Delta-like ligand 4 regulates central nervous system T cell accumulation during experimental autoimmune encephalomyelitis - PubMed (original) (raw)

Delta-like ligand 4 regulates central nervous system T cell accumulation during experimental autoimmune encephalomyelitis

Nathanael D Reynolds et al. J Immunol. 2011.

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated inflammatory demyelinating disease of the CNS that serves as a model for multiple sclerosis. Notch receptor signaling in T lymphocytes has been shown to regulate thymic selection and peripheral differentiation. In the current study, we hypothesized that Notch ligand-receptor interaction affects EAE development by regulating encephalitogenic T cell trafficking. We demonstrate that CNS-infiltrating myeloid dendritic cells, macrophages, and resident microglia expressed Delta-like ligand 4 (DLL4) after EAE induction. Treatment of mice with a DLL4-specific blocking Ab significantly inhibited the development of clinical disease induced by active priming. Furthermore, the treatment resulted in decreased CNS accumulation of mononuclear cells in the CNS. Anti-DLL4 treatment did not significantly alter development of effector cytokine expression by Ag-specific T cells. In contrast, anti-DLL4 treatment reduced T cell mRNA and functional cell surface expression of the chemokine receptors CCR2 and CCR6. Adoptive transfer of Ag-specific T cells to mice treated with anti-DLL4 resulted in decreased clinical severity and diminished Ag-specific CD4(+) T cell accumulation in the CNS. These results suggest a role for DLL4 regulation of EAE pathogenesis through modulation of T cell chemokine receptor expression and migration to the CNS.

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Figures

Figure 1

Figure 1

Expression of DLL4 during EAE development. SJL mice were immunized with PLP139–151in CFA and APC were isolated from naïve mice 7 days after immunization, at disease onset, and at peak of clinical disease. Cell surface DLL4 expression was determined by flow cytometry analysis of five individual mice at each timepoint. DLL4 expression was significantly (*p<0.05) increased on B cells in the lymph nodes and macrophages in the lymph nodes, spleen, and spinal cord during the development of EAE when compared to naïve mice. Spinal cord-infiltrating CD11c+ dendritic cells showed increased DLL4 expression while microglia expression of DLL4 was significantly decreased during the development of EAE. Data are presented as mean percent expression (±S.D.) of DLL4 by cells within a distinct APC population isolated from five individual mice. The results are representative of 3 similar experiments.

Figure 2

Figure 2

Anti-DLL4 treatment reduced clinical EAE. SJL mice were immunized with PLP139–151in CFA and treated with anti-DLL4 serum or control rabbit serum at days 4 and 7 post-immunization. A, Treatment with anti-DLL4 serum significantly (*p<0.05) decreased clinical disease. The data are representative of three individual experiments. B, Photomicrographs (200x) of hematoxylin and eosin stained lumbar spinal cord sections (8μm OCT-embedded) from anti-DLL4 treated mice at peak clinical severity (day 13 post-immunization) show decreased CNS inflammation when compared to control-treated CNS. Data presented are representative of three individual mice from each group. C, The number of CNS lesions in each of 3 mice per group was determined by evaluating 3 sections per mouse. Decreased lesion numbers were found in anti-DLL4-treated lumbar spinal cord sections when compared to control-treated mice at peak clinical score (*p<0.05). The results are representative of 3 similar experiments.

Figure 3

Figure 3

Decreased mononuclear cell accumulation in the spinal cords of anti-DLL4-treated mice during EAE. Spinal cords were removed from 3 individual mice at peak clinical severity (day 13 post-immunization) for isolation and analysis of infiltrating mononuclear cells by flow cytometry. A, Representative flow cytometry dot plots from control- and anti-DLL4-treated spinal cords stained with anti-CD11b, -CD11c, -CD45, -CD4 and -CD8. B, Quantification of individual leukocyte populations indicated a significant (*p<0.05) decrease in cell numbers of macrophages, CD11c+ DC, CD4+ and CD8+ T cells in anti-DLL4-treated spinal cords compared to controls. Data presented are derived from three individually analyzed mice per group. The results are representative of 3 similar experiments.

