs) and their myelin membranes. The selectivity of this injury could be conferred either by the properties of the effectors or the targets. The former would involve antigen specific recognition by either antibody or T cell receptor of the adaptive immune system. OLs are also susceptible to non antigen restricted injury mediated by components of the innate immune system including macrophages/microglia and NK cells. Target related selectivity could reflect the expression of death inducing surface receptors (such as Fas or TNFR-1) required for interaction with effector mediators and subsequent intracellular signaling pathways, including the caspase cascade. Development of therapeutic delivery systems, which would reach the site of disease activity within the CNS, will permit the administration of inhibitors either of the cell death pathway or of effector target interaction and opens new avenues to neuroprotection approach.