Down-regulation of microglia and NG2-positive cells reaction in trimethyltin-injured hippocampus of rats treated with myelin basic protein-reactive T cells: possible contribution to the neuroprotective effect of T cells.

In our previous investigations, we demonstrated that CD4(+) antimyelin basic protein (MBP) T cells protect hippocampal neurons against trimethyltin-induced damage. We hypothesized involvement of T cells, interacting with the various glial populations activated during the neurodegeneration process. In this study, we employ immunocytochemical methods to investigate the influence of administration of T cells on the response of microglia and of NG2(+) cells to trimethyltin (TMT)-induced damage. Female Lewis rats were treated with anti-MBP CD4(+) T cells (4 million per animal, i.v) 24 hr after TMT (8 mg/kg, i.p) intoxication. TMT caused degeneration of CA4 hipppocampal neurons and evoked an abundant reaction of microglial and NG2(+) cells in the injured region. The cells changed morphology into the activated state, and the number of OX42(+) and NG2(+) cells increased about 4.5-fold and 3-fold, respectively, relative to controls as assessed on day 21 after TMT treatment. Additionally, the cells of ameboid morphology, which expressed NG2 or microglial antigens, appeared in the zone of neurodegeneration. Furthermore, certain cells of ameboid phenotype shared both antigens. In rats treated with T cells, down-regulation of the activation of both glial classes and reduction of formation of their ameboid forms was observed. The number of the total OX42(+) and NG2(+) cells decreased by 21% and 54%, respectively, and the number of their ameboid forms decreased by 46% and 73%, respectively. Our data suggest that the diminished activation of microglia and NG2(+) cells, particularly the reduced number of their ameboid forms, may contribute to the neuroprotective effect of T cells.

Trimethyltin-evoked apoptosis of murine hippocampal granule neurons is accompanied by the expression of interleukin-1beta and interleukin-1 receptor antagonist in cells of ameboid phenotype, the majority of which are NG2-positive.

Interleukin-1beta (IL-1beta) has been implicated in various neuropathologies, while IL-1 receptor antagonist (IL-1ra) has been shown to reduce neuronal injury. We investigated the pattern of expression of both cytokines in murine hippocampus after trimethyltin (TMT) intoxication. Using a ribonuclease protection assay, we demonstrated induction of transcription of IL-1beta and IL-1ra 3 days following TMT treatment which correlated with the peak of neuronal apoptosis. At this time, immunocytochemical staining revealed enhanced expression of both cytokines in NG2 proteoglycan expressing ameboid cells located at the site of neurotoxic insult, some of which bound also the microglial marker, lectin. There was some overlap between NG2 and lectin staining. Our results suggest that the two cytokines are involved in apoptotic processes in dentate granule cells and indicate that the pro-apoptotic effect of IL-1beta prevails over the presumed protective action of IL-1ra. The novel finding of expression of both cytokines in NG2(+) cells of ameboid phenotype indicates that these cells, through the regulatory roles of pro- and anti-inflammatory cytokines, may be involved in control of neuronal death or survival after injury.

The ameboid phenotype of NG2 (+) cells in the region of apoptotic dentate granule neurons in trimethyltin intoxicated mice shares antigen properties with microglia/macrophages.

NG2+, stellate cells present in the adult central nervous system (CNS) have been recently recognized as a distinct glial class, identified as multipotent progenitor cells. Antigenically, they are indistinguishable from oligodendroglia progenitor cells. In response to a variety of CNS insults, these cells become rapidly activated and undergo morphological changes accompanied by increased cellular proliferation. The role they play with respect to injured neurons is not clear. In our studies, we performed immunocytochemical investigations and identified a response of NG2-expressing cells in the model of selective neurodegeneration of murine dentate gyrus granule cells induced by systemic administration of trimethyltin. Dying neurons exhibited features of apoptotic cells. Around the region of neurodegeneration, we observed activation of NG2+ stellate cells and microglia. During the peak of apoptosis, we detected the appearance of NG2+ cells of the ameboid phenotype, intermingled with granule neurons. These cells also expressed markers of microglia/macrophages, OX42- and ED1-recognized antigens, an antigen recognized by O4 antibody-a marker of more differentiated cells of the oligodendroglia lineage and, in some cases, also a protein of mature oligodendroglia adenomatus polyposis coli. They also expressed nestin. Our results suggest that the injury induces a parallel transformation of both the activated glial classes: NG2+ stellate cells and resident microglia, into ameboid cells, sharing properties of both oligodendrocyte and monocyte lineages. These cells may play a role in the phagocytosis. If this assumption is verified by electron microscopy, it would indicate a novel function of NG2 transformed cells under CNS injury conditions.

Anti-myelin basic protein T cells protect hippocampal neurons against trimethyltin-induced damage.

We investigated the influence of administration of autoimmune T cells on trimethyltin-induced degeneration of hippocampal neurons. Female Lewis rats received 8 mg/kg trimethyltin intraperitoneally alone, or followed 24 h later by a second intravenous injection of anti-myelin basic protein T cells (green fluorescent protein-tagged). Neurodegeneration was assessed by NeuN and Nissl cell counts 21 days after trimethyltin injection. We found that neurodegeneration in the CA4 region of the hippocampus was significantly reduced in the group receiving T cells. T cells also caused an augmentation of trimethyltin-induced hippocampal astrocytic activation and astrocytic TrkA expression, which was particularly intense in the CA4 region. Our study provides the first evidence of neuroprotection evoked by transferred T cells following a neurotoxic brain insult. The data suggest that mediation of the neuroprotective effects of T-cell-released nerve growth factor occurs mainly via hippocampal astroglial TrkA receptors.