Distinct microglial and macrophage distribution patterns in the concentric and lamellar lesions in Baló's disease and neuromyelitis optica spectrum disorders

Abstract

TMEM119 and purinergic receptor P2Y12 (P2RY12), which are not expressed by recruited peripheral blood macrophages, are proposed to discriminate microglia from macrophages. Therefore, we investigated the distribution patterns of microglia and macrophages in 10 concentric lesions from four autopsied Baló's disease cases and one neuromyelitis optica spectrum disorder (NMOSD) case, using quantitative immunohistochemistry for the markers TMEM119, P2RY12, CD68, CD163 and GLUT5. Three cases with Baló's disease had distal oligodendrogliopathy (DO) showing preferential loss of myelin-associated glycoprotein and early active demyelination in the outermost demyelinating layer (termed DMY-MO). In DMY-MO with DO, TMEM119-positive activated microglia expressing upregulated GLUT5 but markedly downregulated P2RY12 were significantly increased. These activated microglia expressed inducible nitric oxide synthase. Oligodendrocytes and their precursors showed apoptotic-like nuclear condensation in DMY-MO. TMEM119-negative and CD68/CD163-positive macrophages were distributed throughout the lesion center of DMY-MO with DO and these cells demonstrated foamy morphology only in the inner portion but not in the outer portion. In concentric demyelinating lesions from another Baló's case and lamellar demyelinating lesions in an NMOSD case, which had late active demyelination without DO, the densities of TMEM119-, GLUT5- and P2RY12-positive microglia with ramified morphology were significantly increased in myelinated layers but not in demyelinating layers. In particular, in the NMOSD case, TMEM119-positive microglia were confined to the outer portion of the myelinated layers. CD68-positive macrophages with foamy morphology also expressing CD163 accumulated in myelinated as well as in demyelinated layers. These findings suggest that activated microglia expressing TMEM119 and GLUT5, but not P2RY12, are associated with apoptosis of oligodendrocytes in the leading edge of Baló's concentric lesions with DO, whereas TMEM119-, GLUT5- and P2RY12-positive microglia with ramified morphology are associated with myelin preservation in concentric lesions without DO in Baló's disease and NMOSD. These two types of microglia appear to play distinct roles in the formation of concentric lesions.

Keywords: Baló's disease; distal oligodendrogliopathy; macrophage; microglia; neuromyelitis optica spectrum disorders.

Figure 1

Distinct distribution patterns of microglia and macrophages in a Baló's lesion. Case Baló‐2 (A–Q). Macroscopic view of a concentric demyelinating lesion (A–E). KB staining shows a concentric demyelinating lesion in the cerebral white matter (A). The myelin is relatively preserved in the outer‐most demyelinating layer (DMY‐MO) (A, arrowheads). The highest numbers of CD68‐ and CD163‐positive cells are found in layer DMY‐MO (B, C, arrowheads), but are also seen throughout the lesion center (B, C). In contrast, GLUT5 and TMEM119 are confined to cells in DMY‐MO (D, E, arrowheads). Immunostaining for MAG shows numerous foamy macrophages or microglia containing myelin debris in DMY‐MO (F). Higher magnification view of the outer layers of the concentric rings indicated by the square in panel A (G–I). In DMY‐MO, indicated by asterisks in panel G–I, expression of MAG is completely absent, whereas KB and MOG are relatively preserved (distal oligodendrogliopathy; DO) (G–I). Higher magnification view of DMY‐MO, indicated by a square, in panel G (K, N, Q). Panels J, M and P show the inner portion of the DMY‐MO, indicated by the dotted square in panels K, N and Q. The dotted lines show the boundary of DMY‐MO and the outermost myelinated layer (MY‐MO). KB staining shows a reduction in the density of myelin (J). CD68‐positive foamy macrophages and TMEM119‐positive activated microglia are abundant in the inner portion of layer DMY‐MO (M, P). Panels L, O and R show the outer portion of layer DMY‐MO, indicated by the square in panels K, N and Q; arc lines indicate the boundary of the DMY‐MO and the peri‐plaque white matter (PPWM). KB staining shows slightly decreased myelin density in the outer portion of layer DMY‐MO (L), while some CD68‐positive macrophages have infiltrated the lesion, but do not display a foamy morphology (O). TMEM119‐positive microglia are markedly activated in the outer portion of DMY‐MO (R). Scale bars: 1 mm (A–E, G–I), 10 µm (E), 50 µm (J, L, M, O, P and R), 200 µm (K, N and Q).

Figure 2

Accumulation of GLUT5‐positive microglia in a DO lesion in Baló's disease. Case Baló‐1 (A–U). Macroscopic view showing a large whiter matter concentric lesion with KB staining (A). Panel B shows KB staining with a higher magnification view of the area in the square in panel A (B). The outermost demyelinating layer (DMY‐MO) has relative preservation of myelin, indicated by an asterisk (B). Expression of MAG is almost completely absent, while the expression of MOG is preserved in DMY‐MO (C, D). CD68‐ and CD163‐positive macrophages have broadly infiltrated the demyelinating layers (E, F). In contrast, TMEM119‐ and GLUT5‐positive microglia are preferentially distributed in layer DMY‐MO (G, H). Immunoreactivity for P2RY12 is weak at the inner and outer portions of DMY‐MO (I). Higher magnification view of the outer portion of DMY‐MO, indicated by a square in panel H (J–M). Double immunofluorescence labeling for GLUT5 and CD68 showing that GLUT5‐positive microglia are markedly accumulated at the outer portion of DMY‐MO, whereas CD68‐positive macrophages are scarce (J–M). Numerous GLUT5‐positive microglia weakly co‐express CD68 at the lesion center of DMY‐MO (N–Q). In contrast, CD68 expression is upregulated on foamy macrophages, while GLUT5 expression is almost negative in DMY‐1 (R–U). Scale bars: 1 mm (A–I), 100 µm (J–M) and 50 µm (N–U).

