Laura Santambrogio

Developmental plasticity of CNS microglia

Microglia arise from CD45+ bone marrow precursors that colonize the fetal brain and play a key role in central nervous system inflammatory conditions. We report that parenchymal microglia are uncommitted myeloid progenitors of immature dendritic cells and macrophages by several criteria, including surface expression of “empty” class II MHC protein and their cysteine protease (cathepsin) profile. Microglia express receptors for stem cell factor and can be skewed toward more dendritic cell or macrophage-like profiles in response to the lineage growth factors granulocyte/macrophage colony-stimulating factor or macrophage colony-stimulating factor. Thus, in contrast to other organs, where terminally differentiated populations of resident dendritic cells and/or macrophages outnumber colonizing precursors, the majority of microglia within the brain remain in an undifferentiated state.

Modulation of experimental autoimmune encephalomyelitis: effect of altered peptide ligand on chemokine and chemokine receptor expression.

Experimental autoimmune encephalomyelitis (EAE) is a T helper 1 (Th1) cell mediated demyelinating disease and the principal animal model for multiple sclerosis. Spinal cords from SJL mice primed with proteolipid protein peptide 139-151 (pPLP) expressed the chemokines RANTES, MCP-1, MIP-2, KC, MIP-1alpha, MIP-1beta, Mig, and fractalkine. We also identified IP-10 in these samples and described a sequence polymorphism in this transcript. Chemokine expression was specific for tissues of the central nervous system. MCP-1, IP-10, and MIP-2 RNA expression significantly correlated with clinical score. Chemokine receptor expression generally correlated with ligand expression. pPLP-primed mice expressed the Th1-associated markers CCR5 and CXCR3 on mononuclear cells. In addition, cells expressing CCR1, CCR2, CCR3, CCR4, CCR8, and CXCR2 were detected. Here we demonstrate that altered peptide ligand (APL)-induced protection from EAE was accompanied by modulation of chemokine and chemokine receptor expression. Spinal cord tissue sections from APL-protected mice showed greatly reduced levels of all chemokines and of CCR1, CCR5, CCR8, CXCR2 and CXCR3. The Th2-associated chemokine receptors CCR3 and CCR4 were found in protected mice, supporting the hypothesis that Th1 but not Th2 cells are down-regulated by APL treatment. This report concludes that chemokines and chemokine receptors can be useful tools to follow modulation of autoimmune disease.

Granulocyte-Macrophage Colony-Stimulating Factor Induces an Expression Program in Neonatal Microglia That Primes Them for Antigen Presentation

Neonatal microglial cells respond to GM-CSF and M-CSF by acquiring different morphologies and phenotypes. To investigate the extent and consequences of this process, a global gene expression analysis was performed, with significant changes in transcript levels confirmed by biochemical analyses. Primary murine microglial cells underwent substantial expression reprogramming after treatment with GM-CSF or M-CSF with many differentially expressed transcripts important in innate and adaptive immunity. In particular, many gene products involved in Ag presentation were induced by GM-CSF, but not M-CSF, thus potentially priming relatively quiescent microglia cells for Ag presentation. This function of GM-CSF is distinct from its primary function in cell proliferation and survival.

IFN Regulatory Factor-1 Regulates IFN-γ-Dependent Cathepsin S Expression

Cathepsin S is a cysteine protease with potent endoproteolytic activity and a broad pH profile. Cathepsin S activity is essential for complete processing of the MHC class II-associated invariant chain within B cells and dendritic cells, and may also be important in extracellular matrix degradation in atherosclerosis and emphysema. Unique among cysteine proteases, cathepsin S activity is up-regulated by IFN-γ. Given its importance, we sought to elucidate the pathway by which IFN-γ increases cathepsin S expression. Our data demonstrate that the cathepsin S promoter contains an IFN-stimulated response element (ISRE) that is critical for IFN-γ-induced gene transcription in a cell line derived from type II alveolar epithelial (A549) cells. IFN response factor (IRF)-2 derived from A549 nuclear extracts associates with the ISRE oligonucleotide in gel shift assays, but is quickly replaced by IRF-1 following stimulation with IFN-γ. The time course of IRF-1/ISRE complex formation correlates with increased levels of IRF-1 protein and cathepsin S mRNA. Overexpression of IRF-1, but not IRF-2, markedly augments cathepsin S promoter activity in A549 cells. Furthermore, overexpression of IRF-1 increases endogenous cathepsin S mRNA levels in 293T epithelial cells. Finally, freshly isolated bone marrow cells from IRF-1−/− mice fail to up-regulate cathepsin S activity in response to IFN-γ. Thus, IRF-1 is the critical transcriptional mediator of IFN-γ-dependent cathepsin S activation. These data elucidate a new pathway by which IRF-1 may affect MHC class II processing and presentation.

