Anne I. Boullerne

Inhibition of microglial inflammatory responses by norepinephrine: effects on nitric oxide and interleukin-1β production

BackgroundUnder pathological conditions, microglia produce proinflammatory mediators which contribute to neurologic damage, and whose levels can be modulated by endogenous factors including neurotransmitters such as norepinephrine (NE). We investigated the ability of NE to suppress microglial activation, in particular its effects on induction and activity of the inducible form of nitric oxide synthase (NOS2) and the possible role that IL-1β plays in that response.MethodsRat cortical microglia were stimulated with bacterial lipopolysaccharide (LPS) to induce NOS2 expression (assessed by nitrite and nitrate accumulation, NO production, and NOS2 mRNA levels) and IL-1β release (assessed by ELISA). Effects of NE were examined by co-incubating cells with different concentrations of NE, adrenergic receptor agonists and antagonists, cAMP analogs, and protein kinase (PK) A and adenylate cyclase (AC) inhibitors. Effects on the NFκB:IκB pathway were examined by using selective a NFκB inhibitor and measuring IκBα protein levels by western blots. A role for IL-1β in NOS2 induction was tested by examining effects of caspase-1 inhibitors and using caspase-1 deficient cells.ResultsLPS caused a time-dependent increase in NOS2 mRNA levels and NO production; which was blocked by a selective NFκB inhibitor. NE dose-dependently reduced NOS2 expression and NO generation, via activation of β2-adrenergic receptors (β2-ARs), and reduced loss of inhibitory IkBα protein. NE effects were replicated by dibutyryl-cyclic AMP. However, co-incubation with either PKA or AC inhibitors did not reverse suppressive effects of NE, but instead reduced nitrite production. A role for IL-1β was suggested since NE potently blocked microglial IL-1β production. However, incubation with a caspase-1 inhibitor, which reduced IL-1β levels, had no effect on NO production; incubation with IL-receptor antagonist had biphasic effects on nitrite production; and NE inhibited nitrite production in caspase-1 deficient microglia.ConclusionsNE reduces microglial NOS2 expression and IL-1β production, however IL-1β does not play a critical role in NOS2 induction nor in mediating NE suppressive effects. Changes in magnitude or kinetics of cAMP may modulate NOS2 induction as well as suppression by NE. These results suggest that dysregulation of the central cathecolaminergic system may contribute to detrimental inflammatory responses and brain damage in neurological disease or trauma.

Indirect evidence for nitric oxide involvement in multiple sclerosis by characterization of circulating antibodies directed against conjugated S-nitrosocysteine.

Converging data suggest that nitric oxide (NO) production by cytokine-induced immune cells in demyelinating lesions is involved in multiple sclerosis (MS). High levels of NO may complex to suitable amino acids, causing an immune response against the formed neo-epitopes. By testing MS sera with chemically defined nitroso-amino acids conjugated to carrier protein in ELISA, we observed a significant antibody reaction against the S-nitroso-cysteine epitope. The MS antibody response was exclusively of IgM isotype with an avidity of 8 x 10(-7) M. Sera of all clinical MS forms showed a significantly elevated antibody titer versus sera from healthy subjects or from patients affected with other neurological and autoimmune diseases. The detection of circulating antibodies to a conjugated S-nitroso-cysteine epitope provides indirect evidence for NO involvement in MS.

Synergism of nitric oxide and iron in killing the transformed murine oligodendrocyte cell line N20.1

Abstract : Nitric oxide (NO) produced in inflammatory lesions may play a major role in the destruction of oligodendrocytes in multiple sclerosis and experimental allergic encephalomyelitis. The transformed murine oligodendroglial line N20.1 is much more resistant than primary oligodendrocytes to killing by the NO generator S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP). This observation prompted investigation of the mechanisms leading to cell death in the N20.1 cells and comparison of SNAP with another NO donor, sodium nitroprusside (SNP). We observed that N20.1 cells were 30 times more sensitive to SNP than to SNAP. The specific NO scavenger 2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO) protected against SNP only, not against SNAP. However, dithiothreitol protected against both SNAP and SNP, indicating that S‐nitrosylation of cysteines plays a major role in the cytotoxicity of both NO donors. We did not observe any formation of peroxynitrite or increase of Ca2+ concentration with either SNAP or SNP, thus excluding their involvement in the mechanisms leading to N20.1 cell death. Based on two observations, (a) potentiation of the cytotoxic effect of SNP when coincubated with ferricyanide or ferrocyanide, but not sodium cyanide, and (b) protection by deferoxamine, an iron cyanide chelator, we conclude that the greater sensitivity of N20.1 cells to SNP compared with SNAP is due to synergism between NO released and the iron cyanide portion of SNP, with the cyanide accounting for very little of the cytotoxicity. Finally, SNP but not SNAP induces some apoptosis, as shown by DNA laddering and protection by a caspase‐3 inhibitor. These results suggest that low levels of NO in combination with increased iron content lead to apoptotic cell death rather than the necrotic cell death seen with higher levels of NO generated by SNAP.