Figure 4

Figure 4

Anti-DLL4 treatment decreased IFN-γ- and IL-17-expressing CD4+ T cell accumulation in the CNS during EAE. Spinal cords were removed from 3 individual mice at peak clinical severity (day 13–15 post-immunization) for isolation and analysis of cytokine expression by CD4+ T cells. CD4+ T lymphocytes were activated in culture with PMA/ionomycin stimulation for 4 hours and analyzed for intracellular IFN-γ and IL-17 expression using flow cytometry. A, Representative flow cytometry dot plot from control- and anti-DLL4-treated spinal cords stained with CD4, CD45, IFN-γ and IL-17 antibody. Data shown is derived from a CD45 and CD4 gate. The results are representative of 2 similar experiments. B, Anti-DLL4 significantly (*p<0.05) increased IFN-γ-expressing CD4+ T cell frequency but significantly decreased (*p<0.05) IL-17-expressing CD4+ T cell frequency in the CNS. C, Quantification of mean cell number indicate a significant (*p<0.05) decrease in IFN-γ- and IL-17-expressing CD4+ T cells in the CNS of anti-DLL4-treated mice when compared to control. Data presented are derived from six individual analyzed mice per group.

Figure 5

Figure 5

Anti-DLL4 treatment does not affect expression of T regulatory cell-associated factors or chemokine expression in the CNS during EAE. Spinal cords were removed from 3 individual mice at peak clinical severity (day 13–15 post-immunization) for quantitative real-time RT-PCR analysis of isolated leukocytes and IL-10 detection from spinal cord homogenate supernatants. A, Quantitative real-time RT-PCR analysis indicates no significant difference in FoxP3 mRNA expression of anti-DLL4-treated mice compared to control. B, ELISA analysis of IL-10 production from spinal cord homogenate supernatants indicate so significant change in cytokine production between control- and anti-DLL4-treated mice. C, ELISA analysis of CCL2, CCL3, CCL20 and CXCL10 expression in the CNS indicates no significant differences between control and anti-DLL4 treatment. Data representative of 2 individual experiments.

Figure 6

Figure 6

Anti-DLL4 treatment does not influence development of IFN-γ or IL-17 cytokine expression by peripheral antigen-specific T cells. PLP139–151-specific CD4+ 5B6 TCR transgenic T cells were parked in naïve SJL mice by i.v. injection prior to immunization. Mice (n=3/group) were immunized with PLP139–151 in CFA and treated with either anti-DLL4 or control serum at days 4 and 7 post-immunization. At day 10 post-immunization, lymphocytes from peripheral lymph nodes and spleens of individual mice were removed, activated in culture with PMA/ionomycin for 4 hours and analyzed for intracellular IFN-γ and IL-17 expression. Using flow cytometry, 5B6 transgenic T cells were gated on using CD90.1 and CD4 and the frequency and absolute number of cells expressing IFN-γ or IL-17 was calculated for lymph node and spleen. The data shows equivalent frequencies (in percent) and numbers of IFN-γ- and IL-17-expressing 5B6 T cells in the lymph nodes and spleen of control- and anti-DLL4-treated mice at day 10 post-immunization. The results are representative of 2 experiments.

Figure 7

Figure 7

Treatment of antigen-specific encephalitogenic T cell - APC culture with anti-DLL4 decreased T cell chemokine receptor mRNA and functional cell surface expression. Draining lymph nodes were removed from day 7 post-immunized SJL mice and cultured in the presence of PLP139–151with control- or anti-DLL4-antibodies for 96 hours. A, T cells were sorted and analyzed for CCR2, CCR5, CXCR3 and CCR6 expression by quantitative real-time RT-PCR analysis. The results indicate a significant decrease (*p<0.05) in T cell mRNA expression of CCR2, CCR5 and CCR6 from anti-DLL4-treated compared to control cultures. B, T cells were sorted and analyzed for functional cell surface expression of CCR2, CCR5, CXCR3 and CCR6 by in vitro migration assay using chemokines specific for the indicated receptors. The results indicate a significant decrease (*p<0.05) in T cell migration to CCL2 and CCL20 thus demonstrating a decrease in functional surface expression of CCR2 and CCR6 from anti-DLL4-treated compared to control cultures. C, T cells were analyzed by flow cytometry for expression of LFA-1. The data show no difference in LFA-1 expression between T cells from control- (solid line) and anti-DLL4-treated (dashed line) cultures. D, T cells were analyzed by flow cytometry for expression of LFA-1. The data show no difference in VLA-4 expression between T cells from control- (solid line) and anti-DLL4-treated (dashed line) cultures. E, ELISA analysis of IFN-γ and IL-17 from antigen-specific T cell culture supernatants indicate no significant change in cytokine production between control- and anti-DLL4-treated cultures. C, Quantitative real-time RT-PCR analysis indicates no significant difference in FoxP3 mRNA expression by anti-DLL4-treated T cells compared to control. Data representative of 3 individual experiments.

