Decreased expression of CD200 and CD200 receptor in Alzheimer's disease: a potential mechanism leading to chronic inflammation

Abstract

Inflammatory activation of microglia in response to neurodegenerative changes in diseases such as Alzheimer's disease (AD) and Parkinson's disease has been extensively described. These observations have suggested that inflammation could be contributing to disease progression. In this paper, the potential role of CD200 and CD200 receptor (CD200R), whose known functions are to activate anti-inflammatory pathways and induce immune tolerance through binding of CD200 to CD200 receptor (CD200R), was studied in AD. Quantitative studies showed a significant decrease in CD200 protein and mRNA in AD hippocampus and inferior temporal gyrus, but not cerebellum. Immunohistochemistry of brain tissue sections of hippocampus, superior frontal gyrus, inferior temporal gyrus and cerebellum from AD and non-demented cases demonstrated a predominant, though heterogeneous, neuronal localization for CD200. Decreased neuronal expression was apparent in brain regions affected by AD pathology. There was also a significant decrease in CD200R mRNA expression in AD hippocampus and inferior temporal gyrus, but not cerebellum. Low expression of CD200R by microglia was confirmed at the mRNA and protein level using cultured human microglia compared to blood-derived macrophages. Treatment of microglia and macrophages with interleukin-4 and interleukin-13 significantly increased expression of CD200R. Expression of these cytokines was not generally detectable in brain. These data indicate that the anti-inflammatory CD200/CD200R system may be deficient in AD brains. Mechanisms aimed at increasing levels of CD200 and CD200R could have therapeutic potential for controlling inflammation in human neurodegenerative diseases.

Figure 1

Characterization of antibodies to CD200 in cell cultures and brain. A) Western immunoblot of control transfected (HEK-Con) and CD200 transfected (HEK-CD200) protein extracts reacted with rabbit custom antibody to CD200. B) Western immunoblot of control or CD200 transfected SH-SY5Y cells, and control and CD200 transfected HEK cells probed with goat polyclonal antibody to recombinant CD200. C) Immunoblot of control human brain protein extract showing absorption of specific reactivity (Abs) when CD200 antibody (1:10,000 dilution) was preabsorbed with 250 μg of immunizing peptide. CD200 specific band (43 kD) present in sample (Con) reacted with unabsorbed custom rabbit CD200 antibody. D). a) Immunohistochemical staining of section of hippocampus with antibody A2522 to CD200. Section was counterstained with neutral red and show heterogeneous neuronal staining. Arrow indicates CD200 reactive neurons, while arrowhead indicates a neuron with no CD200 immunoreactivity. b). Immunohistochemical staining of subset of round cells with antibody A2522 to CD200 (presumptive lymphocytes) in vessel of AD brain section. c) Immunohistochemical staining of hippocampus with monoclonal antibody MAB2724 showing similar neuronal staining pattern as antibody A2522 to CD200. d) Staining of section of inferior temporal gyrus with antibody A2522 preabsorbed with control peptide. e) Staining of parallel section of inferior temporal gyrus with antibody A2522 preabsorbed with CD200 immunizing peptide. Absorption of CD200 antibody A2522 with immunizing peptide removes neuronal staining. Scale bars represent 10 micron.

Figure 2

Confocal micrographs showing colocalization of CD200 immunoreactivity with cell type markers. Left hand panels (green) represent sections of inferior temporal gyrus from an ND case stained with antibody to identify CD200. Top panels show that most CD200 immunoreactivity colocalizes with cells demonstrating reactivity to neuronal marker NeuN (red)(see merge). Middle panels show that CD200 immunoreactivity (green) colocalizes with some GFAP positive astrocytes (red) (see merge). Lower panels show that CD200 immunoreactivity (green) does not colocalize with HLA-DR immunoreactive (red) microglia, though there is a close association between some of these cells.

Figure 3

Decreased CD200 protein in AD brains in regions with AD pathology. A: Relative levels of CD200 protein in hippocampus (HPC) (n=23 cases; ND - 11 cases; AD - 12 cases), inferior temporal gyrus (ITG) (n-21 cases; ND - 10 cases; AD - 11 cases) and cerebellum (CBM) (n-21 cases; ND - 10 cases; AD - 11 cases) in tissue samples from non-demented (ND) and Alzheimer's disease (AD) cases. Results were normalized for total amount of protein present in each lane as measured by densitometry of Ponceau-S stained membranes. B: Western blots of CD200 and corresponding stained membranes show representative results for some of the samples from hippocampus and inferior temporal gyrus. (lane designation: A; AD case; N; ND case).

Figure 4

Relative expression of CD200 mRNA in hippocampus (HPC) (n=20 cases; ND - 9 cases; AD - 11 cases), inferior temporal gyrus (ITG) (n-21 cases; ND - 10 cases; AD - 11 cases) and cerebellum (CBM) (n-22 cases; ND - 10 cases; AD - 12 cases) in tissue samples from non-demented (ND) and Alzheimer's disease (AD) cases. Levels of CD200 mRNA were measured by real time PCR with expression levels normalized for 18S RNA. Linear regression analysis indicated that there was a significant negative correlation between ITG mRNA levels for CD200 and total plaque score (ITG-plaque)(P=0.0019)(n=21 cases), and between ITG mRNA CD200 mRNA levels and total tangle scores (ITG-tangle)(P=0.0116)(n = 21 cases).

