Chemokine-like receptor-1 expression by central nervous system-infiltrating leukocytes and involvement in a model of autoimmune demyelinating disease

Abstract

We examined the involvement of chemokine-like receptor-1 (CMKLR1) in experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis. Upon EAE induction by active immunization with myelin oligodendrocyte glycoprotein amino acids 35-55 (MOG(35-55)), microglial cells and CNS-infiltrating myeloid dendritic cells expressed CMKLR1, as determined by flow cytometric analysis. In addition, chemerin, a natural ligand for CMKLR1, was up-regulated in the CNS of mice with EAE. We found that CMKLR1-deficient (CMKLR1 knockout (KO)) mice develop less severe clinical and histologic disease than their wild-type (WT) counterparts. CMKLR1 KO lymphocytes proliferate and produce proinflammatory cytokines in vitro, yet MOG(35-55)-reactive CMKLR1 KO lymphocytes are deficient in their ability to induce EAE by adoptive transfer to WT or CMKLR1 KO recipients. Moreover, CMKLR1 KO recipients fail to fully support EAE induction by transferred MOG-reactive WT lymphocytes. The results imply involvement of CMKLR1 in both the induction and effector phases of disease. We conclude that CMKLR1 participates in the inflammatory mechanisms of EAE and represents a potential therapeutic target in multiple sclerosis.

FIGURE 1

Reduced clinical EAE in CMKLR1 KO mice. EAE was induced by active immunization and mice were monitored daily for clinical disease as described in Materials and Methods. Data are pooled from five independent experiments, each consisting of 4–10 mice per group, and are presented as mean clinical score ± SEM vs time. *, p < 0.05, as determined by Mann-Whitney U test.

FIGURE 2

Reduced histological EAE in CMKLR1 KO mice. Representative spinal cord sections are shown from actively immunized WT (left panels) or CMKLR1 KO mice (right panels) that were killed at 13 (A and B) or 46 (C and D) days p.i. A, Meningeal and parenchymal mononuclear cell infiltrates typical of acute EAE in the spinal cord of a WT mouse sacrificed on day 13 p.i. B, Less meningeal infiltration in the spinal cord of CMKLR1 KO mouse with EAE at day 13 p.i. C, Typical meningeal and parenchymal mononuclear cell infiltrates in the spinal cord of a WT mouse with chronic EAE. D, Meningitis and mild parenchymal inflammation are present in the spinal cord of a CMKLR1 KO mouse sacrificed at day 46 p.i. Sections from paraffin-embedded WT (E) and CMKLR1 KO (F) brain and spinal cord tissue harvested at day 46 p.i. were subjected to immunostaining with anti-F4/80 mAb. Reactions were developed with diaminobenzidine chromogen and counter-stained with hematoxylin. White arrows highlight microglia, black arrows indicate foamy macrophages. Magnification = 160×. Bar = 50 µm.

FIGURE 3

Recall proliferation and cytokine responses of lymphocytes from CMKLR1 KO mice. Mice were immunized with MOG_35–55 emulsified in CFA. After 10 days, spleen cells and draining LN cells were restimulated in vitro with the indicated concentrations of MOG_35–55. A, After 72 h of stimulation, proliferation of LN (left panel) or spleen cells (right panel) was assessed by [³H]thymidine incorporation assay. Data are presented as a stimulation index (mean cpm with Ag ÷ mean cpm without Ag); bars represent SEM. B, Culture supernatants were collected after 72 h and levels of the indicated cytokines produced by LN (left panels) or spleen cells (right panels) were measured by ELISA. *, p < 0.05, as determined by Student’s t test. Data shown are representative of two independent experiments with similar results.

FIGURE 4

Induction of EAE by adoptive transfer of MOG-reactive lymphocytes. EAE was induced in WT or CMKLR1 KO mice by passive transfer of WT or CMKLR1 KO MOG_35–55-reactive lymphocytes as described in Materials and Methods. Data shown are pooled from adoptive transfers that were performed at various times (eight independent experiments, n = 1–4 recipient mice per group). Values are presented as mean clinical score vs time.

FIGURE 5

Detection of CMKLR1⁺ cells in CNS of mice with EAE. Mononuclear cells were isolated from the spinal cords of mice with acute EAE as described in Materials and Methods. Cells were stained with anti-mouse CMKLR1 mAb BZ186 or DREG200 isotype control mAb, followed by incubation with PE-conjugated anti-mouse IgGl. Lastly, mAbs directly conjugated to cell surface Ags were added and cells were analyzed by flow cytometry. For all histograms, the red line represents isotype control mAb staining. A, CD3⁻CD19⁻CD11b⁺CD45^low microglia were analyzed for expression of CMKLR1. B, Cells were gated as CD45^high and cells in the CD3⁻CD19⁻ gate were analyzed for expression of CD11b and CD11c. Leukocytes were gated on macrophages (R1), pDC (R2), or mDC (R3), which were then analyzed for expression of CMKLR1. C, A subset of CD11c⁻CD11b⁺ macrophages (R1 gate) expresses CMKLR1 (indicated by the arrow). D, CD11c^intCD11b⁻B220⁺ pDC (R2 gate) are CMKLR1-negative. E, CD11c^highCD11b⁺B220⁻ mDC (R3 gate) express CMKLR1. CNS cells were pooled from three mice for analysis; data shown are representative of three independent experiments with similar results. FSC, forward scatter.