Kinetics of inflammatory response of astrocytes induced by TLR 3 and TLR4 ligation.

Toll-like receptors (TLRs) are sentinels of the innate immune system that recognize an array of exogenous and endogenous pathogenic molecules. The ligation of the receptors triggers inflammatory response necessary for pathogen elimination and for the healing process. In the present study we examined inflammatory response of astrocytes elicited by the ligation of TLR3 and TLR4. Astrocytic cultures established from newborn rat brains were exposed to double stranded RNA (dsRNA) and lipopolysaccharide (LPS), the ligands for TLR3 and TLR4, respectively. The expression of cytokine genes was determined by RNase protection assay, and the generation of nitric oxide (NO) was measured by Griess technique. Both ligands upregulated the expression of several cytokines (i.e., IL-1alpha, IL-1beta, IL-6, TNFalpha, GM-CSF, LTbeta, and TGFbeta3) and downregulated the expression of MIF, but have no effect on the expression of IL-2, IL-3, IL-4, IL-5, IL-10, TGFbeta1, TGFbeta2, TNFbeta, and IFNgamma. Although dsRNA upregulated the expression of IFNbeta, LPS did not indicating that the TRIF-dependent branch of TLR4 signaling is inactive in astrocytes. Proinflammatory response as seen from upregulated cytokine expression and NO generation reached a peak within the first day of exposure, and was subsequently abrogated. The cells also became refractory to subsequent stimulation by the ligands indicating the existence of negative feedback mechanisms that control proinflammatory response in astrocytes.

Nucleotides induce higher order chromatin degradation.

Higher order chromatin degradation (HOCD) is a stepwise dismantling of the genome through the excision of chromatin loops and their oligomers at matrix attachment regions (MARs) during the early stages of programmed cell death. Although HOCD ultimately leads to the inactivation of the genome and cell death, a partial HOCD in cells receiving sublethal signals may result in the loss of genetic stability leading to neoplasia, degeneration, and aging. The present study was undertaken to determine the role of protein poly(ADP-ribosyl)ation in HOCD. Nuclei isolated from rat glioma C6 cells were able to carry poly(ADP-ribosyl)ation as assessed by the incorporation of (32)P-NAD(+) into TCA-insoluble fraction. Under the same experimental conditions, millimolar NAD(+) induced rapid HOCD in nuclei. However, while poly(ADP-ribosyl)ation was totally abrogated by specific inhibitor, benzamide, NAD(+)-induced HOCD was unaffected. Benzamide also failed to inhibit HOCD induced by H(2)O(2) exposure in intact cells. These results indicate that HOCD is not mediated through chromatin poly(ADP-ribosyl)ation, and that NAD(+) activates MAR-associated endonuclease or facilitates the access of the enzyme to DNA by other mechanisms. Furthermore, other nucleotides including NADP(+), ATP, UTP, GTP, and CTP were also found to induce HOCD in isolated nuclei indicating that HOCD is controlled by nucleotide-related ligands.