Immune cells contribute to myelin degeneration and axonopathic changes in mice overexpressing proteolipid protein in oligodendrocytes

Abstract

Overexpression of the major myelin protein of the CNS, proteolipid protein (PLP), leads to late-onset degeneration of myelin and pathological changes in axons. Based on the observation that in white matter tracts of these mutants both CD8+ T-lymphocytes and CD11b+ macrophage-like cells are numerically elevated, we tested the hypothesis that these cells are pathologically involved in the primarily genetically caused neuropathy. Using flow cytometry of mutant brains, CD8+ cells could be identified as activated effector cells, and confocal microscopy revealed a close association of the T-cells with MHC-I+ (major histocompatibility complex class I positive) oligodendrocytes. Crossbreeding the myelin mutants with mice deficient in the recombination activating gene-1 (RAG-1) lacking mature T- and B-lymphocytes led to a reduction of the number of CD11b+ cells and to a substantial alleviation of pathological changes. In accordance with these findings, magnetic resonance imaging revealed less ventricular enlargement in the double mutants, partially because of more preserved corpora callosa. To investigate the role of CD8+ versus CD4+ T-lymphocytes, we reconstituted the myelin-RAG-1 double mutants with bone marrow from either CD8-negative (CD4+) or CD4-negative (CD8+) mice. The severe ventricular enlargement was only found when the double mutants were reconstituted with bone marrow from CD8+ mice, suggesting that the CD8+ lymphocytes play a critical role in the immune-related component of myelin degeneration in the mutants. These findings provide strong evidence that a primary glial damage can cause secondary immune reactions of pathological significance as it has been suggested for some forms of multiple sclerosis and other leukodystrophies.

Figure 1.

Elevated numbers of CD8+ T-lymphocytes in the CNS of PLP mutants. A, Quantification of CD8+ T-lymphocytes in corpus callosum (CC), cerebellum (Cb), white matter of the spinal cord (SCw), and gray matter of the spinal cord (SCg) and in the optic nerve (ON) of wt and PLP mutants (PLP-tg) at 2, 4, 8, and 12 months (mo) of age. Note age-dependent increase of the T-lymphocytes in the different regions of the CNS of the mutants. Mean values + SEM are indicated as well as the number of individuals investigated (n). Asterisks indicate statistical significance between values from the mutants compared with the values from wild-type mice: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. B–D, Double-immunofluorescence staining for CD8+ cells (B, green color) and CD3+ T-lymphocytes (C, red color) in the corpus callosum of a PLP-tg mouse at 12 months of age. Note that all CD8+ cells are also CD3+, proving that the infiltrating CD8+ cells are T-lymphocytes (D, arrows). Scale bar, 50 μm.

Figure 2.

CD3+ T-lymphocytes directly contact MHC-I+ profiles that resemble oligodendrocytic cell somata. Confocal microscopic images of triple-immunohistochemical stainings for CD3 (A), MHC-I (B), and pan-neurofilament (C, green in merged image). Note the direct contact of MHC-I+ profiles with CD3+ T-lymphocytes (C, arrowheads). The MHC-I+ profiles are not identical with neurofilament+ structures (C, arrows) but are closely associated with them being highly reminiscent of oligodendrocytic cell somata. Scale bar, 10 μm.

Figure 3.

Flow cytometry of spleen and brain lymphocytes from PLP-tg mice. Spleen and brain lymphocytes were stained for CD8, CD62L and either CD44 (A) or CD69 (B). Only CD8-positive cells were gated and analyzed for CD44 (effector cell marker), CD69 (effector cell marker), and CD62L (marker for immature lymphocytes in this context) expression. Left blots, Isotype control. Right blots, CD44, CD69, and CD62L staining. Percentages of cells per quadrant are provided in each blot.

Figure 4.

Elevated numbers of macrophage-like cells in the CNS of PLP mutants. Quantification of CD11b+ macrophages in corpus callosum (CC), cerebellum (Cb), white matter of the spinal cord (SCw), and gray matter of the spinal cord (SCg) and in the optic nerve (ON) of wt and PLP mutants (PLP-tg) at 2, 4, 8, and 12 months (mo) of age. Note the age-dependent increase of the macrophages in the different regions of the CNS of the mutants. Mean values + SEM are indicated as well as the number of individuals investigated (n). Asterisks indicate statistical significance between values from the mutants in comparison to the values from wild-type mice: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 5.

