N-acetyl-L-cysteine ameliorates the inflammatory disease process in experimental autoimmune encephalomyelitis in Lewis rats

Abstract

We report that N-acetyl-L-cysteine (NAC) treatment blocked induction of TNF-alpha, IL-1beta, IFN-gamma and iNOS in the CNS and attenuated clinical disease in the myelin basic protein induced model of experimental allergic encephalomyelitis (EAE) in Lewis rats. Infiltration of mononuclear cells into the CNS and induction of inflammatory cytokines and iNOS in multiple sclerosis (MS) and EAE have been implicated in subsequent disease progression and pathogenesis. To understand the mechanism of efficacy of NAC against EAE, we examined its effect on the production of cytokines and the infiltration of inflammatory cells into the CNS. NAC treatment attenuated the transmigration of mononuclear cells thereby lessening the neuroinflammatory disease. Splenocytes from NAC-treated EAE animals showed reduced IFN-gamma production, a Th1 cytokine and increased IL-10 production, an anti-inflammatory cytokine. Further, splenocytes from NAC-treated EAE animals also showed decreased nitrite production when stimulated in vitro by LPS. These observations indicate that NAC treatment may be of therapeutic value in MS against the inflammatory disease process associated with the infiltration of activated mononuclear cells into the CNS.

Figure 1

The protective effect of NAC on the clinical signs of MBP induced EAE in female Lewis rats. EAE was induced as described in Materials and Methods. Data are given as average clinical disease score where 0 = normal; 1 piloerection; 2 = loss in tail tonicity; 3 = hind leg paralysis; 4 = paraplegia and 5 = moribund. Each group i.e., MBP (closed squares), MBP + NAC (open circles) treated and control group (closed diamonds) had 15 animals (n = 15). EAE animals reached a peak clinical score of 4 or 5 on or around the 11^th day after first immunization and were sacrificed according to approved protocol. NAC treated animals had milder clinical signs (average clinical score of 3 as compared to 4 or 5 for EAE). Control group did not show any clinical symptoms (clinical score = 0).

Figure 2

Inflammation and demyelination in sections of lumbar spinal cord from control, EAE and EAE + NAC (n = 12) treated Lewis rats. The spinal cords were isolated at peak manifestation of the disease (i.e. clinical score 5 in EAE and 3 in EAE + NAC treated animals). Photomicrographs represent regions from a) anterior cord b) central region and c) lateral cord. BV-denotes blood vessel. A. H&E staining of cross-sections of lumbar spinal cord. Compared to the control group, Lewis rats with EAE demonstrated gliosis (single arrow) and perivascular (double arrows), meningeal and interstitial chronic inflammatory infiltrates. These effects were attenuated in sections from EAE+NAC treated animals. B. LFB-PAS staining of cross sections of lumbar spinal cord from control, EAE, and EAE+NAC treated Lewis rats. Compared to the control animals, the interface of normal to demyelinating plaque (arrowhead) is notable in sections from the EAE group of animals. Myelin persists in the plaque as globules in the cytoplasm of macrophages. The EAE+NAC group showed demyelination, but to a lesser degree than that seen in the untreated group.

Figure 3

Quantification of the inflammatory infiltrates by immunostaining of Lewis rat spinal cord (n = 12). Top panel: The spinal cords were isolated when the animals were showing maximum clinical symptoms (i.e. for the EAE group clinical score of 5 and EAE+NAC clinical score of 3). The sections were stained for ED1 (macrophage/monocyte -green) and nuclei labeled with DAPI (blue) as described in materials and methods. Spinal cords of rats induced for EAE demonstrated increased numbers of ED1 positive cells (green) and other glial and inflammatory cells (blue) in the CNS. Original magnification 200×. Bottom panel: Quantification of the infiltrates showed significant numbers of glial and inflammatory cells (A: DAPI; nuclei stained blue) of which many also were positive for macrophage/monocyte (B: ED1; stained green) in the spinal cord of EAE animals as compared to control and EAE + NAC treated animals.

Figure 4

Immunofluorescent detection of IFN-γ, TNF-α, IL-1β, iNOS and nitrotyrosine in the CNS of female Lewis rats. The spinal cords were isolated when the animals were showing maximum clinical symptoms (i.e. for the EAE group clinical score of 5 and EAE+NAC clinical score of 3). Immunostaining was performed in spinal cord sections (n = 12) of female Lewis rats as described in Materials and Methods. EAE sections showed intense staining for IFN-γ, TNF-α, iNOS and nitrotyrosine with less intense staining for IL-1 β. Control and EAE + NAC sections showed minimal staining (original magnification 200×).

Figure 5

IFN-γ, IL-10 and nitrite production by splenocytes from control, EAE and treated animals. Splenocytes (8 × 10⁵ cells per well) were obtained from Control, EAE, and EAE + NAC treated rats when they were showing maximum clinical signs, and were stimulated in vitro with PHA (10 μg/ml, a & b), MBP (20 μg/ml, a & b) or LPS (1 μg/ml, c) for 48 hrs. Each treatment was performed in triplicate for each animal group (n = 6). a and b: The levels (pg/ml) of IFN-γ and IL-10 in culture supernatants were measured using ELISA kits. There was a significant increase in IFN-γ and decrease in IL-10 in splenocytes from untreated EAE animals stimulated with PHA and MBP. EAE + NAC splenocytes showed reduced IFN-γ production whereas IL-10 production was increased. c: Culture supernatants were collected and accumulated nitrite, a stable product of NO production, was measured using Griess reagent. LPS-stimulated EAE splenocytes showed significantly higher levels of nitrite as compared to control and this was reduced with NAC treatment.