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. 2008 Jul 8;105(27):9325-30.
doi: 10.1073/pnas.0711175105. Epub 2008 Jun 30.

CD73 is required for efficient entry of lymphocytes into the central nervous system during experimental autoimmune encephalomyelitis

Jeffrey H Mills  1 , Linda F Thompson, Cynthia Mueller, Adam T Waickman, Sirpa Jalkanen, Jussi Niemela, Laura Airas, Margaret S Bynoe
Affiliations

CD73 is required for efficient entry of lymphocytes into the central nervous system during experimental autoimmune encephalomyelitis

Jeffrey H Mills et al. Proc Natl Acad Sci U S A. .

Abstract

CD73 is a cell surface enzyme of the purine catabolic pathway that catalyzes the breakdown of AMP to adenosine. Because of the strong immunosuppressive and antiinflammatory properties of adenosine, we predicted that cd73(-/-) mice would develop severe experimental autoimmune encephalomyelitis (EAE), an animal model for the central nervous system (CNS) inflammatory disease, multiple sclerosis. Surprisingly, cd73(-/-) mice were resistant to EAE. However, CD4 T cells from cd73(-/-) mice secreted more proinflammatory cytokines than wild-type (WT) mice and were able to induce EAE when transferred into naïve cd73(+/+) T cell-deficient recipients. Therefore, the protection from EAE observed in cd73(-/-) mice was not caused by a deficiency in T cell responsiveness. Immunohistochemistry showed that cd73(-/-) mice had fewer infiltrating lymphocytes in their CNS compared with WT mice. Importantly, susceptibility to EAE could be induced in cd73(-/-) mice after the transfer of WT CD73(+)CD4(+) T cells, suggesting that CD73 must be expressed either on T cells or in the CNS for disease induction. In the search for the source of CD73 in the CNS that might facilitate lymphocyte migration, immunohistochemistry revealed a lack of CD73 expression on brain endothelial cells and high expression in the choroid plexus epithelium which regulates lymphocyte immunosurveillance between the blood and cerebrospinal fluid. Because blockade of adenosine receptor signaling with the A(2a) adenosine receptor-specific antagonist SCH58261 protected WT mice from EAE induction, we conclude that CD73 expression and adenosine receptor signaling are required for the efficient entry of lymphocytes into the CNS during EAE development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
cd73−/− mice are resistant to EAE. EAE was induced, disease activity was monitored daily, and the mean EAE score was calculated for cd73−/− (open diamonds, n = 11) and WT (cd73+/+) (filled squares, n = 13) mice. The results shown are representative of 11 separate experiments.
Fig. 2.
Fig. 2.
cd73−/− T cells produce elevated levels of IL-1β and IL-17 and mediate EAE susceptibility when transferred to cd73+/+tcrα−/− mice. (A) CD4 and FoxP3 expression was measured on splenocytes from naïve mice and from cd73−/− and WT mice day 13 after EAE induction. (B) Splenocytes from naïve mice and from WT mice day 13 after MOG immunization were analyzed for CD4 and CD73 cell surface expression by flow cytometry. (C) Sorted cells from immunized WT or cd73−/− mice were cultured with 1 × 104 irradiated splenocytes and 0 or 10 μg/ml MOG peptide. Supernatants were taken at 18 h and run on a cytokine Bio-plex assay. Results represent the fold change in cytokine levels between the 0 and 10 μg/ml MOG peptide groups. Samples were pooled from four mice and are representative of one of three similar experiments. (D) CD4+ T cells from the spleen and lymph nodes from MOG-immunized cd73−/− mice (open diamonds, n = 5) or WT mice (filled squares, n = 5) were adoptively transferred into T cell-deficient cd73+/+tcrα−/− mice. EAE was induced, and disease progression was monitored daily. Results are representative of two separate experiments.
Fig. 3.
Fig. 3.
cd73−/− mice display little or no CNS lymphocyte infiltration after EAE induction; donor cd73−/− T cells infiltrate the CNS of cd73+/+tcrα−/− recipient mice after EAE induction. (A–F) Frozen tissue sections from WT mice (A–C) and cd73−/− mice (D–F) day 13 after EAE induction were labeled with a CD4 antibody. (G) The mean number of CD4+-infiltrating lymphocytes in the brain and spinal cord was quantified per field in frozen tissue sections from WT and cd73−/− mice day 13 after EAE induction. Eight anatomically similar fields per brain and four fields per spinal cord per mouse were analyzed at ×10 magnification (n = 5 mice per group). Error bars represent the standard error of the mean. (H–L) Frozen tissue sections of hippocampus (H, I, and K) and cerebellum (J and L) labeled with a CD4 antibody from EAE-induced tcrα−/− mice that received CD4+ cells from WT (H–J) or cd73−/− (K and L) mice at day 12 (K), 18 (H and L), or 22 (I and J) after EAE induction. Immunoreactivity was detected with HRP anti-rat Ig plus AEC (red) against a hematoxylin-stained nuclear background (blue). Arrows indicate sites of lymphocyte infiltration. (Scale bars: 500 μm.)
Fig. 4.
Fig. 4.
Adoptively transferred CD73+ T cells from WT mice can confer EAE susceptibility to cd73−/− mice. (A) CD4+ T cells from the spleen and lymph nodes of MOG-immunized WT mice were enriched and adoptively transferred into WT (filled squares, n = 5) or cd73−/− (open diamonds, n = 5) mice followed by concomitant EAE induction. Results are shown from one of two independent experiments. (B) T cells from the spleen and lymph nodes of immunized WT and cd73−/− mice were sorted based on CD4 and CD73 expression and adoptively transferred into cd73−/− mice followed by concomitant EAE induction (n = 5 in each group). Filled squares represent donor cells from WT mice that express CD73; open squares represent donor cells from WT mice that lack CD73 expression; open diamonds represent donor cells from cd73−/− mice. (C and D) Frozen tissue sections of the CNS choroid plexus from naïve WT (C Left) and cd73−/− (C Right) mice and WT mice day 12 after EAE induction (D) were stained with a CD73- (C) or CD45- (D) specific antibody. Immunoreactivity was detected with HRP anti-rat Ig plus AEC (red) against a hematoxylin-stained nuclear background (blue). Brackets indicate CD73 staining. Arrows indicate CD45 lymphocyte staining. (Scale bars: 500 μm.)
Fig. 5.
Fig. 5.
AR blockade protects mice from EAE development. (A) EAE was induced, disease activity was monitored daily, and the mean EAE score was calculated in WT (squares) and cd73−/− (diamonds) mice given either drinking water (filled symbols) alone or drinking water supplemented with 0.6 g/ml broad-spectrum AR antagonist caffeine (open symbols). Results are from one experiment (n = 5 mice per group). (B) AR mRNA expression levels relative to the GAPDH housekeeping gene in the Z310 murine choroid plexus cell line. Samples were run in triplicate; error bars represent the standard error of the mean. (C) Mice were treated with the A2a AR antagonist SCH58261 at 2 mg/kg (1 mg/kg s.c. and 1 mg/kg i.p.) in 45% DMSO (filled squares, n = 4 mice per group) or 45% DMSO alone (open squares, n = 5 mice per group) 1 day before and daily up to day 30 after EAE induction. These results are representative of two experiments. (D) The mean number of CD4+-infiltrating lymphocytes in the brain and spinal cord quantified per field in frozen tissue sections from day 15 after EAE induction in SCH58261- and DMSO-treated mice are shown. Eight anatomically similar fields per brain and four fields per spinal cord per mouse were analyzed at ×10 magnification (n = 4 mice). Error bars represent the standard error of the mean.
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