Macrophage-like cells in the optic nerves of wild-type and PLP mutants. A–D, Longitudinal, consecutive frozen sections of retinas (left) and optic nerves from wt mice (A, B) and PLP mutants (PLP-tg; C, D) immunolabeled with antibodies to CNPase (A, C) and with antibodies to the macrophage-specific marker CD11b (B, D). Note that particularly in the rostral part of the mutant optic nerve, there is a substantially increased number of CD11b+ cells compared with the optic nerves of the wild-type mice. In this region, myelin damage is particularly prominent, as reflected by a more patchy CNPase labeling. Arrows in A and C mark the unmyelinated part of the optic nerve, which is clearly confined by myelin labeling in the wild-type mice but diffusely in the mutants. Labeling of meninges and connective tissue of the eye cup in A and C is attributable to unspecific binding of the corresponding secondary antibody. The black appearance associated with the eye cup in all micrographs corresponds to the pigment epithelium. Scale bar, 200 μm.

Figure 6.

Histopathological changes in optic nerves of PLP mutants. Semithin sections of optic nerves (A) of immune-competent (RAG-1+/?) and of immune-deficient PLP mutants (RAG-1−/−) and quantification of macrophages (B) in optic nerves of wt, PLP-tg RAG-1+/?, and PLP-tg RAG-1−/− mutants at 12 months of age. Note the reduced number of periaxonal vacuoles (A) and macrophages (B) in RAG-1-deficient PLP mutants. ∗ p < 0.05. n, Number of individuals investigated. Scale bar, 50 μm.

Figure 7.

Pathological changes in optic nerves of PLP mutants. A–C, Electron microscopy of mutant optic nerves in the rostral (A, C) and in the caudal region (B). Note that demyelination and axonopathic changes are more pronounced in the rostral region, as reflected by many demyelinated axons (A, arrowheads), thin myelin, and periaxonal vacuoles (asterisks). C, Periaxonal vacuoles often contain the remnants of degenerating axons and are often in close vicinity of macrophage-like cells containing myelin debris (M). D–F, Electron microscopy of representative wt (D), PLP-tg RAG-1+/? (E), and PLP-tg RAG-1−/− (F) optic nerves from the rostral region. PLP-tg RAG-1−/− mice (F) show fewer demyelinating/axonopathic profiles (white arrows) than PLP-tg RAG-1+/? mutants (E). Wild-type optic nerves (D) do not show pathological changes. Scale bars: A–C, 5 μm; D–F, 2 μm.

Figure 8.

Ventricular sizes in wt mice and PLP mutants. A–D, MRI scans of brains from wt mice (A, B) and from PLP mutants (C, D). Note the abnormally extended ventricular sizes of the mutants, with sizes 3 (C) and 4 (D). Arrows, Lateral ventricles; arrowheads, third ventricle. E, Ventricular sizes of wt mice (black bars), of immune-competent PLP mutants (PLP-tg/RAG-1+/?; stippled white bars), and of immune-deficient PLP mutants (PLP-tg RAG-1−/−; gray bars) at 2–12 months of age. Ventricular sizes of immune-deficient PLP mutants reconstituted with bone marrow (BM) from wt, CD8+ (CD4−/−), and CD4+ (CD8−/−) mice are also indicated at 6 months of age. Note the enlarged ventricular sizes exceeding the normal size 2 of PLP mutants with intact immune systems and of immune-deficient PLP mutants reconstituted with bone marrow from wild-type mice or CD8+ (CD4−/−) mutants but not when reconstituted with bone marrow from CD4+ (CD8−/−) mutants. As a control experiment, reconstitution with wt BM (comprising CD8+/CD4+ stem cells) into wt mice also fails to increase ventricular sizes (score of 2 in 3 of 3 animals). Error bars indicate mean values + SEMs. The asterisks on the left side indicate a significant enlargement of ventricles in the PLP mutants with intact immune system (RAG-1+/?) with increasing age (p < 0.05, Kruskal–Wallis test), whereas asterisks on the right side (bone marrow chimera) indicate significant enlargement of ventricles [p < 0.05 compared with CD4+ (CD8−/−)] in bone marrow reconstituted immune-deficient mutants.