Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood-Brain Barrier

Abstract

The infiltration of immune cells into the central nervous system mediates the development of autoimmune neuroinflammatory diseases. We previously showed that the loss of either Fabp5 or calnexin causes resistance to the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of multiple sclerosis (MS). Here we show that brain endothelial cells lacking either Fabp5 or calnexin have an increased abundance of cell surface CD200 and soluble CD200 (sCD200) as well as decreased T-cell adhesion. In a tissue culture model of the blood-brain barrier, antagonizing the interaction of CD200 and sCD200 with T-cell CD200 receptor (CD200R1) via anti-CD200 blocking antibodies or the RNAi-mediated inhibition of CD200 production by endothelial cells increased T-cell adhesion and transmigration across monolayers of endothelial cells. Our findings demonstrate that sCD200 produced by brain endothelial cells regulates immune cell trafficking through the blood-brain barrier and is primarily responsible for preventing activated T-cells from entering the brain.

Keywords: CD200; CD200R1; brain endothelial cells; calnexin; fatty acid binding protein 5; neurodegenerative diseases; sCD200.

Figure 1

CD200 on brain endothelial cells. (A) Relative quantification shows higher CD200 mRNA abundance in Canx^−/− or Fabp5^−/− cells compared to wild-type cells (based on 7 experiments performed in duplicates). (B) Representative histograms of negative control (dashed line), unstimulated cells (grey histogram), and cytokine stimulated cells (blue histogram). (C) CD200⁺ cells in wild-type, Canx^−/−, and Fabp5^−/− bEND.3 cell populations. (D) Cell surface abundance (mean fluorescence intensity, MFI) of CD200 on unstimulated and cytokine-treated wild-type, Canx^−/−, and Fabp5^−/− cells. (E) Percentage of CD200 in CD11b⁻PECAM-1⁺ endothelial cells isolated from brains of Canx^−/− and wild-type mice. (F) MFI values of gated CD200⁺CD11b⁻PECAM-1⁺ brain endothelial cells. All data shown are from 7 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Transmigration of activated T-cells across wild-type, Canx^−/−, or Fabp5^−/− bEND.3 cell monolayers. (A) Percentage of activated T-cells migrated through Canx^−/− or Fabp5^−/− bEND.3 cell monolayers vs. wild-type bEND.3 cell monolayers (white bars). The use of anti-CD200 blocking antibody increased T-cell transmigration across wild-type, Canx^−/−, or Fabp5^−/− bEND.3 cell monolayers (blue bars), while the use of the control isotype antibody did not (grey bars). (B) Representative histograms of wild-type, Canx^−/−, or Fabp5^−/− bEND.3 cells treated with CD200 siRNA and non-targeting siRNA. (C) The siRNA-mediated downregulation of CD200 in wild-type, Canx^−/−, or Fabp5^−/− bEND.3 cell monolayers also increased the transmigration of T-cells across wild-type, Canx^−/−, and Fabp5^−/− bEND.3 cell monolayers. Data representative of 8 independent experiments analyzed in triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

CD200-dependent binding of T-cells to wild-type, Canx^−/−, and Fabp5^−/− bEND.3 cells. (A) Relative quantification of T-cells adhered to wild-type, Canx^−/−, and Fabp5^−/− bEND.3 cells. (B) Representative images of T-cells (green) adhered to wild-type, Canx^−/−, and Fabp5^−/− bEND.3 cells (red). (C) Increased T-cell adherence to wild-type, Canx^−/−, and Fabp5^−/− bEND.3 cells in the presence of anti-CD200 antibodies. * p = 0.0325, ** p = 0.006, *** p = 0.0011, **** p < 0.0001. Data shown are the combined results of 3 experiments performed in triplicates. Blue, DAPI staining.

Figure 4

Endothelial cell-derived soluble CD200. (A) Soluble CD200 (sCD200) in conditioned media of wild-type, Canx^−/−, or Fabp5^−/− endothelial bEND.3 cells. (5 experiments in duplicates). (B) Abundance of sCD200 in serum collected from wild-type and Canx^−/− mice. (n = 3 per group). (C) Representative images (n = 3 views per experiment) of T-cells (green) adhered to wild-type bEND.3 monolayers cultured in the absence or presence of serum collected from wild-type and Canx^−/− mice. * p = 0.0325, ** p = 0.0057, **** p < 0.0001. The experiment was repeated 3 times in triplicates. Blue, DAPI staining (D) Ratio of T-cells to wild-type bEND.3 cells in cultures incubated with serum collected from wild-type (black dots) or Canx^−/− mice (red symbols). (E) A schematic representation of how the binding of sCD200 produced by endothelial cells to CD200R1 on T-cells antagonizes the adhesion of T-cells with endothelial cells and thereby inhibits the process of transmigration. Wild-type endothelial cells of the blood–brain barrier (left panel) produce a basal amount of CD200 and sCD200. By contrast, Canx^−/− or Fabp5^−/− brain endothelial cells produce elevated levels of CD200 and sCD200. The amount of sCD200 produced saturates the CD200R1 on activated T-cells, preventing them from interacting with endothelial cells, thus attenuating T-cell adhesion and transmigration across the blood–brain barrier (middle panel). The addition of excess anti-CD200 antibodies antagonizes the binding of CD200 and sCD200 produced by Canx^−/− or Fabp5^−/− brain endothelial cells to CD200R1 on T-cells, thus enabling them to interact with CD200 on endothelial cells and subsequently cross the blood–brain barrier (right panel).

Figure 5

CD200 in brain endothelial cells from MS lesions. Acute MS brain lesions demonstrate low levels of CD200 (asterisk) and thus lack co-localization with PECAM-1⁺ brain endothelial cells (A–C). Unaffected brain white matter shows high CD200 abundance and co-localization with PECAM-1⁺ brain endothelial cells (D–F). Scale bar: 50 µm. Representative images of samples from 2 unaffected and 6 MS donors.

Figure 6

CD200 is lacking in MS lesions compared with unaffected white matter. Representative image of an MS lesion in white matter, left to the lesion border, adjacent to grey matter, right to the lesion border. (A–D) shows a juxtacortical lesion, a well-recognized type of lesion in MS. Samples were stained for CD200 (green) (A,B,D,F) and neuronal marker NeuN (red) (C,E,F). CD200 was not detected in the endothelial layer (open arrows in D,E and solid arrows in F) in the blood vessels marked with an asterisk within the lesion, while neurons showed immunopositivity for CD200 outside the lesion area (B). Positive immunostaining for CD200 in unaffected brain blood capillaries (open arrows) and neuronal cell bodies (open arrowheads) (A,D). Scale bar: 100 µm for A, 50 µm for B–F. Representative images of samples from 2 unaffected and 6 MS donors.