Glucocorticosteroids modulate antigen-induced T cell apoptosis in experimental autoimmune neuritis and cause T cell proliferation in situ.

Treatment of experimental autoimmune disorders of the nervous system with high doses of glucocorticosteroids (GC) or with administration of the specific antigen is effective and associated with marked T cell apoptosis in situ. Here we investigated in adoptive transfer-experimental autoimmune neuritis (AT-EAN) of the Lewis rat whether induction of T cell apoptosis resulting from T cell activation by antigen therapy can be further augmented by glucocorticosteroids (GC). AT-EAN was induced by intravenous injection of P2-specific T cell blasts. At the maximum of disease two pulses of the antigen recombinant human P2 (rhP2) were given within 12 h. Methylprednisolone was administered simultaneously or 2 h after the antigen and animals were killed 6 h after the second antigen injection. Using an in situ tailing technique followed by immunocytochemical analysis, the presence of DNA fragmentation in T lymphocytes was confirmed. The bromodeoxyuridine (BrdU) technique was employed to detect in situ proliferating cells. T cell apoptosis in sciatic nerve was enhanced after monotherapy with either antigen or GC compared to the control group receiving an irrelevant myelin protein, recombinant human P0. In combination therapy, a synergistic effect on T cell apoptosis in sciatic nerve was obtained when methylprednisolone was injected sequentially, 2 h after rhP2 protein. BrdU incorporation in the sciatic nerve as well as in the spleen, a major lymphoid organ, was significantly enhanced in animals treated with antigen followed by GC 2 h later as compared to rats receiving rhP2 only, speaking for T cell proliferation in situ associated with T cells undergoing apoptosis. Our findings underscore that different proapoptotic stimuli may act synergistically, depending on the timing of the second treatment. In this scenario even local T cell proliferation in the inflamed nervous system occurs. These results support the paradigm of antigen presentation in the nervous system.

Association of increased bcl-2 expression with rescue from tumor necrosis factor-alpha-induced cell death in the oligodendrocyte cell line OLN-93.

The present study investigated the effects of flupirtine (Katadolon) on tumor necrosis factor (TNF)-alpha-mediated cell death and Bcl-2 expression in the permanent rat oligodendrocyte cell line OLN-93 (OLN cells). TNF-alpha (500 U/ml) induced apoptosis of OLN cells, which was confirmed by DNA fragmentation using an in situ end-labeling technique and ultrastructural analysis. Flupirtine significantly reduced the rate of spontaneous cell death of OLN cells already at low concentrations; TNF-alpha-mediated apoptosis was suppressed only with higher concentrations of flupirtine (100 microM:). Expression of Bcl-2 protein and mRNA in OLN cells was detected by immunocytochemistry, western blot, and RT-PCR. Quantitative analysis of western blots revealed an approximately 2. 5-fold up-regulation of Bcl-2 protein during TNF-alpha treatment. Furthermore, addition of 10 or 100 microM: flupirtine before incubation with TNF-alpha led to an approximately threefold increase of Bcl-2 expression. Exposure of OLN cells to flupirtine alone moderately augmented the expression of Bcl-2 protein. Our data demonstrate that flupirtine up-regulates the expression of Bcl-2 protein in OLN cells; this Bcl-2 induction is associated with a reduced rate of TNF-alpha-induced cell death.

Interleukin-6 expression in human multiple sclerosis lesions.

The present study investigated interleukin-6 (IL-6) expression in 36 multiple sclerosis (MS) cases by immunocytochemistry. The numbers of IL-6 expressing cells were correlated to the stage of demyelinating activity and the pattern of oligodendrocyte pathology. IL-6 positive cells were identified as macrophages and astrocytes by morphological criteria. Approximately 10-17% of the astrocytes and up to 2% of the macrophages within the lesion expressed IL-6. Highest numbers of IL-6 expressing cells were found in inactive demyelinating lesions. There was a significant increase of IL-6 positive cells in lesions with oligodendrocyte preservation, whereas absence of IL-6 expression correlated with oligodendrocyte loss. These observations indicate a possible important role for IL-6 in oligodendrocyte protection and survival in MS lesions.

CNTF and its receptor subunits in human gliomas.

Ciliary neurotrophic factor (CNTF) promotes the survival of various neuronal cell populations. It is produced by astrocytes and influences the development and differentiation of glial cells. CNTF and related neuropoietic cytokines affect growth and differentiation of various neoplasms. Moreover, they induce the reactive transformation of astrocytes (gliosis) and influence growth and differentiation of neuroectodermal tumor cell lines in vitro. However, their role in gliomas is largely unknown. We studied the expression of CNTF and its receptor subunits in human astrocytomas and glioblastomas. In more than 95% of the tumors, CNTF transcripts were found by RNAase protection assay; in more than 80% of the cases, tumor cells were CNTF immunoreactive. CNTF receptor alpha (CNTFR alpha), the specific component of the tripartite CNTF receptor system, was detectable by Northern blot analysis in 80% of the cases. In situ hybridization revealed CNTFR alpha mRNA in the cytoplasm of neoplastic cells. Transcripts of the remaining two components of the CNTF receptor system, gp130 and LIFR beta, were found by Northern blotting in 83% and 70% of the tumors, respectively. Simultaneous expression of CNTF and all its receptor components was detected in approximately half of the tumors. These results indicate that CNTF and its receptor components are expressed by human glioma cells. The simultaneous expression of ligands and receptor subunits suggests that CNTF might act on human glioma cells via an auto- or paracrine mechanism.

Identification of glial cell proliferation in early multiple sclerosis lesions.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system which leads to destruction of myelin sheaths. The patterns of cell proliferation in the early course of the disease are largely unknown. The present study used immunohistochemical identification of proliferating glial cells in stereotactic brain biopsy material of eight patients with early chronic MS. Double-labelling with the proliferation marker MIB-1 detected proliferating oligodendrocytes (MOG), astrocytes (GFAP) and microglia/macrophages (Ki-M1P). The majority of proliferating cells were macrophages/microglia when compared with oligodendrocytes (P > 0.005) or astrocytes (P > 0.0005); only a minor proportion of microglia/macrophages, however, proliferated in situ. Astrocytic and oligodendroglial proliferation was sparse to absent and showed significant variations between different patients. There were statistically significant differences when comparing the amount of proliferation between lesions of different demyelinating activity: highest numbers of proliferating cells were found in early active lesions compared with demyelinated and early remyelinated lesions (P > 0.05) or the periplaque white matter (P > 0.01). MOG-positive oligodendrocytes proliferated occasionally in the early stages of lesion formation; this proliferation occurred in four cases but was independent of the stage of the disease. Since MOG is expressed by mature oligodendrocytes, and not by immature precursors, this might suggest a potential role for the proliferation of mature surviving oligodendrocytes with subsequent remyelination.