Regulation of experimental autoimmune encephalomyelitis by natural killer (NK) cells

Abstract

In this report, we establish a regulatory role of natural killer (NK) cells in experimental autoimmune encephalomyelitis (EAE), a prototype T helper cell type 1 (Th1)-mediated disease. Active sensitization of C57BL/6 (B6) mice with the myelin oligodendrocyte glycoprotein (MOG)35-55 peptide induces a mild form of monophasic EAE. When mice were deprived of NK cells by antibody treatment before immunization, they developed a more serious form of EAE associated with relapse. Aggravation of EAE by NK cell deletion was also seen in beta 2-microglobulin-/- (beta 2m-/-) mice, indicating that NK cells can play a regulatory role in a manner independent of CD8+ T cells or NK1.1+ T cells (NK-T cells). The disease enhancement was associated with augmentation of T cell proliferation and production of Th1 cytokines in response to MOG35-55. EAE passively induced by the MOG35-55-specific T cell line was also enhanced by NK cell deletion in B6, beta 2m-/-, and recombination activation gene 2 (RAG-2)-/- mice, indicating that the regulation by NK cells can be independent of T, B, or NK-T cells. We further showed that NK cells inhibit T cell proliferation triggered by antigen or cytokine stimulation. Taken together, we conclude that NK cells are an important regulator for EAE in both induction and effector phases.

Figure 1

Effect of anti-NK1.1 mAb on MOG_35-55 induced EAE in wild-type B6 mice. Mice were immunized two times with the MOG_35-55 for EAE induction. They were intravenously injected with PBS, 500 μg of control mAb (M-11; A), or 500 μg of anti-NK1.1 mAb (PK136; B) 1 d before first immunization with the MOG peptide. Clinical score of individual mice at each observation time point is shown by different marks. This is a representative of two experiments with similar results. The result of pretreatment with PBS did not differ significantly from that with control mAb.

Figure 1

Effect of anti-NK1.1 mAb on MOG_35-55 induced EAE in wild-type B6 mice. Mice were immunized two times with the MOG_35-55 for EAE induction. They were intravenously injected with PBS, 500 μg of control mAb (M-11; A), or 500 μg of anti-NK1.1 mAb (PK136; B) 1 d before first immunization with the MOG peptide. Clinical score of individual mice at each observation time point is shown by different marks. This is a representative of two experiments with similar results. The result of pretreatment with PBS did not differ significantly from that with control mAb.

Figure 2

NK cell deletion by anti-NK1.1 mAb. B6 mice were intraperitoneally injected with 500 μg of control mAb (M-11; top) or 500 μg of anti-NK1.1 mAb (PK136; bottom). On days 5, 16, and 36, the spleen cells were stained with anti-NK1.1-PE and anti-CD3-FITC mAbs. Note the persistent deletion of NK cells (NK1.1⁺CD3⁻ cells) in the total spleen cells after anti-NK1.1 mAb treatment: 2.68–5.63% in mice treated with control mAb versus 0.3–0.6% in mice treated with anti-NK1.1 mAb.

Figure 3

Effect of anti-NK1.1 mAb on MOG_35-55 induced EAE in β2m^−/− mice. The mice were immunized two times with MOG_35-55 peptide for EAE induction. On the day before first immunization, control mAb (A) or anti-NK1.1 mAb (B) was intravenously injected. This is a representative of three experiments with similar results.

Figure 3

Effect of anti-NK1.1 mAb on MOG_35-55 induced EAE in β2m^−/− mice. The mice were immunized two times with MOG_35-55 peptide for EAE induction. On the day before first immunization, control mAb (A) or anti-NK1.1 mAb (B) was intravenously injected. This is a representative of three experiments with similar results.

Figure 4

Effect of anti-NK1.1 mAb on MOG_35-55 induced EAE in IFN-γ^−/− mice. The mice were immunized two times with MOG_35-55 peptide for EAE induction. 1 d before first immunization, the mice were intravenously injected with control mAb (A) or anti-NK1.1 mAb (B). The result of pretreatment with PBS did not differ from results shown in A or B.

Figure 4

Effect of anti-NK1.1 mAb on MOG_35-55 induced EAE in IFN-γ^−/− mice. The mice were immunized two times with MOG_35-55 peptide for EAE induction. 1 d before first immunization, the mice were intravenously injected with control mAb (A) or anti-NK1.1 mAb (B). The result of pretreatment with PBS did not differ from results shown in A or B.

Figure 5

Effect of NK cell deletion on T cell response to MOG_35-55. (A) LN cell proliferative response. 11 d after immunization with MOG_35-55, draining LN cells were prepared and their proliferative responses to MOG_35-55, PLP_136-150 (PLP), and rat myelin basic protein_89-101 (MBP) (reference 9) were assayed by a standard method. 1 d before immunization, mice were injected intravenously either with control mAb or with anti-NK1.1 mAb. Data represent mean ± SD of the mean cpm obtained by triplicate cultures in four independent experiments. Each column shows the data of wild-type B6 mice pretreated with control M-11 mAb (B6,control), B6 pretreated with anti-NK1.1 mAb (B6,anti-NK1.1), β2m^−/− mice pretreated with control M-11 mAb (b2m−/−, control), and β2m^−/− mice pretreated with anti-NK1.1 mAb (b2m−/−, anti-NK1.1). (B) IFN-γ production by LN cells. 11 d after immunization with MOG_35-55, the LN cells from control mAb– (control) or anti-NK1.1 mAb– treated (anti-NK1.1) B6 or β2m^−/− mice were cultured for 40 h with (MOG+) or without MOG_35-55 (MOG−) and the supernatants were collected for measurement of IFN-γ, IL-2, IL-4, and IL-10 by ELISA. Although IL-2, IL-4, and IL-10 were not detectable in this experimental setting, significant production of IFM-γ was measured as shown here. Data represent mean ± SD of the mean value obtained by duplicate assays in four independent experiments.

Figure 5

Effect of NK cell deletion on T cell response to MOG_35-55. (A) LN cell proliferative response. 11 d after immunization with MOG_35-55, draining LN cells were prepared and their proliferative responses to MOG_35-55, PLP_136-150 (PLP), and rat myelin basic protein_89-101 (MBP) (reference 9) were assayed by a standard method. 1 d before immunization, mice were injected intravenously either with control mAb or with anti-NK1.1 mAb. Data represent mean ± SD of the mean cpm obtained by triplicate cultures in four independent experiments. Each column shows the data of wild-type B6 mice pretreated with control M-11 mAb (B6,control), B6 pretreated with anti-NK1.1 mAb (B6,anti-NK1.1), β2m^−/− mice pretreated with control M-11 mAb (b2m−/−, control), and β2m^−/− mice pretreated with anti-NK1.1 mAb (b2m−/−, anti-NK1.1). (B) IFN-γ production by LN cells. 11 d after immunization with MOG_35-55, the LN cells from control mAb– (control) or anti-NK1.1 mAb– treated (anti-NK1.1) B6 or β2m^−/− mice were cultured for 40 h with (MOG+) or without MOG_35-55 (MOG−) and the supernatants were collected for measurement of IFN-γ, IL-2, IL-4, and IL-10 by ELISA. Although IL-2, IL-4, and IL-10 were not detectable in this experimental setting, significant production of IFM-γ was measured as shown here. Data represent mean ± SD of the mean value obtained by duplicate assays in four independent experiments.

Figure 6

Effect of NK cell deletion on passive EAE in wild-type B6 mice. After activation with MOG_35-55 for 3 d, ZB-1 line cells (3 × 10⁶) were intravenously transferred into wild-type B6 mice pretreated on day −1 with control mAb (A) or with anti-NK1.1 mAb (B). The mice had been x irradiated (450 rad) shortly before cell transfer, and received 500 ng of PT immediately after cell transfer.

Figure 6

Effect of NK cell deletion on passive EAE in wild-type B6 mice. After activation with MOG_35-55 for 3 d, ZB-1 line cells (3 × 10⁶) were intravenously transferred into wild-type B6 mice pretreated on day −1 with control mAb (A) or with anti-NK1.1 mAb (B). The mice had been x irradiated (450 rad) shortly before cell transfer, and received 500 ng of PT immediately after cell transfer.

Figure 7

Effect of NK cell deletion on passive EAE in β2m^−/− mice. After activation with MOG_35-55 for 3 d, ZB-1 line cells (10⁷) were intravenously transferred into β2m^−/− mice pretreated on day −1 with control mAb (A) or with anti-NK1.1 mAb (B). The mice had been x irradiated (450 rad) shortly before cell transfer, and received 500 ng of PT immediately after cell transfer.

Figure 7

Effect of NK cell deletion on passive EAE in β2m^−/− mice. After activation with MOG_35-55 for 3 d, ZB-1 line cells (10⁷) were intravenously transferred into β2m^−/− mice pretreated on day −1 with control mAb (A) or with anti-NK1.1 mAb (B). The mice had been x irradiated (450 rad) shortly before cell transfer, and received 500 ng of PT immediately after cell transfer.

Figure 8

Effect of NK cell deletion on passive EAE in RAG-2^−/− mice and the treatment with spleen cells. 5 × 10⁵ of activated ZB-1 line cells were intravenously transferred into (A) RAG-2^−/− mice pretreated on day −1 with control mAb (A) or with anti-NK1.1 mAb (B, C, and D). The mice were injected with 500 ng PT after cell transfer. Although mice in B did not receive any further manipulation, 2 × 10⁷ of spleen cells from RAG-2^−/− mice were intravenously transferred to mice in C on day 2 and the same number of spleen cells from RAG-2^−/− mice which had been pretreated with anti-NK1.1 mAb on day −1, were intravenously transferred to mice in D. This is a representative of two experiments with similar results.

Figure 8

Effect of NK cell deletion on passive EAE in RAG-2^−/− mice and the treatment with spleen cells. 5 × 10⁵ of activated ZB-1 line cells were intravenously transferred into (A) RAG-2^−/− mice pretreated on day −1 with control mAb (A) or with anti-NK1.1 mAb (B, C, and D). The mice were injected with 500 ng PT after cell transfer. Although mice in B did not receive any further manipulation, 2 × 10⁷ of spleen cells from RAG-2^−/− mice were intravenously transferred to mice in C on day 2 and the same number of spleen cells from RAG-2^−/− mice which had been pretreated with anti-NK1.1 mAb on day −1, were intravenously transferred to mice in D. This is a representative of two experiments with similar results.

Figure 8

Effect of NK cell deletion on passive EAE in RAG-2^−/− mice and the treatment with spleen cells. 5 × 10⁵ of activated ZB-1 line cells were intravenously transferred into (A) RAG-2^−/− mice pretreated on day −1 with control mAb (A) or with anti-NK1.1 mAb (B, C, and D). The mice were injected with 500 ng PT after cell transfer. Although mice in B did not receive any further manipulation, 2 × 10⁷ of spleen cells from RAG-2^−/− mice were intravenously transferred to mice in C on day 2 and the same number of spleen cells from RAG-2^−/− mice which had been pretreated with anti-NK1.1 mAb on day −1, were intravenously transferred to mice in D. This is a representative of two experiments with similar results.

Figure 8

Effect of NK cell deletion on passive EAE in RAG-2^−/− mice and the treatment with spleen cells. 5 × 10⁵ of activated ZB-1 line cells were intravenously transferred into (A) RAG-2^−/− mice pretreated on day −1 with control mAb (A) or with anti-NK1.1 mAb (B, C, and D). The mice were injected with 500 ng PT after cell transfer. Although mice in B did not receive any further manipulation, 2 × 10⁷ of spleen cells from RAG-2^−/− mice were intravenously transferred to mice in C on day 2 and the same number of spleen cells from RAG-2^−/− mice which had been pretreated with anti-NK1.1 mAb on day −1, were intravenously transferred to mice in D. This is a representative of two experiments with similar results.

Figure 9

Effect of NK cell deletion on antigen-induced proliferation of T line cells. ZB-1 T line cells (4 × 10⁴ cells/well) were stimulated with MOG_35-55 in the presence of x irradiated spleen cells (8 × 10⁵ cells/well) from wild-type B6 (A) or β2m^−/− mice (B). In each experiment, spleen cells from mice pretreated with control mAb (control) and those pretreated with anti-NK1.1 mAb (anti-NK1.1) were compared in their accessory function. Data represent mean cpm ± SD of triplicate cultures. This is a representative of three experiments with similar results.

Figure 9

Effect of NK cell deletion on antigen-induced proliferation of T line cells. ZB-1 T line cells (4 × 10⁴ cells/well) were stimulated with MOG_35-55 in the presence of x irradiated spleen cells (8 × 10⁵ cells/well) from wild-type B6 (A) or β2m^−/− mice (B). In each experiment, spleen cells from mice pretreated with control mAb (control) and those pretreated with anti-NK1.1 mAb (anti-NK1.1) were compared in their accessory function. Data represent mean cpm ± SD of triplicate cultures. This is a representative of three experiments with similar results.

Figure 10

Effect of insolubilized anti-NK1.1 mAb, IFN-γ, and anti–IFN-γ on ZB-1 line proliferation. (A) Anti-NK1.1 and control M-11 mAb dissolved in PBS were added into relevant wells at various concentrations (shown in micrograms per milliliter), incubated overnight, and then washed with PBS intensively. ZB-1 line cells (4 × 10⁴/well) were stimulated with MOG_35-55 (25 μg/ml) in the presence of irradiated spleen APCs (8 × 10⁵/well) in the wells coated with M-11 (ins. M11) or with anti-NK1.1 mAb (ins.NK1.1). (B) ZB-1 line cells were cultured with spleen cell APCs in the absence of the MOG peptide in the antibody-coated wells in parallel with experiment A. (C) ZB-1 line cells were stimulated with MOG_35-55 using spleen APCs in the presence of different concentrations of recombinant mouse IFN-γ (PharMingen). (D) ZB-1 line cells were stimulated with MOG_35-55 using spleen APCs in the presence of different concentrations of anti–mouse IFN-γ mAb (PharMingen). All the data represent mean cpm ± SD of triplicate cultures.

Figure 10

Effect of insolubilized anti-NK1.1 mAb, IFN-γ, and anti–IFN-γ on ZB-1 line proliferation. (A) Anti-NK1.1 and control M-11 mAb dissolved in PBS were added into relevant wells at various concentrations (shown in micrograms per milliliter), incubated overnight, and then washed with PBS intensively. ZB-1 line cells (4 × 10⁴/well) were stimulated with MOG_35-55 (25 μg/ml) in the presence of irradiated spleen APCs (8 × 10⁵/well) in the wells coated with M-11 (ins. M11) or with anti-NK1.1 mAb (ins.NK1.1). (B) ZB-1 line cells were cultured with spleen cell APCs in the absence of the MOG peptide in the antibody-coated wells in parallel with experiment A. (C) ZB-1 line cells were stimulated with MOG_35-55 using spleen APCs in the presence of different concentrations of recombinant mouse IFN-γ (PharMingen). (D) ZB-1 line cells were stimulated with MOG_35-55 using spleen APCs in the presence of different concentrations of anti–mouse IFN-γ mAb (PharMingen). All the data represent mean cpm ± SD of triplicate cultures.

Figure 10

Effect of insolubilized anti-NK1.1 mAb, IFN-γ, and anti–IFN-γ on ZB-1 line proliferation. (A) Anti-NK1.1 and control M-11 mAb dissolved in PBS were added into relevant wells at various concentrations (shown in micrograms per milliliter), incubated overnight, and then washed with PBS intensively. ZB-1 line cells (4 × 10⁴/well) were stimulated with MOG_35-55 (25 μg/ml) in the presence of irradiated spleen APCs (8 × 10⁵/well) in the wells coated with M-11 (ins. M11) or with anti-NK1.1 mAb (ins.NK1.1). (B) ZB-1 line cells were cultured with spleen cell APCs in the absence of the MOG peptide in the antibody-coated wells in parallel with experiment A. (C) ZB-1 line cells were stimulated with MOG_35-55 using spleen APCs in the presence of different concentrations of recombinant mouse IFN-γ (PharMingen). (D) ZB-1 line cells were stimulated with MOG_35-55 using spleen APCs in the presence of different concentrations of anti–mouse IFN-γ mAb (PharMingen). All the data represent mean cpm ± SD of triplicate cultures.

Figure 10

Effect of insolubilized anti-NK1.1 mAb, IFN-γ, and anti–IFN-γ on ZB-1 line proliferation. (A) Anti-NK1.1 and control M-11 mAb dissolved in PBS were added into relevant wells at various concentrations (shown in micrograms per milliliter), incubated overnight, and then washed with PBS intensively. ZB-1 line cells (4 × 10⁴/well) were stimulated with MOG_35-55 (25 μg/ml) in the presence of irradiated spleen APCs (8 × 10⁵/well) in the wells coated with M-11 (ins. M11) or with anti-NK1.1 mAb (ins.NK1.1). (B) ZB-1 line cells were cultured with spleen cell APCs in the absence of the MOG peptide in the antibody-coated wells in parallel with experiment A. (C) ZB-1 line cells were stimulated with MOG_35-55 using spleen APCs in the presence of different concentrations of recombinant mouse IFN-γ (PharMingen). (D) ZB-1 line cells were stimulated with MOG_35-55 using spleen APCs in the presence of different concentrations of anti–mouse IFN-γ mAb (PharMingen). All the data represent mean cpm ± SD of triplicate cultures.