Microglial recruitment, activation, and proliferation in response to primary demyelination

Abstract

We have characterized the cellular response to demyelination/remyelination in the central nervous system using the toxin cuprizone, which causes reproducible demyelination in the corpus callosum. Microglia were distinguished from macrophages by relative CD45 expression (CD45(dim)) using flow cytometry. Their expansion occurred rapidly and substantially outnumbered infiltrating macrophages and T cells throughout the course of cuprizone treatment. We used bromodeoxyuridine incorporation and bone marrow chimeras to show that both proliferation and immigration from blood accounted for increased microglial numbers. Microglia adopted an activated phenotype during demyelination, up-regulating major histocompatibility class I and B7.2/CD86. A subpopulation of CD45(dim-high) microglia that expressed reduced levels of CD11b emerged during demyelination. These microglia expressed CD11c and were potent antigen-presenting cells in vitro. T cells were recruited to the demyelinated corpus callosum but did not appear to be activated. Our study highlights the role of microglia as a heterogeneous population of cells in primary demyelination, with the capacity to present antigen, proliferate, and migrate into demyelinated areas.

Figure 1

Cuprizone treatment induces demyelination and increases cellularity in the corpus callosum. Luxol fast blue staining of coronal brain sections from unmanipulated mice (A), 6-week cuprizone-treated mice (B), and mice treated with cuprizone for 6 weeks followed by 3 weeks of recovery (6 + 3* weeks) (C) revealed demyelination in 6-week treated mice and remyelination in recovered animals. D: Quantification of the Luxol fast blue stains showed that animals treated for 3 weeks with cuprizone had incomplete demyelination, whereas after 6 weeks of treatment, demyelination was more complete. Six weeks of cuprizone treatment followed by 3 weeks of recovery showed myelination scores returning to those found in unmanipulated animals. E: Flow cytometry of corpus callosa from mice during cuprizone treatment showed changes in the number of live-gated cells recovered, which peaked at 4.5 weeks. Significance of data was analyzed using a one-way analysis of variance with a Dunnett posthoc test. *P < 0.05, **P < 0.01.

Figure 2

Flow cytometric analysis of CD45- and CD11b-positive cells isolated from corpus callosa. Corpus callosa were microdissected from animals treated with cuprizone at 0, 1, 3, 4.5, and 6 weeks as well as from mice undergoing recovery (6 + 3*). Dissociated corpus callosa were analyzed by FACS to quantify the number of microglia and macrophages. Both microglia (A) and macrophages (B) significantly increased in number during cuprizone treatment compared with the number found in unmanipulated mice. Each data point represents the number of cells from one mouse. Data were analyzed using a one-way analysis of variance with a Dunnett posthoc test. **P < 0.01.

Figure 3

Expression of markers of activation on microglia at 4.5 weeks of cuprizone treatment. Microglia were analyzed for expression of activation markers using flow cytometry at either 0 weeks (gray histograms) or 4.5 weeks (black histograms) after cuprizone treatment. For analysis, samples were gated on CD11b-positive CD45^dim cells for microglia. The mean fluorescence intensity was examined for B7.2/CD86 (A), B7.1/CD80 (B), MHC class I (C), MHC class II (G), and CD40 (H). Isotype controls for each histogram are shown in white. The percentage of microglia expressing each marker was also examined for B7.2/CD86 (D), B7.1/CD80 (E), and MHC class I (F). Significance of data was analyzed using a two-tailed unpaired Student’s t-test. *P < 0.05, ***P < 0.001.

