Glatiramer acetate increases IL-1 receptor antagonist but decreases T cell-induced IL-1beta in human monocytes and multiple sclerosis

Abstract

Mechanisms of action as well as cellular targets of glatiramer acetate (GA) in multiple sclerosis (MS) are still not entirely understood. IL-1beta is present in CNS-infiltrating macrophages and microglial cells and is an important mediator of inflammation in experimental autoimmune encephalitis (EAE), the MS animal model. A natural inhibitor of IL-1beta, the secreted form of IL-1 receptor antagonist (sIL-1Ra) improves EAE disease course. In this study we examined the effects of GA on the IL-1 system. In vivo, GA treatment enhanced sIL-1Ra blood levels in both EAE mice and patients with MS, whereas IL-1beta levels remained undetectable. In vitro, GA per se induced the transcription and production of sIL-1Ra in isolated human monocytes. Furthermore, in T cell contact-activated monocytes, a mechanism relevant to chronic inflammation, GA strongly diminished the expression of IL-1beta and enhanced that of sIL-1Ra. This contrasts with the effect of GA in monocytes activated upon acute inflammatory conditions. Indeed, in LPS-activated monocytes, IL-1beta and sIL-1Ra production were increased in the presence of GA. These results demonstrate that, in chronic inflammatory conditions, GA enhances circulating sIL-1Ra levels and directly affects monocytes by triggering a bias toward a less inflammatory profile, increasing sIL-1Ra while diminishing IL-1beta production. This study sheds light on a mechanism that is likely to participate in the therapeutic effects of GA in MS.

Fig. 1.

sIL-1Ra levels are elevated in sera of EAE mice treated with GA. (A) GA ameliorates EAE. C57BL/6 mice were injected s.c. daily with GA (150 μg) or vehicle (PBS solution) 7 d before immunization with 10 μg myelin oligodendrocyte glycoprotein 35–55 peptide (dpi, day post-immunization). (B) EAE mice treated (GA) or not (vehicle) were killed at disease peak and their serum analyzed for IL-1β and sIL-1Ra content. IL-1β was not detected (not shown).

Fig. 2.

sIL-1Ra levels are elevated in sera of patients with MS treated with GA. The levels of sIL-1Ra and IL-1β were measured in sera of patients with RRMS treated with GA or not treated, and age-matched healthy controls, as described in Table 1. IL-1β was under the detection limit (15 pg/mL) in all individuals. There was no significant difference between healthy controls and untreated patients with RRMS. Results are presented as a box plot (GraphPad Prism 4).

Fig. 3.

GA differentially regulates IL-1β and sIL-1Ra production in human monocytes. (A) Monocytes (5 × 10⁴ cells/200 μL/well; 96-well plates) were activated with the indicated dose of GA. After 48 h, supernatants were harvested and the production of IL-1β (open circles) and sIL-1Ra (filled circles) were measured in triplicate wells and represented as mean ± SD. Results are representative of 3 different experiments. (B) Monocytes (5 × 10⁴ cells/200 μL/well; 96-well plates) were preincubated for 1 h with or without 25 μg/mL GA and then cultured for 48 h in the presence or absence of CE_sHUT (1 μg/mL) or LPS (100 ng/mL). sIL-1Ra was measured in culture supernatants (mean ± SD, n = 3 different experiments). (C) Monocytes (5 × 10⁴ cells/200 μL/well; 96-well plates) were preincubated for 1 h with or without 25 μg/mL GA and then cultured for 48 h in the presence or absence of CE_sHUT (6 μg/mL) or LPS (100 ng/mL). IL-1β was measured in culture supernatants (mean ± SD, n = 3 different experiments, i.e., monocytes prepared from 3 different blood donors).

Fig. 4.

GA affects sIL-1Ra and IL-1β mRNA in both CE_sHUT-activated and resting monocytes. (A) Monocytes (2 × 10⁶ cells/well/3 mL) were preincubated for 1 h with the indicated dose of GA and then cultured for 3 h in the presence of CE_sHUT (6 μg/mL). Total mRNA was isolated and analyzed by duplex real-time quantitative PCR (see Materials and Methods) for the presence of IL-1β (open circles) and sIL-1Ra (filled circles) transcripts. Results are presented as percentage of mRNA expression level, 100% being transcript expression measured after 3 h of monocyte activation by CE_sHUT in the absence of GA; mean ± SD of 3 different experiments. (B) Monocytes (2 × 10⁶ cells/well/3 mL) were activated for 18 h with the indicated dose of GA. Total mRNA was isolated and analyzed by duplex real-time quantitative PCR (see Materials and Methods) for the presence of IL-1β (open circles) and sIL-1Ra (filled circles) transcripts. Results are presented as percentage of mRNA expression level, 100% being the transcript level at 50 μg/mL GA; mean ± SD of 3 different experiments, i.e., monocytes prepared from 3 different blood donors.