Figure 5

Immunohistochemistry of ND and AD brain sections demonstrating distribution of neuronal immunoreactivity of CD200. A and B: Lower power magnifications of inferior temporal gyrus showing distribution of staining of CD200 in ND (A) and AD (B) section through cortical layers I to V. Scale bars represent 20 micron. C and D: Higher power magnifications of corresponding inferior temporal gyrus sections. Scale bars represent 10 micron. E and F: CD200 immunoreactivity in hippocampus sections (CA1-2) of ND and AD case showing heterogeneous distribution of immunoreactivity. Scale bars represent 10 micron.

Figure 6

A and B Figures demonstrate similar immunoreactivity of CD200 in cerebellum of ND (A) and AD (B). Positive staining is observed in cells in molecular and granular layers, as well as Purkinje neurons. C: Strong immunoreactivity for CD200 in white matter (WM) region of inferior temporal cortex compared to gray matter (GM). D: Higher magnification shows CD200 staining of fibers of white matter. Scale bars represent 10 micron (panels A, B, and D) and 20 micron (panel C).

Figure 7

A-C: Relationship of CD200 stained neurons with Aβ plaques. Inferior temporal gyrus sections from A) ND and B) AD double stained for CD200 (purple) and Aβ (brown). Both sections show layers IV and V. In panel B, neuronal staining for CD200 was not reduced in pyramidal neurons closely opposed to Aβ plaques, shown at higher magnification in panel C. Scale bars represent 20 micron in panels A and B and 10 micron in panel C. D and E: Sections of hippocampus from ND (D) and AD (E) showing double staining for CD200 (purple) and HLA-DR as a marker for activated microglia (brown). (D) Weakly stained, less reactive microglia closely associated with CD200 immunoreactive neurons in ND case (arrow), but in AD case (E), strongly reactive microglial clusters (arrow) is not associated with CD200 immunoreactive neurons. Scale bars represent 10 micron.

Figure 8

Fractionation of brain tissue samples (inferior temporal gyrus) into soluble (sol), Triton X100 extractable (memb), and SDS extractable (insol) fractions demonstrate majority of CD200 localized to Triton X100 extractable membrane fraction. Equal amounts of protein in each fraction was analyzed by western blot for CD200 and normalized for β-actin.

Figure 9

Decreased expression of CD200R mRNA in human brains affected by AD pathology. A: Real time PCR analyses of CD200R mRNA expression in brain samples hippocampus (HPC) (n=27 cases; ND - 11 cases; AD - 16 cases), inferior temporal gyrus (ITG) (n-27 cases; ND - 13 cases; AD - 14 cases) and cerebellum (CBM) (n-22 cases; ND - 10 cases; AD - 10 cases) in tissue samples from non-demented (ND) and Alzheimer's disease (AD) cases showed significant decrease in expression in HPC and ITG, brain regions affected by AD pathology, but not in CBM, a brain region usually spared significant pathology. Regression analysis demonstrated a significant negative correlation between HPC levels of CD200R and total brain tangle score (B) (P=0.034), and between ITG levels of CD200R and total brain plaque score (C) (P=0.027) (n=27 cases).

Figure 10

Human macrophages express significantly greater amounts of CD200R mRNA and protein than human microglia. A: Characterization of custom made antibody to CD200R (A2524) using HEK cell line transfected with control vector (Con) or CD200R expression plasmids. Antibody recognized band only in CD200R plasmid transfected cells. Molecular weight of CD200R varied between different HEK isolates. B: CD200R mRNA is expressed by microglia (MG), macrophages (MAC) and differentiated SH-SY5Y neuronal cells. Expression of CD200R mRNA by microglia is noticeably less than by macrophages. C: Detection of CD200R protein with molecular weight of 66-68 kD by western blot in extracts of human microglia (MG), macrophages (MAC), and SH-SY5Y neuronal cells, and in CD200R transfected HEK cells (CD200R-HEK - at 1% of protein level compared to other cells). Protein bands were detected with custom antibody to CD200R (A2524). D: Quantitative measurements of western immunoblots showed that macrophages express significantly greater amounts of CD200R protein than human microglia.

Figure 11

Induction of CD200R mRNA expression by interleukin-4 and interleukin-13 in human macrophages and microglia. A: Bar chart showing results of real time PCR analyses for CD200R mRNA expression in human macrophages treated with 10 ng/ml and 100 ng/ml of IL-4, IL-13 and IL-10, and 100 ng/ml of IL-1β and IFN-γ for 24 hours. CD200R expression values were normalized for expression of β-actin mRNA. Results show significant induction of CD200R mRNA in a dose response manner for IL-4 and IL-13, but not IL-10, IL-1β or IFN-γ. B: Representative gel image showing induction of CD200R expression by macrophages with IL-4 treatment. C: Bar chart showing results of real time PCR analyses for CD200R mRNA expression in human microglia treated with 10 ng/ml and 100 ng/ml of IL-4, 100 ng/ml IL-13, and 10 ng/ml of IL-12 and IL-18 for 24 hours. CD200R expression values were normalized for expression of β-actin mRNA. Results show significant induction of CD200R mRNA expression with IL-4 and IL-13 treatments. D: Representative gel image showing induction of CD200R expression by human microglia with IL-4 treatment. (***; P <0.001; ** P< 0.01 as compared to control groups).

Figure 12

Comparison of expression of interleukin-4 mRNA expression in AD and ND inferior temporal gyrus samples compared to blood cells. PCR analyses demonstrated that IL-4 mRNA expression was not detectable in most of the ND and AD samples tested (n = 8 in each group). Levels of expression in the positive expressing samples were low compared to sample derived from white blood cells (Blood) (representative image shown), which demonstrated strong expression of both transcript variants of IL-4. IL-4 PCR was carried out for 35 cycles; β-actin PCR was carried out for 24 cycles. (-rt: shows one representative brain sample that was processed without reverse transcriptase).