Role of immune cells in animal models for inherited neuropathies: facts and visions

Abstract

Mice heterozygously deficient in the peripheral myelin adhesion molecule P0 (P0+/- mice) are models for some forms of Charcot-Marie-Tooth (CMT) neuropathies. In addition to the characteristic hallmarks of demyelination, elevated numbers of CD8-positive T-lymphocytes and F4/80-positive macrophages are striking features in the nerves of these mice. These immune cells increase in number with age and progress of demyelination, suggesting that they might be functionally related to myelin damage. In order to investigate the pathogenetic role of lymphocytes, the myelin mutants were cross-bred with recombination activating gene 1 (RAG-1)-deficient mice, which lack mature T- and B-lymphocytes. The immunodeficient myelin mutants showed a less severe myelin degeneration. The beneficial effect of lymphocyte-deficiency was reversible, since demyelination worsened in immunodeficient myelin-mutants when reconstituted with bone marrow from wild-type mice. Ultrastructural analysis revealed macrophages in close apposition to myelin and demyelinated axons. We therefore cross-bred the P0+/- mice with spontaneous osteopetrotic (op) mutants deficient in the macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the corresponding double mutants the numbers of macrophages were not elevated in the peripheral nerves, and the demyelinating phenotype was less severe than in the genuine P0+/- mice, demonstrating that macrophages are also functionally involved in the pathogenesis of genetically mediated demyelination. We also examined other models for inherited neuropathies for a possible involvement of immune cells. We chose mice deficient in the gap junction component connexin 32, a model for the X-linked form of CMT. Similar to P0-deficient mice, T-lymphocytes and macrophages were elevated and macrophages showed a close apposition to degenerating myelin. We conclude that the involvement of T-lymphocytes and macrophages is a common pathogenetic feature in various forms of slowly progressive inherited neuropathies.

Fig. 1

Semi-thin sections of ventral spinal roots from P0+/− /RAG-1−/− mice that received no bone marrow (A), or bone marrow from wild-type mice (B), or bone marrow from RAG-1−/− mice (C). For comparison, ventral spinal roots from genuine P0+/− mice are shown (D). Note that demyelination is mild when no bone marrow (A) or bone marrow from RAG-1−/− mice had been transplanted (C), whereas in P0+/− /RAG-1−/− mutants that had received bone marrow from wt mice (B) demyelination is similarly severe as in genuine P0+/− mice (D). Bv, blood vessel. Scale bar in D (for A-D): 20 μm.

Fig. 2

(A,B) Immunohistological localization of macrophages in femoral quadriceps nerves of P0+/− (A) and P0+/+ mice (B) at the age of 6 months using antibodies to F4/80. In quadriceps nerves of P0+/− mice the number of macrophages is clearly elevated when compared to P0+/+ mice. Note the larger size of the cells and the close vicinity of two cells to an endoneurial blood vessel in the nerve of the mutant (A). (C,D) Immunoelectron microscopic localization of F4/80-positive macrophages in peripheral nerves of 6-month-old P0+/− mice. (C) An F4/80-positive macrophage (M), containing myelin debris, is in close apposition to a demyelinated axon. Arrows indicate electron-dense immunoreaction product. Axon (Ax), Schwann cell (S). (D) A slender, immunoreactive process (arrows) of an F4/80-positive macrophage (M) has penetrated in between the pericaryon of a Schwann cell (S) and its normal appearing myelin sheath (My). Scale bars: 20 μm (for A and B); 1.5 μm (for C and D).

Fig. 3

(A,B) Electron microscopy of ventral roots of 6-month-old P0+/− op/wt (heterozygous osteopetrotic (op) mice with wildtype-like M-CSF expression; A) and of P0+/− op/op littermates (homozygous op mice which are M-CSF-deficient; B). In many fibres of P0+/− op/wt mice, there is profound demyelination when compared to P0+/− op/op littermates deficient in M-CSF. Scale bars: 5 μm.

Fig. 4

Cellular localization of the M-CSF receptor (MCSFR, red) immunoreactivity in teased fibre preparations from ventral roots of P0+/+ and P0+/− mice using antibodies to αMβ2 integrin (green) as a marker for peripheral nerve macrophages. αMβ2-negative cells, such as the adjacent Schwann cells, were never labelled. Note the particularly strongly labelled M-CSFR-immunoreactive macrophage in the P0+/− mutant (arrow). Scale bar: 50 μm.

Fig. 5

Immunoelectron microscopy of F4/80-positive macrophages in ventral roots of 6-month-old Cx32−/− mice. (A) A demyelinated axon (Ax) is in close contact to an F4/80-positive macrophage. Arrows demarcate F4/80-related immunoprecipitate. Note that the surface and the basement membrane of the Schwann cell (S; arrowheads) and the axon-Schwann cell interface are not labelled. (B) An F4/80-positive macrophage (M) has penetrated the Schwann cell basal lamina and contacts poorly preserved and partially vacuolized myelin (My). Note well preserved myelin of the fibres not in contact with the macrophage. Arrows demarcate F4/80-related immunoprecipitate. Ax, myelinated axon; S, F4/80-negative Schwann cell. Scale bars: 1 μm.

Fig. 6

Hypothetical sequence of events in peripheral nerves of mice with inherited demyelination leading to superimposed immune attack. 1, pathogenesis starts with myelin instability due to myelin-related mutations in Schwann cell genes. Schwann cells react to myelin instability by unknown signals and secrete chemokines/cytokines. 2, Schwann cell-derived chemokine secretion may chemotactically attract monocytes/macrophages, in part by activating adjacent endothelial cells, and lead to proliferation of resident macrophages (Mö). As a novel player, endoneurial fibroblasts (F) may be also activated by the Schwann cell-derived chemokine secretion. 3, macrophages may interact with endoneurial fibroblasts (F) and become activated by fibroblast-derived M-CSF. 4, activated macrophages present antigens and/or secrete cytokines that attract and activate T-lymphocytes (T) which in turn activate more macrophages and T-cells. 5, macrophages penetrate Schwann cell basal laminae (Sc-BM), phagocytose myelin and secrete gliotoxic factors.