Differential tumor necrosis factor alpha expression by astrocytes from experimental allergic encephalomyelitis-susceptible and -resistant rat strains

Tumor Necrosis Factor Expression by Astrocytes TNF-a production was assessed. Treatment of cultures with indomethacin increased TNF-a production by both BN and Lewis astrocytes, but did not alter the overall pattern of TNF-a expression in response to LPS or IFN-y/LPS (Table 1) . Similar results were obtained for IFN-y/IIr1a-induced TNF-a production (data not shown) . Most striking is the consistent low level of TNF-a production by Lewis rats in response to LPS plus indomethacin, and the enhancement with IFN-,y pretreatment . These results suggest that differential TNF-a production by astrorytes from BN and Lewis rats is not due to suppression by endogenous PGE2 . TNF-a mRNA Expression by Astrocytes from EAE-resistant and -susceptible Strains. We next examined TNF-a mRNA levels from stimulated BN and Lewis astrocytes to assess if differences in TNF-a protein expression were reflected at the mRNA level. We had previously used the sensitive technique of reverse transcription (RT PCR) to demonstrate levels of TNF-a mRNA in astrorytes because very low levels of this specific RNA are expressed (11) . The astrorytes from BN and Lewis were incubated with LPS (10 jig/ml) for 4 h, IFN--Y (100 U/ml) for 8 h followed by LPS for an additional 4 h, or IFN-y (100 U/ml) for 8 h followed by IIAR (100 U/ml) for an additional 4 h; then RNA was isolated. Using this RNA, we initially synthesized the corresponding cDNA by RT, and then used PCR to amplify a specific sequence of the TNF-ci cDNA as described in Materials and Methods. As shown in Fig. 4 , the amplified TNF-ot cDNA sequence is detected in astrocytes stimulated with LPS alone, IFNy/LPS, or IFN-y/11,10, but not in unstimulated astrocyte cultures . The amplified sequence, hybridizing with a mouse TNF-cx cDNA probe, had the expected size of 275 bp. To insure linearity of the assay, varying amounts of the PCR product (1-16 p,l) were run on a Southern blot . Scanning ofthe autoradiographs was performed on the exposure shown in Fig . 4 , as well as ones developed for less time. Differences in TNF-ct mRNA expression were observed within each rat strain, depending on the stimulus used, as well as between BN and Lewis astrocytes. Comparing TNF-ci mRNA levels in BN astrorytes, our results indicate that these cells express TNF-oi mRNA in response to LPS alone, and slightly more upon pretreatment with IFN-y (1.5-fold increase) . Low levels of TNF-ot mRNA are expressed in response to stimulation with IFN-y/IL1# compared with mRNA levels from LPS-80 6 Tumor Necrosis Factor Expression by Astrocytes or IFN-y/LPS-treated astrocytes . When examining TNF-ci mRNA expression by Lewis astrorytes, we observed that low levels of TNF-ot mRNA are detected in cells stimulated with LPS alone, enhanced expression with the combined stimuli of IFN-y/LPS (2.5-fold increase), and even higher levels of TNT-ot mRNA upon stimulation with IFN-y/IIr1/3. These differences within each strain are consistent with the TNF-a protein results (Figs. 1 and 2) , although the differences in the mRNA levels are not as pronounced . When TNF-cx mRNA levels are compared between BN and Lewis astrocytes, the most striking difference is in response to IFN-.y/11,10 ( Fig . 5) . TNFca Protein Production by Peritoneal Macrophages_from EAEresistant and -susceptible Rat Strains. To determine if the differences in TNF-ot production by Lewis and BN astrorytes were restricted to this cell type, we examined TNF-ot production by peritoneal macrophages from these