Cannabinoid CB1 and CB2 receptors and fatty acid amide hydrolase are specific markers of plaque cell subtypes in human multiple sclerosis

Abstract

Increasing evidence supports the idea of a beneficial effect of cannabinoid compounds for the treatment of multiple sclerosis (MS). However, most experimental data come from animal models of MS. We investigated the status of cannabinoid CB1 and CB2 receptors and fatty acid amide hydrolase (FAAH) enzyme in brain tissue samples obtained from MS patients. Areas of demyelination were identified and classified as active, chronic, and inactive plaques. CB1 and CB2 receptors and FAAH densities and cellular sites of expression were examined using immunohistochemistry and immunofluorescence. In MS samples, cannabinoid CB1 receptors were expressed by cortical neurons, oligodendrocytes, and also oligodendrocyte precursor cells, demonstrated using double immunofluorescence with antibodies against the CB1 receptor with antibodies against type 2 microtubule-associated protein, myelin basic protein, and the platelet-derived growth factor receptor-alpha, respectively. CB1 receptors were also present in macrophages and infiltrated T-lymphocytes. Conversely, CB2 receptors were present in T-lymphocytes, astrocytes, and perivascular and reactive microglia (major histocompatibility complex class-II positive) in MS plaques. Specifically, CB2-positive microglial cells were evenly distributed within active plaques but were located in the periphery of chronic active plaques. FAAH expression was restricted to neurons and hypertrophic astrocytes. As seen for other neuroinflammatory conditions, selective glial expression of cannabinoid CB1 and CB2 receptors and FAAH enzyme is induced in MS, thus supporting a role for the endocannabinoid system in the pathogenesis and/or evolution of this disease.

Figure 1.

Distribution of CB₂-positive cells (right column) compared with that of HLA-DR-positive cells (left column) in the three different types of MS plaques (delineated with dashed lines). A–F, The morphology and distribution of CB₂-positive cells in an active (B) and chronic (D) plaques were nearly identical to those of HLA-DR-positive microglia (see insets; A, C). No CB₂-positive cells were evident in inactive plaques (E, F). Luxol fast blue staining (blue) combined with immunostaining (brown). Scale bar: A–F, 500 μm; insets, 20 μm.

Figure 2.

Double-immunofluorescence assays of CB₂-positive cells (left column) and phenotypic markers (middle columns). A–O, CB₂ was observed in HLA-DR-positive microglia (A–C), CD68-positive macrophages (D–F), MBP-containing macrophages (G–I), astrocytes (GFAP positive; J–L) and T-lymphocytes (M–O). Scale bar: 20 μm.

Figure 3.

CB₁ distribution in MS samples. A, B, D, Subcortical white matter cells exhibited CB₁ immunoreactivity (A, B); CB₁-positive cells (D) were identified as neurons by double staining with MAP-2 (E). G–I, Adult oligodendrocytes exhibited cytoplasmic staining for CB₁. J, K, Scarce OPCs in active plaques were positive for CB₁ (J) and PDGFR-α (K). Round-shaped macrophages (CD68 positive; N) also showed CB₁ immunostaining (C, M). Perivascular T-lymphocytes (Q; see vessel wall, arrow in P) expressed CB₁ receptors (P). Scale bars: A, 500 μm; B, 200 μm; C–F, 100 μm; G–R, 20 μm.

Figure 4.

FAAH is expressed by hypertrophic astrocytes in active MS plaques. A–E, FAAH-positive cells were abundant in active plaques (A) and exhibited morphological (B) and phenotypic (GFAP positive; C–E) characteristics of astrocytes. Scale bars: A, 500 μm; B, 20 μm; C–E, 100 μm.