CLM-1 is expressed on myeloid cells in CNS inflammatory lesions. (A) CLM-1 mRNA expression in spinal cord after MOG_35-55 immunization. Fold induction is calculated based on day 0 values. Values are expressed as means ± SEM. (B) CLM-1 expression on CD11b⁺CD11c⁺ cells in the spinal cord of naive and MOG_35-55-immunized mice. (C) Clusters of CLM-1⁺ CD11c⁺ myeloid cells in the ventrolateral funiculi and adjacent to leptomeninges (dotted line) of the cervical spinal cord (rectangle enlarged in middle and right). Bar, 10 µm. (D) TNF, iNOS, and CLM-1 expression on CD45^loCD11b⁺-resident microglia (top row) and on CD45^hiCD11b⁺-infiltrating BM-derived cells. Open histograms represent antigen-specific staining and shaded histograms represent isotype control. Results shown are from one experiment out of three, with five (A), three (C), or two (B and D) mice per experimental group.

Lack of CLM-1 on BM-recruited cells or treatment with a CLM-1-Fc fusion protein increases EAE disease severity. (A) CLM-1 protein expression in BMDCs obtained from CLM-1 WT and KO mice. (B) Surface-expression of CLM-1 on CD11c⁺ myeloid cells isolated from inflamed spinal cords of CLM-1 WT and KO mice. Shaded histograms represent isotype control. (C) CLM-1 staining on CD11c⁺ myeloid cells in inflamed spinal cords from WT and KO mice. Bar, 10 µm. (D) Clinical scores in CLM-1 WT and KO mice. (E) Clinical scores in WT mice treated with CLM-1-Fc or control fusion protein. (F and G) Clinical scores and mortality in irradiated chimeras consisting of CLM-1 WT or KO mice reconstituted with BM cells from CLM-1 WT or CLM-1 KO mice. Results shown in A–C are from one of two experiments with two mice per group. Values shown in D–G are means ± SEM from one experiment out of three (D and E) or two (F and G) with 14–18 mice per group. *, P < 0.05; **, P < 0.01. AUC, area under curve.

Lack of CLM-1 results in increased release of myeloid-specific inflammatory mediators and exacerbated demyelination. (A) Production of Th1, Th17, and myeloid cell–specific cytokines by leukocytes isolated from spinal cord 15 d after immunization. (B) CLM-1–positive cells apposed to MOG-positive myelin in the dorsal funiculus of the thoracic spinal cord on day 15 after immunization. (C) MOG and CD11c staining in the dorsal funiculus of the thoracic spinal cord on day15 after immunization. The lines delineate the area of demyelination. (D) Quantification of demyelinated regions in CLM-1 WT and KO mice. (E) Quantification of infiltrating leukocytes in the spinal cords of WT and KO mice 15 d after MOG immunization. Values in E are expressed as means ± SEM of six mice per group. Each symbol represents one individual mouse (A [top row] and D) or a pool of two to three mice (A, bottom row). Experiments were repeated twice (A–D) or three times (E) with at least six mice per group. n.s., not significant. *, P < 0.05; **, P < 0.01. Bars: (B) 10 µm; (C) 50 µm.

CLM-1 expression on irradiation-sensitive myeloid cells infiltrating the CNS and on inflammatory monocytes in the circulation. (A) CLM-1 expression on recipient (CD45.1⁺) or donor (CD45.2⁺)-derived myeloid cells as determined by immunohistochemistry (left) and flow cytometry (right). (B) CLM-1 expression on CX₃CR1^loCD11b⁺ inflammatory monocytes and CX₃CR1^hiCD11b⁺-resident monocytes 7 d after MOG_35-55 immunization. (C) CX₃CR1 and CLM-1 expression in a thoracic spinal cord section 14 d after immunization. CX₃CR1^loCLM-1⁺ cells are localized in the ventromedian funiculi (arrowheads) close to the leptomeninges (dotted line). CX₃CR1^hiCLM-1⁻ cells (arrows) are present throughout the white and gray matter of the ventral horn. Enlarged views of the rectangle show nonoverlapping staining of CLM-1 on a cell with a dendritic-like morphology and high CX3CR1-GFP expression on a ramified microglia cell. (D) CLM-1 expression on CD45^hiCX3CR1^lo BM-derived myeloid cells and CD45^loCX3CR1^hi microglia. Open histograms in A, B, and D represent antigen-specific staining and shaded histograms represent isotype controls. Results shown are from one experiment out of two (A and C) or three (B and D), with four (A) or two (B–D) mice per experimental group. Bars: (A and enlarged views in C) 10 µm; (C) 100 µm.

CLM-1 absence increases disease severity without affecting T cell priming. (A) T cells isolated from DLNs 7 d after immunization were restimulated with MOG_35-55 peptide and proliferation was measured by ³H thymidine incorporation. (B) Quantification of Foxp3-expressing CD4⁺ T cells obtained from DLNs of naive CLM-1 WT and KO mice or 14 d after immunization. (C) Cytokine responses of CD4⁺ T cells obtained from MOG_35-55-immunized mice restimulated with MOG_35-55 peptide ex vivo. (D) Adoptive EAE by transfer of encephalitogenic CD4⁺ T cells isolated from DLNs of CLM-1 WT or KO mice into CLM-1 WT recipients. (E) Adoptive EAE by transfer of encephalitogenic CD4⁺ T cells isolated from DLNs of CLM-1 WT mice into CLM-1 WT or KO recipients. Values are expressed as means ± SEM and experiments have been repeated three times with 6 (A and C), 15–18 (B), 10 (D), and 11 (E) mice per group. n.s., not significant. *, P < 0.05; **, P < 0.01. AUC, area under curve.