This study has investigated the cellular mechanism by which LPS induces injury to oligodendrocyte precursors, the cell type predominantly damaged in the diffuse white matter lesion of PVL. To investigate the mechanism underlying LPS-induced selective loss of preOLs in primary mixed glial cultures, various single and co-cultures were prepared. Consistent with previous report (Lehnardt et al., 2002
; Li et al., 2005
), we found that activation of microglia, but not astrocytes, is absolutely required for LPS toxicity. In preOL-microglia co-cultures, a diffusible potent oxidant, peroxynitrite, was identified as the primary underlying toxic factor killing preOLs (Li et al., 2005
). Blocking peroxynitrite formation by preventing either NO production or superoxide production or enhancing peroxynitrite decomposition abolished preOL death in these co-cultures. However, when the mechanism of LPS-induced toxicity to preOLs was reexamined in mixed glial cultures in which astrocytes were also present, paradoxically, LPS-induced toxicity was independent of peroxynitrite but instead relied on TNFα signaling, even though copious amount of NO was produced. This apparent paradox was reconciled by the fact that astrocytes efficiently block peroxynitrite-mediated toxicity to preOLs.
The determination of a peroxynitrite-independent cell death pathway when astrocytes are present is based on the evidence that: 1) blocking NO production with the NOS inhibitors, L-NMMA or 1400W, had minimal effect on LPS toxicity in mixed glial cultures; 2) a peroxynitrite decomposition catalyst and superoxide scavenger, FeTMPyP, as well as other antioxidants did not protect preOLs; 3) disruption of genes encoding iNOS or the gp91phox
NADPH oxidase, two enzymes that we identified previously to be indispensable for LPS-induced microglial killing of preOLs in co-cultures with microglia (Li et al., 2005
), did not prevent LPS-induced toxicity in mixed glial cultures; and 4) astrocytes efficiently block peroxynitrite toxicity to preOLs.
Microglia activated by endotoxin LPS have been shown to release proinflammatory cytokines such as TNFα and IL-1β (Hanisch, 2002
). As a potent source of immunologically relevant cytokines, including TNFα, astrocytes also play a pivotal role in the type and extent of CNS immune and inflammatory responses. A previous study showed that induction of iNOS in astrocytes by IFNγ and IL-1β potentiates NMDA-receptor mediated excitotoxicity (Hewett et al., 1994
). Activated microglia also enhance TNFα production and glutamate release from astrocytes, resulting in amplified neurotoxicity (Bezzi et al., 2001
). Neuropathological studies have revealed that in human PVL cases, but not age-matched controls, abundant hypertrophic reactive astrocytes and activated microglia populate diffuse white matter lesions (Haynes et al., 2003
). Therefore, it is most likely that intercellular communication among these activated glia may play an important role in the pathogenesis of PVL. Our data demonstrate that with the peroxynitrite cell death pathway inhibited by astrocytes, a cell death pathway orchestrated by TNFα/TNFR signaling becomes dominant for LPS-triggered injury to preOLs in culture.
