No abstract

We recently demonstrated that interleukin-1β (IL-1β) increases system xc− (cystine/glutamate antiporter) activity in mixed cortical cell cultures, resulting in an increase in hypoxic neuronal injury when glutamate clearance is impaired. Herein, we demonstrate that neurons, astrocytes and microglia all express system xc− subunits (xCT, 4F2hc, RBAT) and are capable of cystine import. However, IL-1β stimulation increases mRNA for xCT— the light chain that confers substrate specificity— in astrocytes only; an effect blocked by the transcriptional inhibitor actinomycin D. Additionally, only astrocytes show an increase in cystine uptake following IL-1β exposure; an effect associated with a change in xCT protein. The increase in cystine uptake that follows IL-1β is lacking in astrocytes derived from mice harboring a mutation in Slc7a11 (sut gene), which encodes for xCT, and in wild-type astrocytes treated with the protein synthesis inhibitor cycloheximide. IL-1β does not regulate the light chain of the amino acid transporter, LAT2, or the expression and function of astrocytic excitatory amino acid transporters (EAATs), demonstrating some target selectivity. Finally, the enhanced neuronal vulnerability to hypoxia that followed IL-1β treatment in our mixed culture system was not observed in chimeric cultures consisting of wild-type neurons plated on top of sut astrocytes. Nor was it observed in wild-type cultures treated with a system xc− inhibitor or an NMDA receptor antagonist. Overall, our data demonstrate that IL-1β selectively regulates system xc− activity in astrocytes and that this change is specifically responsible for the deleterious, excitotoxic effects of IL-1β found under hypoxic conditions.

No abstract

Tumor necrosis factor-alpha (TNF-alpha) and blood neutrophils (polymorphonuclear leukocytes; PMNs) have been implicated in the pathogenesis of endotoxin (lipopolysaccharide, LPS) hepatotoxicity. However, the mechanism by which these factors mediate liver injury during LPS exposure is uncertain. The objective of this study was to test the hypothesis that TNF-alpha contributes to LPS hepatotoxicity by an indirect, PMN-dependent mechanism. Pretreatment of rats with an antiserum to TNF-alpha afforded protection against liver injury 6 h after LPS exposure. Pretreatment with pentoxifylline (100 mg/kg i.v.), which attenuated the increase in circulating TNF-alpha concentration 1.5 h after administration of LPS, also afforded protection against liver injury. Neither antiserum to TNF-alpha nor pentoxifylline affected hepatic PMN accumulation 1.5 h after LPS exposure. Depletion of circulating PMNs, which protects against LPS hepatotoxicity, enhanced circulating TNF-alpha concentration compared with control rats 1.5 h after LPS exposure. These results suggest that TNF-alpha contributes to liver injury after LPS exposure, but in the absence of circulating PMNs it is insufficient for full manifestation of liver injury. TNF-alpha apparently contributes to the pathogenesis of LPS-induced liver injury by an indirect, PMN-dependent mechanism.

Both transforming growth factor-beta1 (TGF-beta1) and nitric oxide synthase-2 (NOS-2) are upregulated under various neuropathological states. Evidence suggests that TGF-beta1 can either attenuate or augment NOS-2 expression, with the prevailing effect dependent on the experimental paradigm employed and the cell-type under study. The purpose of the present study was to determine the effect of TGF-beta1 on astrocytic NOS-2 expression. In purified astrocyte cultures, TGF-beta1 alone did not induce NOS-2 or NO production. However, NO production induced by lipopolysaccharide (LPS) plus IFNgamma was enhanced by TGF-beta1 in a concentration-dependent manner between 10 and 1,000 pg/mL. The presence of IFNgamma was not necessary for this effect to occur, as TGF-beta1 enhanced NO production induced by LPS in a similar fashion. In cultures stimulated with LPS plus IFNgamma, the enhancement of NO production by TGF-beta1 was associated with a corresponding increase in NOS-2 mRNA and protein expression. Interestingly, immunocytochemical assessment of NOS-2 protein expression demonstrated that TGF-beta1 augmented astrocytic NO production, specifically by increasing the pool of astrocytes capable of expressing NOS-2 induced by either LPS (approximately threefold) or LPS plus IFNgamma (approximately sevenfold). In a broader sense, our results suggest that TGF-beta1 recruits a latent population of astrocytes to respond to stimulation by pro-inflammatory mediators.