Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis

Abstract

Recent findings in the animal model for multiple sclerosis (MS), experimental autoimmune encephalomyelitis, implicate a novel CD4+ T-cell subset (TH17), characterized by the secretion of interleukin-17 (IL-17), in disease pathogenesis. To elucidate its role in MS, brain tissues from patients with MS were compared to controls. We detected expression of IL-17 mRNA (by in situ hybridization) and protein (by immunohistochemistry) in perivascular lymphocytes as well as in astrocytes and oligodendrocytes located in the active areas of MS lesions. Further, we found a significant increase in the number of IL-17+ T cells in active rather than inactive areas of MS lesions. Specifically, double immunofluorescence showed that IL-17 immunoreactivity was detected in 79% of T cells in acute lesions, 73% in active areas of chronic active lesions, but in only 17% of those in inactive lesions and 7% in lymph node control tissue. CD8+, as well as CD4+, T cells were equally immunostained for IL-17 in MS tissues. Interestingly, and in contrast to lymph node T cells, no perivascular T cells showed FoxP3 expression, a marker of regulatory T cells, at any stage of MS lesions. These observations suggest an enrichment of both IL-17+CD4+ and CD8+ T cells in active MS lesions as well as an important role for IL-17 in MS pathogenesis, with some remarkable differences from the experimental autoimmune encephalomyelitis model.

Figure 1

IL-17 mRNA expression in chronic active MS lesions. A: Low magnification with light microscopy of IL-17 mRNA expression in a chronic active lesion visualized by in situ hybridization with a specific probe for IL-17. Dotted line denotes the edge of an active border. B–D: High-magnification images of IL-17 mRNA expression in the active area of MS lesions with perivascular cells consistent with T cells [arrowhead in B and in high magnification (see inset)], parenchymal cells with astrocytic morphology (arrowhead in C) and cells with characteristic oligodendrocyte sizes and locations (arrowheads in D), all showing evidence of IL-17 mRNA expression. E: Negative control using the sense IL-17 riboprobe on a consecutive section counterstained with toluene blue (arrow in E). The figure shows that IL-17⁺ cells are located predominantly at the active border of a chronic active lesion (right side of dotted line in A), and that T cells (B), astrocytes (C), and oligodendrocytes (D) express IL-17 mRNA. Scale bars = 30 μm.

Figure 2

IL-17 production in inflammatory and glial cells in active areas of MS lesions. Double-immunofluorescence staining analyzed by confocal microscopy. Staining for IL-17 (A, D, G, and J; green), CD3 (B; for T cells, red), MHC class II (E; macrophages/microglia, red), GFAP (H; astrocytes, red), or CAII (K; oligodendrocytes, red) taken from active areas of an MS lesion. Overlays demonstrate expression of IL-17 in T cells (C; arrowhead demonstrates IL-17⁺ T cells, arrows demonstrate T cells negative for IL-17), astrocytes (I; arrowheads), and in oligodendrocytes (L; arrowheads), but not in macrophages/microglia, which are IL-17-negative (F; arrows). Scale bars = 10 μm.

Figure 3

IL-17 immunohistochemistry in perivascular cuffs from lesions with different activity. Light microscopic analysis of A, C, E, G, I, and K: CD3 immunostaining on consecutive sections. B, D, F, and H: IL-17 immunostaining in perivascular cells in MS lesions of different activity. A and B: Acute lesion. C and D: Active border of a chronic active lesion. E and F: Inactive center of a chronic active lesion. B, D, and F: The fibrillary staining beyond the perivascular spaces represents immunostaining for IL-17 in the processes of astrocytes. G and H: An inactive lesion is shown. I and J: CD3 and IL-17 immunostaining in NAWM from a case of MS. K and L: CD3 and IL-17 immunostaining in control brain. The figure shows more IL-17⁺ perivascular cells in the active (B and D) than the inactive (F and H) areas of the lesions. Arrowheads in B and D indicate abundance of IL-17 cells, whereas J and L indicate sparse IL-17⁺ cells. All sections were counterstained with hematoxylin. Scale bar = 30 μm.

Figure 4

Density of IL-17⁺ T cells in MS lesions of different activity. Quantitative analysis of perivascular CD3⁺ (A) and IL-17⁺ (B) T-cell density in areas of MS lesions with different activity, in NAWM, and in control brain. Histograms demonstrate a significantly higher density of T cells (A) and IL-17⁺ T cells (B) within perivascular active areas (acute lesions/active border of chronic active lesions) than in inactive lesions/inactive areas of chronic active lesions or NAWM and control tissue (n = 4). In contrast, there is no significant difference in the density of IL-17⁺ T cells within inactive areas and control tissue. The proportion of T cells that are IL-17⁺ was significantly higher in active than inactive areas of chronic active lesions (P = 0.0005).

Figure 5

IL-17⁺ in CD4⁺ and in CD8⁺ T cells. Confocal microscopy, high-magnification image from an inflammatory cuff, demonstrating a single cell. A and D: IL-17 staining (green); B: CD4 staining (red); E: CD8 staining (red). C and F: Overlays show production of IL-17 in CD4⁺ T cells (arrowhead in C, yellow) and in CD8⁺ T cells (arrowhead in F, yellow). G: Bars demonstrate higher frequency of IL-17⁺ both in CD4⁺ and CD8⁺ T-cell subsets within active lesions and in active borders of chronic active lesions. In contrast, in inactive areas of inactive and chronic active lesions there is a very low frequency of IL-17⁺ CD4⁺ (P = 0.002) and CD8⁺ (P = 0.03) T cells. Scale bar = 10 μm.

Figure 6

FoxP3⁺ T cells are absent in MS lesions. Double-immunofluorescent staining, analyzed by confocal microscopy. A, D, G, and J: staining for CD3 (red) and B, E, H, and K: FoxP3 (blue). C, F, and I: Overlays demonstrate that FoxP3 is not expressed in CD3⁺ cells in perivascular cuffs from acute (C) and inactive (F) MS lesions and control brain (I). L: Overlay demonstrates in lymph nodes that some T cells express FoxP3. M: IL-17⁺ (green) and N: FoxP3⁺ (blue) cells. O: Overlay demonstrates absence of FoxP3 expression from IL-17⁺ cells (arrowheads demonstrate IL-17⁺ cells and arrows demonstrate FoxP3⁺ cells). Scale bar = 10 μm.