Multiple lines of evidence suggest that several proinflammatory cytokines, including IFNγ (Folkerth et al., 2004
), TNFα (Deguchi et al., 1996
) (Kadhim et al., 2001
) and IL-6 (Yoon et al., 1997
) and IL-2 (Kadhim et al., 2002
) are elevated in PVL and may play pivotal roles in perinatal white matter injury (Pang et al., 2006
; Smith et al., 2007
; Rezaie and Dean, 2002
; Dammann and Leviton, 1997
). Proinflammatory cytokines such as TNFα are also potent regulators of glial activation and iNOS induction (John et al., 2003
). TNFα is a prototypical proinflammatory cytokine that plays a central role in initiating inflammatory reactions of the innate immune system, in part through the induction of expression and release of other cytokines (Wajant et al., 2003
). TNFα exerts its biologic functions by binding to and signaling through TNFR1 and TNFR2 in an autocrine and/or paracrine fashion. In situ
immunohistochemical studies revealed locally increased TNFR1/2 expression and TNFα production in both reactive astrocytes and microglia in human PVL lesions (Deguchi et al., 1996
; Yoon et al., 1997
; Kadhim et al., 2001
). TNFα/TNFR1 was also responsible for optical nerve OL death and subsequent retinal ganglion cell loss in a mouse model of glaucoma (Nakazawa et al., 2006
). Transgenic overexpression of TNFα in astrocytes, but not neurons, results in demyelination and selective OL apoptosis (Akassoglou et al., 1998
). It is not clear whether preOLs are damaged in these transgenic mice during early development. Other in vivo
studies also revealed critical roles of the TNF/TNFR signaling pathway in CNS inflammation and white matter degeneration (Akassoglou et al., 1997
; Probert et al., 2000
). Our data demonstrate that TNFα/TNFR signaling is necessary for LPS-initiated death of preOLs in mixed glial cultures. The cellular source for LPS-induced TNFα production in mixed glia was not defined in current study, but activated microglia are most likely responsible for LPS-induced initial TNFα production, given our observation that microglia respond robustly to LPS and produce NO and TNFα in culture, whereas astrocytes respond poorly (Li et al, unpublished). However, astrocytes, microglia and preOLs all express TNFα receptors (Dopp et al., 1997
) and thus all are capable of engaging in TNFα/TNFR signaling. Therefore, TNFα released from activated microglia may activate astroglial TNFα receptors, resulting in further production of this cytokine and/or other toxic factors and amplification of microglial responses to LPS. Interestingly, TNFα itself had minimal effect on preOL viability in preOL monocultures, suggesting a non-cell autonomous cell death pathway. The actual mediator(s) regulated by TNFα signaling and responsible for preOL death in mixed glial cultures remains to be identified.
Our data do not support a role for iNOS in LPS-induced preOL death in mixed glial cultures. However, this does not necessarily mean that reactive nitrogen species, in particular peroxynitrite, play no role in white matter injury in vivo
. Immunoreactive nitrotyrosine, a footprint of peroxynitrite formation, is found in astrocytes and preOLs in diffuse human PVL lesions (Haynes et al., 2003
). Interestingly, morphologically identified microglia/macrophages within the subacute necrotic foci, but not in the diffuse lesions, are immunostained positively for nitrotyrosine (Haynes et al., 2003
), indicating that a robust and significant burst of oxidative/nitrative stress may be present in the necrotic foci. In culture, peroxynitrite is highly toxic to preOLs and is indeed the molecule directly responsible for the death of preOLs triggered by acutely activated microglia (Li et al., 2005
). However, the presence of astrocytes switches the death mechanism from a peroxynitrite-dependent to a TNFα-dependent pathway. These results have several important implications. First, peroxynitrite generated by activated microglia/macrophages within the necrotic foci in PVL may play a role there in direct killing of preOLs. On the other hand, in the diffuse white matter lesion, which is populated by abundant reactive astrocytes as well as microglia, a different mechanism of toxicity, such as that mediated by TNFα signaling, may be more important. Second, understanding how astrocytes communicate with activated microglia and influence the mechanism of toxicity and identifying TNFα-regulated mediators of preOL injury should provide us with mechanistic insights that can be exploited to augment protective signals while suppressing deleterious signals. It should be noted that the role of reactive nitrogen species in neonatal white matter injury has yet to be established in animal models. Blocking iNOS may have only a limited beneficial effect. In fact, in inflammatory demyelination models of multiple sclerosis, ablation of the iNOS gene actually exacerbates OL injury and clinical symptoms (Fenyk-Melody et al., 1998
; Sahrbacher et al., 1998
). The role of iNOS and its product NO in neonatal white matter injury in vivo
therefore remains unknown. Multiple pathogenic mechanisms of preOL destruction are likely to exist. Combinatorial approaches such as blocking TNFα signaling and NO production may prove beneficial in preventing white matter injury.
In summary, this study highlights the importance of both astrocytes and microglia in mediating and regulating injury to oligodendrocyte precursors, and identifies a distinct cellular mechanism by which endotoxin-activated microglia kill preOLs in an environment in which all three types of CNS glial cells interact. Although peroxynitrite is upregulated, its toxicity to preOL is blunted by astrocytes. Instead, activation of TNFα/TNFR signaling results in preOL death when astrocytes are present. Our study provides new mechanistic insights into inflammatory injury to preOLs and underscores the necessity to consider cell-cell interactions when developing new strategies for the prevention and treatment of white matter injury.