RANTES-Induced Chemokine Cascade in Dendritic Cells

Dendritic cells (DC) are the most potent APCs and the principal activators of naive T cells. We now report that chemokines can serve as activating agents for immature DC. Murine bone marrow-derived DC respond to the CC chemokine RANTES (10–100 ng/ml) by production of proinflammatory mediators. RANTES induces rapid expression of transcripts for the CXC chemokines KC and macrophage inflammatory protein (MIP)-2, the CC chemokines MIP-1β and MIP-1α, and the cytokines TNF-α and IL-6. Synthesis of KC, IL-6, and TNF-α proteins were also demonstrated. After 4 h, autoinduction of RANTES transcripts was observed. These responses are chemokine specific. Although DC demonstrated weak responses to eotaxin, DC failed to respond to other chemokines including KC, MIP-2, stromal-derived factor-1α, MIP-1β, MIP-1α, monocyte chemoattractant protein-1, T cell activation gene 3, or thymus-derived chemotactic agent 4. In addition, RANTES treatment up-regulated expression of an orphan chemokine receptor termed Eo1. Chemokine induction was also observed after treatment of splenic DC and neonatal microglia with RANTES, but not after treatment of thymocytes or splenocytes depleted of adherent cells. TNF-α-treated DC lose responsiveness to RANTES. DC from mice deficient for CCR1, CCR3, and CCR5 respond to RANTES, indicating that none of these receptors are exclusively used to initiate the chemokine cascade. RANTES-mediated chemokine amplification in DC may prolong inflammatory responses and shape the microenvironment, potentially enhancing acquired and innate immune responses.

ALTERED PEPTIDE LIGAND MODULATION OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS : IMMUNE RESPONSES WITHIN THE CNS

An altered peptide ligand (analog) of the encephalitogenic epitope of proteolipid protein residues 139-151 (p139-151) in which residues 144 and 147 are substituted with leucine and arginine, respectively (LR), protects from clinical but not histological experimental allergic encephalomyelitis (EAE). To understand in situ events associated with this protection, T cells from brains of mice immunized with either native p139-151, the analog LR or a combination of the two were isolated and characterized. High proportions of cells from co-immunized mice (38%) and LR-immunized mice (58%) reacted to both p139-151 and LR, whereas fewer cells from p139-151 immunized mice (7%) were cross-reactive. T cell clones derived from brains of LR- and co-immunized mice were also cross-reactive in vitro. By reverse transcriptase-based polymerase chain reaction, higher levels of TGF-beta mRNA, and lower levels of TNF-alpha and IFN-gamma mRNA were found in the central nervous system (CNS) tissue of LR and co-immunized mice. Immunohistochemistry demonstrated greater TGF-beta immunoreactivity in CNS inflammatory foci in co-immunized and LR-immunized mice. There were no significant differences in CD4+ or CD8+ cell infiltrates among the groups and differences in other cytokines were not identified by immunocytochemistry. Protection from clinical EAE in LR and co-immunized mice was partially abolished by anti-TGF-beta antibody treatment. Thus, protection from clinical disease following immunization with the analog LR is associated with infiltration into the CNS of a T cell population that could potentially recognize the native PLP peptide and with enhanced TGF-beta production by cells within CNS inflammatory foci.

Copolymer effects on microglia and T cells in the central nervous system of humanized mice

The random amino acid copolymers FYAK and VWAK ameliorate EAE in a humanized mouse model expressing both a human transgenic myelin basic protein (MBP)85–99‐specific T cell receptor and HLA‐DR2. Here we show that microglia isolated from the central nervous system (CNS) of humanized mice with EAE induced by MBP85–99 and treated with these copolymers had reduced expression of HLA‐DR, and thus reduced capacity to present MBP85–99 and activate transgenic T cells. In vitro microglia up‐regulated empty HLA‐DR2 upon activation with GM‐CSF with or without LPS or IFN‐γ, but not with IL‐4 or IL‐10. Correspondingly, gene chip arrays showed that the CNS of untreated and YFAK‐treated mice differentially expressed pro‐ and anti‐inflammatory molecules during MBP85–99‐induced EAE. Interestingly, microglia expressed the full‐length γβ and αβ subunits of the tetrameric adaptor protein complexes AP‐1 and AP‐2 respectively, but after treatment with GM‐CSF these complexes were cleaved, as had been found in immature dendritic cells derived from bone marrow. Strikingly, in vivo the perivascular lymphocyte infiltration seen in untreated mice immunized with MBP85–99 was composed of equal numbers of hVβ2+ MPB85–99‐specific transgenic and hVβ2– endogenous T cells, while the much smaller infiltration seen after treatment with YFAK was composed predominantly of hVβ2– endogenous T cells.

Antigen presenting capacity of brain microvasculature in altered peptide ligand modulation of experimental allergic encephalomyelitis

Co-immunization with an altered peptide ligand (LR) partially protects SJL mice from proteolipid protein peptide 139-151-induced experimental allergic encephalomyelitis [Kuchroo, V.K., Greer, J.M., Kaul, D., Ishioka, G.Y., Franco, A., Sette, A., Sobel, R.A., Lees, M.B., 1994. A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis mediated by a diverse T cell repertoire. J. Immunol. 153, 3326-3336; Santambrogio, L., Lees, M.B., Sobel, R.A., 1998. Altered peptide ligand modulation of experimental allergic encephalomyelitis: immune responses within the CNS. J. Neuroimmunol. 81, 1-13]. Clinical protection was noted despite extensive central nervous system inflammation observed after co-immunization with native and altered peptides. To extend our previous reports on this model, we now compare MHC class II expression and antigen presenting cell activity of cells associated with the blood-brain barrier in diseased and protected mice. Immunohistochemical studies identified MHC class II products on both the endothelial and microglial/macrophage populations. Ex vivo experiments suggested a correlation between the reduced clinical disease observed in the co-immunized mice and the antigen presenting activity of cells at the blood-brain barrier. The results suggest that antigen presenting activity is primarily mediated by macrophage-lineage cells of the central nervous system.