Circulating Ly-6C+ myeloid precursors migrate to the CNS and play a pathogenic role during autoimmune demyelinating disease

Abstract

Mature myeloid cells (macrophages and CD11b(+) dendritic cells) form a prominent component of neuroinflammatory infiltrates in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). The mechanism by which these cells are replenished during relapsing and chronic neuroinflammation is poorly understood. Here we demonstrate that CD11b(+)CD62L(+)Ly6C(hi) monocytes with colony-forming potential are mobilized into the bloodstream by a granulocyte-macrophage colony-stimulating factor-dependent pathway immediately before EAE relapses. Circulating Ly6C(hi) monocytes traffic across the blood-brain barrier, up-regulate proinflammatory molecules, and differentiate into central nervous system dendritic cells and macrophages. Enrichment of Ly6C(hi) monocytes in the circulating pool is associated with an earlier onset and increased severity of clinical EAE. Our studies indicate that granulocyte-macrophage colony-stimulating factor-driven release of Ly6C(hi) precursors from the bone marrow prevents exhaustion of central nervous system myeloid populations during relapsing or chronic autoimmune demyelination, suggesting a novel pathway for therapeutic targeting.

Figure 1

Myeloid precursor cells expand in the blood before clinical episodes of EAE and are contained within the CD11b⁺Ly-6C^hi population. (A) SJL mice were immunized with PLP_139-151 in CFA. Mice were bled at various time points during relapsing-remitting EAE, and peripheral blood leukocytes were plated in methylcellulose cultures supplemented with GM-CSF and stem cell factor. GM-CFUs were counted on day 7. The data shown represent the mean (± SD) of 3 similar experiments. The frequency of GM-CFU at each time point represents the mean of at least 3 mice/group (*P < .05 comparing the frequency of GM-CFU before onset of EAE with their frequency during disease). (B) C57/B6 mice were immunized with MOG/CFA. Six days later, peripheral blood cells were stained for CD11b and Ly-6C and sorted into Ly-6C^hi, Ly-6C^int, and Ly-6⁻ subsets. (C) Sorted cells were plated in methylcellulose cultures as described in panel A, and GM-CFUs were counted on day 7. The data represent mean (± SE) of five experiments. (D) Hematoxylin and eosin staining of sorted CD11b⁺Ly-6C^hi cells. (E) CD115, CD62L, and Ly6G levels were measured on gated CD11b⁺Ly-6C^hi cells by flow cytometric analysis. Broken lines represent isotype controls. (B-E) Data are representative of at least 2 experiments.

Figure 2

Ly-6C⁺ cells accumulate in the blood and CNS before the onset of EAE. (A) PBMCs from naive and PLP_139-151-immunized SJL mice were analyzed by flow cytometry. Immunized mice were bled on the day before expected clinical onset and during peak EAE. Dot plots are gated on CD11b⁺CD115⁺ cells; percentages of each subset among total blood leukocytes are indicated. (B) Absolute numbers of circulating monocytes per milliliter of blood in naive and PLP_139-151-immunized SJL mice (*P < .05, **P < .02 by comparison to naive). (C) Ly-6C⁺ monocytes were sorted from immunized SJL mice and analyzed for MHC class II and CD11c expression either immediately ex vivo or after a 48-hour culture with GM-CSF. Data shown are representative of 5 separate experiments. (D) Spinal cord mononuclear cells were harvested from naive and PLP_139-151-immunized SJL mice and subjected to flow cytometric analysis. The lowest panels show CD11c and MHC class II staining of CD11b⁺LyC^hi-gated PBMCs from mice in the preclinical (left) or symptomatic (right) stages of EAE. The gates used are depicted in the middle panels. Data shown are representative of 4 separate experiments.

Figure 3

Ly-6C⁺ monocytes migrate to the CNS during EAE and up-regulate CD11c and MHC class II. Mice were treated with clodronate or PBS liposomes 24 hours after transfer of encephalitogenic T cells. Eighteen hours after liposome treatment, animals were injected with FITC-labeled microspheres. (A) Left: Expression of FITC and CD11b by CD115⁺-gated cells 5 days after liposome treatment and 4 days after FITC microsphere injection. Right: Ly-6C expression on FITC⁺CD115⁺ blood monocytes from clodronate versus PBS liposome-treated animals. Histograms are based on cells that fall within the R1 gate (left panel). (B) Spinal cord mononuclear cells were harvested from mice treated with clodronate (left) or PBS liposome (right) during peak EAE and analyzed for FITC expression. (C) The cell surface phenotype of CNS-infiltrating FITC⁺ cells (shown in the gate of panel B left) was determined by FACS. (D) CD11b⁺MHC class II⁺Ly-6C⁺ cells were sorted from the blood and CNS of mice with EAE and analyzed by real-time RT-PCR for the genes shown. The data are shown as fold expression in CNS-infiltrating cells over circulating cells. (A-D) All experiments shown were repeated 3 times with similar results. *P < .05, comparing mRNA levels in CNS versus blood Ly-6C⁺ monocytes.

Figure 4

Enrichment of Ly-6C⁺ cells in the circulating monocyte pool enhances EAE. (A) The absolute number of total monocytes and Ly6C⁺ monocytes per milliliter of blood in adoptive transfer recipients at serial time points after treatment with clodronate liposomes. (B) The absolute number of circulating Ly-6C⁺ monocytes per milliliter of blood 5 days after treatment with either clodronate or PBS liposomes. Mean (± SD) of 5 independent experiments; **P < .01 comparing frequency of Ly-6C⁺ monocytes in PBS versus clodronate treated mice. (C) Clinical course of mice treated with a single dose of clodronate or PBS liposomes 18 hours after the adoptive transfer of encephalitogenic cells. Mice in a third group received clodronate liposomes on days 8, 10, and 12 after T-cell transfer. Data shown represent the results from 3 separate experiments. Mean (± SD) of 3 independent experiments; P < .05 by comparison to PBS treated group.

Figure 5

GM-CSF triggers accelerated myelopoiesis during EAE. (A) CD11b⁺CD115⁺Ly-6C⁺ or Ly-6C⁻ blood cells (left) were enumerated 7 days after immunization of C57BL/6 WT and GM-CSF^−/− mice with MOG peptide in CFA. The data are presented as the fold increase in each subset over their frequency in unimmunized counterparts. Frequencies of circulating Ly-6C⁺ and Ly-6C⁻ monocytes did not differ significantly between unimmunized WT and unimmunized GM-CSF^−/− mice. Clinical scores (right) of MOG-immunized WT and GM-CSF^−/− mice. (B) Left: The number of circulating Ly-6C⁺ monocytes/mL of blood in anti–GM-CSF versus control antibody-treated WT mice on day 7 after immunization with MOG peptide. Mean (± SD) of 4 independent experiments; *P < .05 comparing frequency of Ly-6C⁺ monocytes in rat IgG versus αGM-CSF treated mice. Right: Clinical scores of MOG-immunized WT mice treated with either control antibody or anti–GM-CSF across a range of doses. (C) Left: Frequency of Ly6C⁺ blood monocytes on day 8 after active immunization of WT or GM-CSF^−/− mice. Some GM-CSF^−/− mice received 5 μg of recombinant mGM-CSF 8 hours before phlebotomy. Mean (± SD) of 3 independent experiments; *P < .05, **P < .01 by comparison to GM^−/− mice. Right: Clinical scores of MOG-immunized WT and GM-CSF^−/− mice. Some GM-CSF^−/− mice received 5 μg of rmGM-CSF every day from days 0 to 16 after immunization.