Reactive microglia and astrocytes are present in lesions of white matter disorders, such as periventricular leukomalacia and multiple sclerosis. However, it is not clear whether they are actively involved in the pathogenesis of these disorders. Previous studies demonstrated that microglia, but not astrocytes, are required for lipopolysaccharide (LPS)-induced selective killing of developing oligodendrocytes (preOLs) and that the toxicity is mediated by microglia-derived peroxynitrite. Here we report that, when astrocytes are present, the LPS-induced, microglia-dependent toxicity to preOLs is no longer mediated by peroxynitrite but instead by a mechanism dependent on tumor necrosis factor-␣ (TNF␣) signaling. Blocking peroxynitrite formation with nitric oxide synthase (NOS) inhibitors or a decomposition catalyst did not prevent LPS-induced loss of preOLs in mixed glial cultures. PreOLs were highly vulnerable to peroxynitrite; however, the presence of astrocytes prevented the toxicity. Whereas LPS failed to kill preOLs in cocultures of microglia and preOLs deficient in inducible NOS (iNOS) or gp91 phox , the catalytic subunit of the superoxide-generating NADPH oxidase, LPS caused a similar degree of preOL death in mixed glial cultures of wild-type, iNOS Ϫ/Ϫ , and gp91 phox؊/؊ mice. TNF␣ neutralizing antibody inhibited LPS toxicity, and addition of TNF␣ induced selective preOL injury in mixed glial cultures. Furthermore, disrupting the genes encoding TNF␣ or its receptors TNFR1/2 completely abolished the deleterious effect of LPS. Our results reveal that TNF␣ signaling, rather than peroxynitrite, is essential in LPS-triggered preOL death in an environment containing all major glial cell types and underscore the importance of intercellular communication in determining the mechanism underlying inflammatory preOL death.

Ischemia/reperfusion (I/R) carries significant injury to endothelial cells in transplanted organs and is an important factor in chronic rejection. Immunosuppressive drugs, notably cyclosporin A (CyA) and FK506, can potentially augment this injury. Here, our goal was to determine the combined effects of I/R and CyA or FK506 on endothelial cells. Transformed mouse endothelial cells (SVEC 4–10) were subjected to ischemia or I/R for 2–24 hours by incubating cells in 100 per cent N2 (ischemia) followed by 5 per cent CO2 and 95 per cent O2 (reperfusion) for 24 hours. In separate experiments, CyA or FK506 was added to cells subjected to ischemia or I/R. Nonviable cells were determined by Trypan blue exclusion assay. All experiments (done in triplicate) were analyzed by Student's t test. Increasing ischemia times resulted in a greater number of nonviable cells (2% nonviable cells at 0 hours and 57% at 24 hours of I/R). Addition of CyA significantly increased the number of nonviable cells when compared with the control (I/R only) group ( P = 0.014). Interestingly, FK506 did not increase the percentage of nonviable cells compared with the control group ( P = 0.2). Unlike FK506, CyA augments I/R injury to endothelial cells in vitro. These findings could be relevant in chronic rejection and transplantation.