Temporal expression and cellular origin of CC chemokine receptors CCR1, CCR2 and CCR5 in the central nervous system: insight into mechanisms of MOG-induced EAE

Abstract

Background: The CC chemokine receptors CCR1, CCR2 and CCR5 are critical for the recruitment of mononuclear phagocytes to the central nervous system (CNS) in multiple sclerosis (MS) and other neuroinflammatory diseases. Mononuclear phagocytes are effector cells capable of phagocytosing myelin and damaging axons. In this study, we characterize the regional, temporal and cellular expression of CCR1, CCR2 and CCR5 mRNA in the spinal cord of rats with myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE). While resembling human MS, this animal model allows unique access to CNS-tissue from various time-points of relapsing neuroinflammation and from various lesional stages: early active, late active, and inactive completely demyelinated lesions.

Methods: The expression of CCR1, CCR2 and CCR5 mRNA was studied with in situ hybridization using radio labelled cRNA probes in combination with immunohistochemical staining for phenotypic cell markers. Spinal cord sections from healthy rats and rats with MOG-EAE (acute phase, remission phase, relapse phase) were analysed. In defined lesion stages, the number of cells expressing CCR1, CCR2 and CCR5 mRNA was determined. Data were statistically analysed by the nonparametric Mann-Whitney U test.

Results: In MOG-EAE rats, extensive up-regulation of CCR1 and CCR5 mRNA, and moderate up-regulation of CCR2 mRNA, was found in the spinal cord during episodes of active inflammation and demyelination. Double staining with phenotypic cell markers identified the chemokine receptor mRNA-expressing cells as macrophages/microglia. Expression of all three receptors was substantially reduced during clinical remission, coinciding with diminished inflammation and demyelination in the spinal cord. Healthy control rats did not show any detectable expression of CCR1, CCR2 or CCR5 mRNA in the spinal cord.

Conclusion: Our results demonstrate that the acute and chronic-relapsing phases of MOG-EAE are associated with distinct expression of CCR1, CCR2, and CCR5 mRNA by cells of the macrophage/microglia lineage within the CNS lesions. These data support the notion that CCR1, CCR2 and CCR5 mediate recruitment of both infiltrating macrophages and resident microglia to sites of CNS inflammation. Detailed knowledge of expression patterns is crucial for the understanding of therapeutic modulation and the validation of CCR1, CCR2 and CCR5 as feasible targets for therapeutic intervention in MS.

Figure 1

Sampling of rats from various clinical stages of MOG-EAE. Mean clinical score in female DA rats (n = 30), evaluated daily 8–29 days after immunization with 20 μg recombinant rat MOG in incomplete Freund's adjuvant. The arrows indicate selected time-points at which subsequent kinetic analyses were performed. Rats (n = 3/time-point) which conformed in the clinical score curve were chosen for histopathology and evaluation of CCR1, CCR2 and CCR5 mRNA expression in the spinal cord. Vertical bars represent mean and standard error of the mean.

Figure 2

Distribution of CCR1, CCR2, CCR5 mRNA expressing cells at different time points in the spinal cord of MOG-EAE rats. In situ hybridization with ³⁵S-labelled antisense cRNA probes encoding rat CCR1, CCR2 and CCR5 to coronal sections from the lumbar segment of spinal cord of rats with MOG-EAE. Cells expressing CCR1, CCR2 and CCR5 mRNA are visualized by dark field illumination of the photo emulsion-dipped slides. Intensive signals for CCR1 and CCR5 mRNA, and moderate signals for CCR2 mRNA, were detected on days 13 (B, E, H) and 24 (C, F, I) post immunization. No signal for CCR1, CCR2 or CCR5 mRNA was detected in healthy control animals (A, D, G). No signal was detected in control sections hybridized with sense probe (not shown).

Figure 3

Histopathological features of MOG-EAE during the acute and remission stages. Spinal cord sections from a rat in the acute stage (day 13 post immunization) of EAE show extensive inflammation involving the white and grey matter (D), with marked demyelination in the inflammatory areas (F). The majority of the infiltrating inflammatory cells are macrophages, as evidenced by positive staining for the ED-1 marker (E). During the remission phase (day 18 post immunization), inflammation (G) and infiltration of macrophages (H), as well as demyelination (I) are substantially reduced. A normal control rat is included for comparison (A, B, C). H&E staining (A, D, G), ED-1 immunohistochemistry (B, E, H), LFB/PAS staining (C, F, I). Magnification: lens × 4.

Figure 4

Cellular phenotype of chemokine receptor mRNA expressing cells in MOG-EAE. High magnification bright-field photomicrographs of spinal cord sections from MOG-EAE rats processed for combined GSA/B4 immunohistochemistry and CCR1, CCR2, CCR5 mRNA in situ hybridization. Cells expressing CCR1 (A), CCR2 (B) or CCR5 (C) mRNA are positively stained with GSA/B4, identifying them as macrophages/microglia.

Figure 5

Quantification of CCR1, CCR2 and CCR5 mRNA expressing cells in defined lesional stages. Mean numbers of CCR1+ cells (A), CCR2+ cells (B) and CCR5+ cells (C) per square unit in spinal cord sections from MOG-EAE rats. Lesions were characterized as EA = early active, LA = late active and IADM = inactive demyelinated. PPWM = periplaque white matter. Bar = mean.