“Tumor necrosis factor-alfa and interleukin1- alfa mediate cell death in process-bearing cells of the L3 cell line.
Tez Türü: Yüksek Lisans
Tezin Yürütüldüğü Kurum: Vrije Universiteit Brussel, Belçika
Tez Danışmanı: Elisabeth Hooghe-Peters,Liliane Tenenbaum
Tezin Onay Tarihi: 1994
Tezin Dili: İngilizce
Desteklendiği Program: Diğer
Özet:
Multiple Sclerosis (MS) is a neurodegenerative disease characterized by focal perivascular inflammatory cell infiltrates and demyelination of the white matter in the Central Nervous System (CNS). Inflammatory cell infiltration by T and B lymphocytes, and macrophages, oligodendrocytes (OLs) loss with breakdown of associated myelin sheats, and astrocytes (AS) proliferation are found at the site of inflammatory demyelination. Cytokines as tumor necrosis factors (TNFs), interleukin 1 (IL1) and interferon regulatory factor-gama (IFN-) are involved in inflammation of the CNS and are found at elevated concentrations at the active edges of MS plaques. The cytotoxicity of these cytokines toward OLs were demonstrated in vitro in primary cultures of embryonic OLs and oligodendrocyte-type 2-astrocyte (O-2A) progenitors, a common precursor of OLs and type 2-AS. In the present work we have showed that rhTNF- and rhIL1- separately or in combination can induce cytotoxicity toward the L3 cell line, a spontaneously immortalized rat glial cell line. The L3 cell line consist of two distinct populations of cells : i) process-bearing cells behaving like O-2A progenitors growing on a monolayer of ii) adherent cells, probably derived from type 1-AS. The main advantage of the L3 cell line is that L3 process-bearing cells similar to the O-2A lineage cells can be propagated and obtained continuously from the adherent monolayer by gentle shaking of the culture plate. The first part of this work consists of the study of differentiation and the phenotypical characterization of the L3 process-bearing cells in two different culture medium by using immunocytochemistry. As expected, in serum-supplemented medium, in which primary cultures of O-2A differentiate into A2B5+ GFAP+ (glial fibrillary acidic protein+) type 2-AS, the L3 process-bearing cells consisted mainly of A2B5+ GFAP+ cells. In B-S (Bottenstein and Sato) chemically defined medium in which O-2A progenitors differentiate into OLs, 20% of the L3 process-bearing cells differentiated into galC+ (galactocerebroside+) cells, the marker of mature OL. We have addressed the sensitivity of L3 process-bearing cells to rhTNF- and rhIL1- mediated cell death by the MTT cell survival assay which provides a biochemical measure of the mitochondrial function. A 50% reduction in the mitochondrial activity of L3 process-bearing cells cultured in B-S medium was observed after 4 days treatment with rhTNF- and rhIL1- We then observed the cytoarchitecture of the L3 cells using acridine orange staining of the nucleic-acids in fixed cells. Several morphological changes were apparent after 24 hrs treatment : the nucleus was swollen and blebbing, the cytoplasm condensed and the processes retracted. These changes were characteristic of an apoptotic rather than a necrotic mechanism of cell death. The cytotoxic activity of rhTNF and rhIL1- toward the L3 process-bearing cells was evident from our study. However, the mechanism of this cell death has not been fully characterized. Identification of the mechanism involved in cytokinesinduced OL cell death will be the future study in our laboratory