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Besides exerting regulatory roles within astrocytes, the Ca2+-modulated protein of the EF-hand type S100B is released into the brain extracellular space, thereby affecting astrocytes, neurons, and microglia. However, extracellular effects of S100B vary, depending on the concentration attained and the protein being trophic to neurons up to nanomolar concentrations and causing neuronal apoptosis at micromolar concentrations. Effects of S100B on neurons are transduced by receptor for advanced glycation end products (RAGE). At high concentrations, S100B also up-regulates inducible NO synthase in and stimulates NO release by microglia by synergizing with bacterial endotoxin and IFN-gamma, thereby participating in microglia activation. We show here that S100B up-regulates cyclo-oxygenase-2 expression in microglia in a RAGE-dependent manner in the absence of cofactors through independent stimulation of a Cdc42-Rac1-JNK pathway and a Ras-Rac1-NF-kappaB pathway. Thus, S100B can be viewed as an astrocytic endokine, which might participate in the inflammatory response in the course of brain insults, once liberated into the brain extracellular space.

The Ca(2+)-modulated protein, S100B, is expressed in high abundance in and released by astrocytes. At the low levels normally found in the brain, extracellular S100B acts as a trophic factor, protecting neurons against oxidative stress and stimulating neurite outgrowth through its binding to the receptor for advanced glycation end products (RAGE). However, upon accumulation in the brain extracellular space, S100B might be detrimental to neurons. At relatively high concentrations, S100B stimulates NO release by microglia in the presence of lipid A or interferon-gamma (IFN-gamma). We analyzed further the S100B-microglia interaction to elucidate the molecular mechanism by which the protein brings about this effect. We found that S100B increased NO release by BV-2 microglia by stimulating reactive oxygen species (ROS) production and activating the stress-activated kinases, p38 and JNK. However, S100B stimulated NO production to the same extent in microglia overexpressing a transduction-incompetent mutant of RAGE and in microglia overexpressing full-length RAGE, with a significantly smaller effect in mock-transfected microglia. This suggests that the RAGE transducing activity has little or no role in S100B-stimulated NO production by microglia, whereas RAGE extracellular domain is important, probably serving to concentrate S100B on the BV-2 cell surface. On the other hand, S100B stimulated NF-kappaB transcriptional activity in BV-2 microglia in a manner that was strictly dependent on RAGE transducing activity, pointing to additional, RAGE-mediated effects of the protein on microglia that remain to be investigated.

Coronavirus infection of mice has been used extensively as a model for the study of acute encephalitis and chronic demyelination. To examine the evolution of coronavirus RNA during chronic demyelinating infection, we isolated RNA from intracerebrally inoculated mice at 4, 6, 8, 13, 20, and 42 days postinfection and used reverse transcription-polymerase chain reaction amplification methods (RT-PCR) to detect viral sequences. RNA sequences from two viral structural genes, the spike gene and the nucleocapsid gene, were detected throughout the chronic infection. In contrast, infectious virus was not detectable from brain homongenates beyond 13 days postinfection. These results indicate that coronavirus RNA persists in the brain at times when infectious virus is not detected. To determine if genetic changes were occurring during viral replication in the host, we cloned and sequenced the RT-PCR products from the spike and nucleocapsid regions and analyzed the sequences for mutations. Sequencing of the cloned products revealed that a variety of mutant forms of viral RNA persisted in the CNS, including point mutants, deletion mutants, and termination mutants. The mutations accumulated during persistent infection in both the spike and the nucleocapsid sequences, with greater than 65% of the mutations encoding amino acid changes. These results show that a diverse population or quasispecies consisting of mutant and deletion variant viral RNAs (which may not be capable of producing infectious virus particles) persists in the central nervous system of mice during chronic demyelinating infection. The implications of these results for the role of persistent viral genetic information in the pathogenesis of chronic demyelination are discussed.

We evaluated the intracellular and extracellular biological role of S100B protein with respect to microglia. S100B, which belongs to the multigenic family of Ca2+-binding proteins, is abundant in astrocytes where it is found diffusely in the cytoplasm and is associated with membranes and cytoskeleton constituents. S100B protein is also secreted by astrocytes and acts on these cells to stimulate nitric oxide secretion in an autocrine manner. However, little is known about the relationship between S100B and microglia. To address this issue, we used primary microglia from newborn rat cortex and the BV-2 microglial cell line, a well-established cell model for the study of microglial properties. S100B expression was assessed by immunofluorescence in primary microglia and by RT-PCR, Western blotting, and immunofluorescence in BV-2 cells. S100B was found in microglia in the form of a filamentous network as well as diffusely in the cytoplasm and associated with intracellular membranes. S100B relocated around phagosomes during BV-2 phagocytosis of opsonized Cryptococcus neoformans. Furthermore, interferon-gamma (IFN-gamma) treatment caused cell shape changes and redistribution of S100B, and downregulation of S100B mRNA expression in BV-2 cells. Treatment of BV-2 cells with nanomolar to micromolar amounts of S100B resulted in increased IFN-gamma-induced expression of inducible nitric oxide synthase mRNA as well as nitric oxide secretion. Taken together, these data suggest a possible role for S100B in the accomplishment/regulation of microglial cell functions.

The demyelinating process in Theiler’s murine encephalomyelitis virus (TMEV) infection in mice requires virus persistence in the central nervous system. Using recombinant TMEV assembled between the virulent GDVII and less virulent BeAn virus cDNAs, we now provide additional evidence supporting the localization of a persistence determinant to the leader P1 (capsid) sequences. Further, recombinant viruses in which BeAn sequences progressively replaced those of GDVII within the capsid starting at the leader NH2 terminus suggest that a conformational determinant requiring homologous sequences in both the VP2 puff and VP1 loop regions, which are in close contact on the virion surface, might underlie persistence.

Although several murine macrophage (m phi) cell lines from different sites have previously been obtained by in vitro infection with the J2 murine retrovirus, which carries the v-raf and v-myc oncogenes, it was not possible to immortalize thioglycolate-elicited peritoneal macrophages (Pm phi s) by this in vitro procedure. A technique utilizing in vivo injection of the J2 virus has been developed to overcome this problem. The J2 virus immortalized Pm phi s in a very efficient manner in vivo because no exogenous growth factors were required for the in vitro proliferation of these cells and numerous continuous cloned cell lines were readily established. In contrast, Pm phi s obtained from uninfected mice or Pm phi s infected in vitro with the J2 virus did not proliferate. The in vivo immortalized cells had many of the morphological and functional characteristics of m phi s. Analysis of two of the clones, PMJ2-PC and PMJ2-R, demonstrated intracellular expression of the product of the v-raf gene, presence of m phi-associated cell surface antigens, interleukin-6 secretion induced by lipopolysaccharide, and biological response modifier-induced cytotoxic activity against tumor cells. In addition, one of the clones, PMJ2-PC, constitutively expressed major histocompatibility complex (MHC) class II antigens, and in the other clone, PMJ2-R, MHC class II antigens expression was induced by recombinant murine interferon-gamma. This method of utilizing the J2 virus in vivo represents a novel technique for obtaining hematopoietic cell lines from cells that are difficult to immortalize in vitro.

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