Figure 3

Microglia and macrophages in lesions without DO in Baló's disease. Case Baló‐4 (A–Aa). KB staining showing a large concentric lesion in the cerebral white matter (A). MAG and MOG expression levels are diminished to the same extent in each demyelinating layer (B, C). MBP‐positive macrophages or microglia are seen in all demyelinating layers (D). CD68‐positive cells are seen in both demyelinating and myelinated layers, as well as in peri‐plaque white matter (PPWM) (E). TMEM119‐ and P2RY12‐positive microglia are observed in the myelinated layers (F, G). In the PPWM, activated microglia are positive for TMEM119, GLUT5, P2RY12 and CD68, whereas CD163 expression is restricted to perivascular macrophages (H–L). In the outermost demyelinating layer (DMY‐MO), immunoreactivities for TMEM119, GLUT5 and P2RY12 are diminished, while CD68 and CD163 are robustly detected in both parenchymal and perivascular (indicated by a star) macrophages (M–Q). In the outermost myelinated layer (MY‐MO), TMEM119, GLUT5 and P2RY12 are expressed by microglia, and some infiltrated macrophages are positive for CD68 and CD163 (R–V). In MY‐2, TMEM119‐positive microglia are rare (W), while GLUT5 and P2RY12 are expressed by microglia/macrophages with a foamy morphology (X, Y). CD68 and CD163 are expressed by foamy macrophages (Z, Aa). Scale bars: 1 mm (A–C, E–G), 10 µm (D) and 50 µm (H–Aa).

Figure 4

Focal accumulation of TMEM119‐positive microglia in preserved myelin layers of an NMOSD case with lamellar lesions. Lamellar demyelinating lesion from the same case of NMOSD described in Figure S5. CD68‐positive cells have infiltrated both demyelinating and myelinated layers (A, B). TMEM119‐positive cells are confined to the outer portion of each myelinated layer in this lesion (C, arrows). P2RY12‐positive microglia are also abundantly present in myelinated layers and have accumulated to some extent in the outer portions of the myelinated layers (D, arrows). KB staining showing the outermost demyelinating layer (DMY‐MO) and the outermost myelinated layer (MY‐MO) in this lesion (E). CD68‐positive cells have predominantly infiltrated DMY‐MO compared with MY‐MO (E, F). In contrast, TMEM119‐and P2RY12‐positive cells have preferentially accumulated in the MY‐MO (G, H). Higher magnification views showing DMY‐MO (I–K) and MY‐MO (L–N). CD68 is expressed by macrophages with a foamy morphology in DMY‐MO (I), and a few TMEM119‐ and P2RY12‐positive microglia are detected (J, K). CD68‐positive cells are diffusely distributed throughout layer MY‐MO (L), whereas TMEM119‐positive microglia are focally localized in the outer portion of MY‐MO (M). P2RY12‐positive microglia are diffusely seen in MY‐MO (N). Scale bars: 1 mm (A–D), 200 µm (E–H), 100 µm (I–K) and 50 µm (L–N).

Figure 5

Quantitative analysis of microglia and macrophages in the concentric demyelinating lesions. (A–F) Concentric demyelinating lesions with distal oligodendrogliopathy (DO). TMEM119‐ and GLUT5‐positive cells are significantly increased in early active (EA) lesions of the outermost demyelinating layer (DMY‐MO) compared with the normal‐appearing white matter (NAWM) (A, B). Conversely, these cells are significantly decreased compared with NAWM in late active (LA) lesions (A, B). Both demyelinating and myelinated layers show a significant reduction in P2RY12‐positive cells compared with NAWM (C). Numerous CD68‐ and CD163‐positive macrophages are still present in LA lesions (E, F). CD163‐positive cells are also significantly increased compared with NAWM in all myelinated layers with macrophage‐rich inactive (IA) lesions (E). (F) A schematic drawing of microglia/macrophage distribution in concentric lesions with DO. TMEM119‐positive, GLUT5‐positive and P2RY12‐negative activated microglia predominantly accumulate in the outer portion of DMY‐MO. These activated microglia in DMY‐MO with DO features express iNOS. Although CD68/CD163‐positive foamy macrophages are distributed in all demyelinating layers, these cells demonstrated foamy morphology in the inner portion but not in the outer portion. (G–L) Concentric demyelinating lesions without DO. All demyelinating layers, including DMY‐MO, show a significant reduction of TMEM119‐, GLUT5‐ and P2RY12‐positive cells compared with NAWM (G–I). In contrast, TMEM119‐ and GLUT5‐positive cells are significantly increased in MY‐MO compared with NAWM (G, H). CD68‐ and CD163‐positive macrophages are significantly increased throughout the demyelinating and myelinated layers (J, K). (L) In concentric lesions without DO, TMEM119‐positive, GLUT5‐positive and P2RY12‐positive activated microglia with ramified morphology are accumulated in the outer portion of the myelinated layers, while CD68/CD163‐positive macrophages with foamy morphology are distributed among all demyelinating layers. The values for TMEM119, GLUT5, P2RY12, CD68 and CD163 are cells per mm²; thus, the numbers in the y‐axes are directly comparable. *P < 0.05; **P < 0.01; ***P < 0.001.