Circulating antibodies directed against conjugated fatty acids in sera of patients with multiple sclerosis

Using an adapted ELISA assay, we have tested sera from multiple sclerosis (MS) patients for antibodies directed against ten fatty acids conjugated to bovine serum albumin. In serum samples from 68 MS patients and 20 patients suffering from rheumatoid arthritis (RA), a significant antibody titer elevation to the ten tested fatty acids was found when compared to sera of 40 healthy subjects and 82 patients with other neurological and autoimmune diseases. G-200 purified IgM of MS patients reacted specifically with the aliphatic chains with an avidity of 3 x 10(-7) M. These results suggest that in MS and RA, autoepitopes on cell membranes that are normally hidden from the immune system become immunogenic. This may arise because of previous membrane disruption by oxidative processes.

Role of calcium in nitric oxide-induced cytotoxicity: EGTA protects mouse oligodendrocytes

Active nitrogen species are overproduced in inflammatory brain lesions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). NO has been shown to mediate the death of oligodendrocytes (OLs), a primary target of damage in MS. To develop strategies to protect OLs, we examined the mechanisms of cytotoxicity of two NO donors, S‐nitroso‐N‐acetyl‐penicillamine (SNAP) and sodium nitroprusside (SNP) on mature mouse OLs. Nitrosonium ion (NO+) rather than NO · mediates damage with both SNAP and SNP, as shown by significant protection with hemoglobin (HbO2), but not with the NO · scavenger PTIO. SNAP and SNP differ in time course and mechanisms of killing OLs. With SNAP, OL death is delayed for at least 6 hr, but with SNP, OL death is continuous over 18 hr with no delay. Relative to NO release, SNP is more toxic than SNAP, due to synergism of NO with cyanide released by SNP. SNAP elicits a Ca2+ influx in over half of the OLs within min. Further, OL death due to NO release from SNAP is Ca2+‐dependent, because the Ca2+ chelator EGTA protects OLs from killing by SNAP, and also from killing by the NONOates NOC‐9 and NOC‐18, which spontaneously release NO ·. SNP does not elicit a Ca2+ influx, and EGTA is not protective. In comparison to the N20.1 OL cell line (Boullerne et al., [1999] J. Neurochem. 72:1050–1060), mature OLs are (1) more sensitive to SNAP, (2) much more resistant to SNP, (3) sensitive to cyanide, but not iron, and (4) exhibit a Ca2+ influx and EGTA protection in response to NO generated by SNAP. J. Neurosci. Res. 63:124–135, 2001.

Seric immune complexes in multiple sclerosis do not contain MBP epitopes.

Immune complexes from sera of MS patients, other neurological diseases, and healthy donors were precipitated using polyethyleneglycol and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Silver staining evidenced additional protein bands whose molecular weights were 14-16, 38, and 43 kDa. These IC proteins were present in most MS patients studied. To identify their nature, immunoblotting was performed with antihuman immunoglobulins A, M, G antibodies. No immunoreactivity was found below a molecular weight of 66 kDa on a nitrocellulose sheet having the transferred protein pattern of MS IC. Using purified human myelin, MS IC transferred to an immobilon sheet and antihuman myelin basic protein antibodies, an immunoreactivity was seen only on purified human MBP. The small proteins of 14-16 kDa and the others of 38, 43 kDa were not immunoreactive. Identification of the nature of these additional proteins in MS IC is in progress.

Raised antibody titre against conjugated S-nitrosocysteine in IgM paraproteinaemic peripheral, neuropathy: possible role of nitric oxide in pathogenesis.

to cause a different syndrome from myasthenia gravis. In myasthenia gravis the progressive weakness is explained by the smaller amounts of ACh released at the neuromuscular junction with each successive nerve impulse. The reduced number of ACh molecules are less likely to activate the few remaining AChRs before they are enzymatically destroyed. In the intermediate syndrome, however, any liberated ACh is likely

The emerging threat of superwarfarins: history, detection, mechanisms, and countermeasures: The emerging threat of superwarfarins

Superwarfarins were developed following the emergence of warfarin resistance in rodents. Compared to warfarin, superwarfarins have much longer half‐lives and stronger affinity to vitamin K epoxide reductase and therefore can cause death in warfarin‐resistant rodents. By the mid‐1970s, the superwarfarins brodifacoum and difenacoum were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raise the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1–2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K–dependent and –independent effects, which are not mitigated by vitamin K therapy. In this review, we summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches.