Mitoxantrone repression of astrocyte activation: relevance to multiple sclerosis

Abstract

Mitoxantrone has been approved by the FDA for the treatment of multiple sclerosis (MS). However, the mechanisms by which mitoxantrone modulates MS are largely unknown. Activated astrocytes produce nitric oxide (NO), TNF-α, and IL-1β, molecules which can be toxic to central nervous system (CNS) cells including oligodendrocytes, thus potentially contributing to the pathology associated with MS. MCP-1 is a chemokine believed to modulate the migration of monocytes to inflammatory lesions present in the CNS of MS patients. IL-12 and IL-23 have been demonstrated to play critical roles in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, by contributing to the development of CD4(+) T cell lineages termed Th1 and Th17, respectively. The current study demonstrates that mitoxantrone inhibits lipopolysachharide (LPS) induction of NO, TNF-α, IL-1β, and MCP-1 production by primary astrocytes. Mitoxantrone also inhibited IL-12 and IL-23 production by these cells. Furthermore, mitoxantrone suppressed the expression of C-reactive protein (CRP). Finally, we demonstrate that mitoxantrone suppressed LPS induction of NF-κB DNA-binding activity, suggesting a novel mechanism by which mitoxantrone suppresses the expression of proinflammatory molecules. Collectively, these studies demonstrate that mitoxantrone represses astrocyte production of potentially cytotoxic molecules, as well as molecules capable of altering T-cell phenotype. These in vitro studies suggest mechanisms by which mitoxantrone may modulate inflammatory diseases including MS.

Figure 1

Mitoxantrone inhibits LPS induction of pro-inflammatory molecules by primary astrocytes. Cells were pre-treated for 1h with the indicated concentrations of mitoxantrone. LPS (2 µg/ml) was added as indicated, and 24h later the concentration of TNF-α in the culture medium was determined by ELISA (A). Cell viability was determined by MTT assay (B). The concentration of nitrite in the culture was determined by Greiss reaction (C). The concentration of IL-1β (D) in the culture supernatant was determined by ELISA. Values represent the mean +/− s.e.m. for triplicate cultures. *p<0.001 vs. LPS treated cultures. Data were analyzed by ANOVA followed by a Bonferroni test to determine the significance of difference. The experiment was performed three times independently.

Figure 2

Mitoxantrone inhibits LPS induction of MCP-1 by primary astrocytes. Cells were pre-treated for 1h with the indicated concentration of mitoxantrone. LPS (2 µg/ml) was added as indicated, and 24h later the concentration of IL-6 (A) and MCP-1 (B) in the culture medium was determined by ELISA. Values represent the mean +/− s.e.m. for triplicate cultures. *p<0.001 vs. LPS treated cultures. Data were analyzed by ANOVA followed by a Bonferroni test to determine the significance of difference. The experiment was performed three times independently.

Figure 3

Mitoxantrone inhibits LPS induction of IL-12 and Il-23 by primary astrocytes. Cells were pre-treated for 1h with the indicated concentration of mitoxantrone. LPS (2 µg/ml) was added as indicated, and 24h later the concentration of IL-12 p40 (A), IL-23 (B), and IL-27 p28 in the culture medium was determined by ELISA. Values represent the mean +/− s.e.m. for triplicate cultures. *p<0.001 vs. LPS treated cultures. Data were analyzed by ANOVA followed by a Bonferroni test to determine the significance of difference. The experiment was performed three times independently.

Figure 4

Mitoxantrone inhibits LPS induction of C-reactive protein by primary astrocytes. Cells were pre-treated for 1h with the indicated concentration of mitoxantrone. LPS (2 µg/ml) was added as indicated, and 24h later the concentration of CRP in the culture medium was determined by ELISA. Values represent the mean +/− s.e.m. for triplicate cultures. *p<0.001 vs. LPS treated cultures. Data were analyzed by ANOVA followed by a Bonferroni test to determine the significance of difference. The experiment was performed three times independently.

Figure 5

Mitoxantrone inhibits NF-κB DNA-binding activity. Nuclear extracts were prepared from untreated primary astrocytes (Control), astrocytes treated with 2 µg/ml LPS (LPS) or astrocytes pre-treated for 1h with 3 µM mitoxantrone followed by an additional treatment for 4h with 2 µg/ml LPS (LPS + Mito). An excess of cold competitor was added to some EMSA assays to demonstrate the specificity of binding (Comp). Specific NF-κB bands are indicated by the arrow. The experiment was performed three times independently.