Figure 8

Figure 8

In vitro anti-DLL4 treatment reduced adoptive EAE severity. Draining lymph nodes were removed from day 7 post-immunized SJL mice and lymphocytes were cultured in the presence of PLP139–151 and either control or anti-DLL4 antibody for 96 hours. A, Adoptive transfer of lymphocytes into normal SJL recipient mice (n=5/group) following culturing conditions resulted in a significant (*, p<0.05) decrease in mean clinical severity of the anti-DLL4-treated cells when compared to control. B, Mean cumulative score was significantly (*, p<0.05) decreased as a result of anti-DLL4-treated culture conditions when compared to control. C, Representative flow cytometry plots of spinal cord tissue at 9–11 days post-transfer stained with CD4 and CD45 indicate equivalent percentages of CD4+ T cells in the CNS of recipient mice receiving anti-DLL4-treated cells when compared to control. The results are derived from 2 similar experiments. D, Quantification of leukocyte populations indicated a significant (*, p<0.05) decrease in CD4+ T cell numbers in recipient spinal cords receiving anti-DLL4-treated cells when compared to control. Data representative of five mice in each group.

Figure 9

Figure 9

Anti-DLL4 treatment reduces adoptively transferred EAE severity and CNS accumulation of antigen-specific T cells. Antigen-specific donor T cells were obtained from PLP139–151-primed SJL.CD90.1 congenic mice, re-activated in vitro with specific antigen, and adoptively transferred to wild type SJL CD90.2 recipient mice. Recipients were treated with either control antibodies or anit-DLL4 2 days after adoptive transfer. A, Clinical disease severity was monitored and the data show a significant (p<0.05) decrease in clinical disease over the the entire course of the experiment. N=10/group. B, CNS mononuclear cell accumulation was assessed by flow cytometric analysis of spinal cord infiltrates and the data show a significant (*, p<0.05) decrease in the number of adoptively transferred donor antigen-specific T cells in the anti-DLL4-treated mice compared to controls. There was no statistical difference in the other CNS mononuclear cell populations between treatment groups. N=3/group. C, CNS CD90.1+CD4+ antigen-specific donor T cells were isolated by magnetic bead cell sorting and analyzed for functional cell surface chemokine receptor expression using an in vitro migration assay. There is no significant difference in CNS-derived T cell migration to CCL2 and CCL20 thus demonstrating no change in functional surface expression of CCR2 and CCR6 from anti-DLL4-treated mice compared to controls. N=3/group. D, Splenic CD90.1+CD4+ antigen-specific donor T cells were isolated by cell sorting and analyzed for functional cell surface chemokine receptor expression using an in vitro migration assay. The results indicate a significant decrease (*p<0.05) in T cell migration to CCL2 and CCL20 thus demonstrating a decrease in functional surface expression of CCR2 and CCR6 from anti-DLL4-treated mice compared to controls. N=3/group. The results are representative of two individual experiments.

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References

    1. Hohlfeld R. Biotechnological agents for the immunotherapy of multiple sclerosis. Principles, problems, and perspectives. Brain. 1997;120:865–916. - PubMed
    1. Skundric DS, Kim C, Tse HY, Raine CS. Homing of T cells to the central nervous system throughout the course of relapsing experimental autoimmune encephalomyelitis in Thy-1 congenic mice. Journal of Neuroimmunology. 1993;46:113–122. - PubMed
    1. Waldner H, Whitters MJ, Sobel RA, Collins M, Kuchroo VK. Fulminant spontaneous autoimmunity of the central nervous system in mice transgenic for the myelin proteolipid protein-specific T cell receptor. Proc Natl Acad Sci USA. 2000;97:3412–3417. - PMC - PubMed
    1. Karpus WJ, Lukacs NW, McRae BL, Strieter RM, Kunkel SL, Miller SD. An important role for the chemokine macrophage inflammatory protein-1 alpha in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis. J Immunol. 1995;155:5003–5010. - PubMed
    1. Fife BT, Kennedy KJ, Paniagua MC, Lukacs NW, Kunkel SL, Luster AD, Karpus WJ. CXCL10 (IFN-gamma-inducible protein-10) control of encephalitogenic CD4+ T cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis. Journal of Immunology. 2001;166:7617–7624. - PubMed

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