Figure 4

A distinct population of microglia express CD11c. A: Throughout the course of cuprizone treatment, the proportion of CD11c-positive microglia increased in the corpus callosum. These cells formed a distinct cluster defined by CD45 and CD11b staining. FACS plots in A show only the CD45-positive (dim + high) cells. CD11c-positive cells are represented by black dots. B: The percentage of microglia expressing CD11c increased during cuprizone treatment with a maximum at 6 weeks. C: At 6 weeks of treatment, CD11c-positive microglia had lower CD11b levels and higher CD45 levels than CD11c-negative microglia. D: CD11c-positive microglia from cuprizone-treated mice at 4.5 weeks had significantly higher expression of MHC class I, B7.2/CD86, and B7.1/CD80 as compared with CD11c-negative microglia. Significance of data in B was analyzed using a one-way analysis of variance with a Dunnett posthoc test. Other panels were analyzed using a two-tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

CD11c-positive microglia accumulate in the corpus callosum after cuprizone treatment. Immunohistochemistry for CD11c (green) counterstained with Hoechst (blue) in coronal sections showed CD11c-positive cells in 6-week cuprizone-treated mice (A) but not in unmanipulated animals (C). B: Higher magnification of the CD11c-positive cells in the corpus callosum of a cuprizone-treated mouse. Double staining for CD11c (D, green) and CD11b (E, red) showing co-localization (yellow, F). Double staining for laminin (red) to visualize blood vessels (G) and CD11c (green) showed that the CD11c-positive cells are not blood vessel-associated. Original magnifications: ×400 (A, C); ×1000 (B).

Figure 6

Microglia increase in the corpus callosum during cuprizone treatment because of recruitment from the blood as well as proliferation. Flow cytometry was used to enumerate blood-derived (CD45.1-positive) microglia in corpus callosa from bone marrow chimeras treated with cuprizone. A: The percentage of CD45.1-positive microglia in untreated chimeras and in chimeras treated with cuprizone for 3 and 4.5 weeks. B: BrdU treatment of cuprizone-treated mice showed that at 4.5 weeks microglia were proliferating. Corpus callosa from cuprizone-treated mice injected with BrdU for 5 days between either 22 to 27 days or 27 to 32 days were isolated for flow cytometry. The graph shows the number of microglia that had incorporated BrdU at either 4.5 weeks of treatments or 0 weeks. Data were analyzed using a one-way analysis of variance with a Bonferroni posthoc test. **P < 0.01, ***P < 0.001.

Figure 7

CCR2 contributes to the increase in CD11c-positive microglia during cuprizone treatment. Total microglial (A, B) and macrophage (C, D) numbers were assessed in CCR2-deficient mice, CCL2-deficient mice, and wild-type controls by FACS. C57BL/6 mice were used as controls in experiments with CCL2-deficient mice, whereas B6.129 mice (F2) were used to control for genetic background in studies with CCR2-deficient mice. E: CCR2^−/− mice (open circles) had fewer CD11c-positive microglia at 4.5 weeks of cuprizone treatment and after recovery (6 + 3*) compared with B6.129 controls (filled circles) at the same time points. Significance of data at each time point was analyzed using a two-tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01.

Figure 8

Antigen-presenting capacity of CD11c-positive microglia versus CD11c-negative microglia. Proliferative response of MOG-specific T cells to MOG p35-55 presented by FACS-sorted microglial subpopulations was measured by [³H]thymidine incorporation. Microglia were plated in a 1:2 ratio to T cells. Results are shown as mean stimulation index in response to CD11c-positive (white) and CD11c-negative (black) microglial cells from 6-week cuprizone-treated corpus callosum and to CD11c-negative microglia from unmanipulated corpus callosum (black). Significance of differences was analyzed using a one-way analysis of variance with a Bonferroni posthoc test. *P < 0.05, **P < 0.01.

Figure 9

T cells infiltrate the demyelinated corpus callosum. The T-cell response to demyelination and remyelination was measured in 0-, 1-, 3-, 4.5-, and 6-week treated mice as well as from mice treated for 6 weeks with cuprizone followed by 3 weeks of recovery (6 + 3*). T cells were identified as TCRβ-positive CD45^high cells. Data were analyzed using a one-way analysis of variance with a Dunnett posthoc test *P < 0.05, **P < 0.01.