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CXCL12 (SDF-1alpha) suppresses ongoing experimental autoimmune encephalomyelitis by selecting antigen-specific regulatory T cells.

Meiron M, Zohar Y, Anunu R, Wildbaum G, Karin N - J. Exp. Med. (2008)

Bottom Line: The beneficial effect included selection of antigen-specific T cells that were CD4(+)CD25(-)Foxp3(-)IL-10(high), which could adoptively transfer disease resistance, and suppression of Th17 selection.However, in vitro functional analysis of these cells suggested that, even though CXCL12-Ig-induced tolerance is IL-10 dependent, IL-10-independent mechanisms may also contribute to their regulatory function.Collectively, our results not only demonstrate, for the first time, that a chemokine functions as a regulatory mediator, but also suggest a novel way for treating multiple sclerosis and possibly other inflammatory autoimmune diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel.

ABSTRACT
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system induced by antigen-specific effector Th17 and Th1 cells. We show that a key chemokine, CXCL12 (stromal cell-derived factor 1alpha), redirects the polarization of effector Th1 cells into CD4(+)CD25(-)Foxp3(-)interleukin (IL) 10(high) antigen-specific regulatory T cells in a CXCR4-dependent manner, and by doing so acts as a regulatory mediator restraining the autoimmune inflammatory process. In an attempt to explore the therapeutic implication of these findings, we have generated a CXCL12-immunoglobulin (Ig) fusion protein that, when administered during ongoing EAE, rapidly suppresses the disease in wild-type but not IL-10-deficient mice. Anti-IL-10 neutralizing antibodies could reverse this suppression. The beneficial effect included selection of antigen-specific T cells that were CD4(+)CD25(-)Foxp3(-)IL-10(high), which could adoptively transfer disease resistance, and suppression of Th17 selection. However, in vitro functional analysis of these cells suggested that, even though CXCL12-Ig-induced tolerance is IL-10 dependent, IL-10-independent mechanisms may also contribute to their regulatory function. Collectively, our results not only demonstrate, for the first time, that a chemokine functions as a regulatory mediator, but also suggest a novel way for treating multiple sclerosis and possibly other inflammatory autoimmune diseases.

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CXCL12-Ig preserves the biological activities of native CXCL12. (A) Purified CXCL12-Ig was separated on 12% SDS-PAGE and subjected to Western blot analysis under reducing and nonreducing conditions (with or without β-mercaptoethanol) using anti-CXCL12 mAb (clone 79014) as a primary antibody (molecular masses are shown). (B) THP-1 cells (human monocytic cell line) were subjected to a migration assay using a Transwell system. Lower chambers were supplemented with culture medium, rCXCL12, CXCL12-Ig, CXCL12-Ig plus anti-CXCR4 mAb, or β-actin–Ig. The number of cells migrating to the lower chamber was counted by FACS 3 h later. Results shown represent three independent experiments and are the mean of the migration percentage (number of cells that migrated to the lower chamber divided by the number of cells originally plated in the upper chamber) ± SE. (C) Freshly isolated peritoneal macrophages were supplemented with PBS, rCXCL12, or CXCL12-Ig. Supernatants were collected 48 h later, and the IL-10 concentration was determined by ELISA. The results shown represent three experiments done in triplicates, and are the mean IL-10 concentration ± SE. (D) Primary spleen cell cultures responding to their target MOGp35-55 antigen were supplemented with PBS, rCXCL12, CXCL12-Ig, or β-actin–Ig. Supernatants were collected 48 h later, and IL-10 levels were determined by standard ELISA. The results represent three experiments done in triplicates, and are the mean IL-10 concentration ± SE. (E) Dose-dependent inhibition of CXCL12-induced migration of anti-CD3/anti-CD28–activated spleen T cells from naive C57BL/6 mice. Results are shown as the mean ± SE of three independent experiments with similar results. (F and G) IL-10 and IL-2 production of anti-CD3/anti-CD28–activated spleen T cells in the presence of 100 ng/ml CXCL12, 100 nM AMD3100, or CXCL12 plus 100 nM AMD3100. The results represent three experiments done in triplicates and are shown as the mean cytokine concentration ± SE.
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fig3: CXCL12-Ig preserves the biological activities of native CXCL12. (A) Purified CXCL12-Ig was separated on 12% SDS-PAGE and subjected to Western blot analysis under reducing and nonreducing conditions (with or without β-mercaptoethanol) using anti-CXCL12 mAb (clone 79014) as a primary antibody (molecular masses are shown). (B) THP-1 cells (human monocytic cell line) were subjected to a migration assay using a Transwell system. Lower chambers were supplemented with culture medium, rCXCL12, CXCL12-Ig, CXCL12-Ig plus anti-CXCR4 mAb, or β-actin–Ig. The number of cells migrating to the lower chamber was counted by FACS 3 h later. Results shown represent three independent experiments and are the mean of the migration percentage (number of cells that migrated to the lower chamber divided by the number of cells originally plated in the upper chamber) ± SE. (C) Freshly isolated peritoneal macrophages were supplemented with PBS, rCXCL12, or CXCL12-Ig. Supernatants were collected 48 h later, and the IL-10 concentration was determined by ELISA. The results shown represent three experiments done in triplicates, and are the mean IL-10 concentration ± SE. (D) Primary spleen cell cultures responding to their target MOGp35-55 antigen were supplemented with PBS, rCXCL12, CXCL12-Ig, or β-actin–Ig. Supernatants were collected 48 h later, and IL-10 levels were determined by standard ELISA. The results represent three experiments done in triplicates, and are the mean IL-10 concentration ± SE. (E) Dose-dependent inhibition of CXCL12-induced migration of anti-CD3/anti-CD28–activated spleen T cells from naive C57BL/6 mice. Results are shown as the mean ± SE of three independent experiments with similar results. (F and G) IL-10 and IL-2 production of anti-CD3/anti-CD28–activated spleen T cells in the presence of 100 ng/ml CXCL12, 100 nM AMD3100, or CXCL12 plus 100 nM AMD3100. The results represent three experiments done in triplicates and are shown as the mean cytokine concentration ± SE.

Mentions: Chemokines possess a very short half-life time in vivo, and therefore, their potential use as drugs is limited. To overcome this, we adopted the strategy of generating Ig-based fusion proteins and constructed a chimeric protein composed of CXCL12 fused to IgG1 (Fc). The fusion protein was expressed as a disulphide-linked homodimer, similar to IgG1, and it had a molecular mass of ∼72 kD, consisting of two identical 36-kD subunits (Fig. 3 A). Next, we determined whether our CXCL12-Ig maintains the functional properties of the chemokine, including its ability to attract human THP-1 monocytic cell line cells (P < 0.001; Fig. 3 B), as well as Jurkat cells (not depicted), in a Transwell system. CXCL12-Ig was also tested for its ability to elicit IL-10 production in LPS-activated peritoneal macrophages (Fig. 3 C), and in primary T cells undergoing antigen-specific in vitro activation (Fig. 3 D). Both the commercially available rCXCL12 and our fusion protein could significantly (P < 0.01) induce IL-10 production in these cells.


CXCL12 (SDF-1alpha) suppresses ongoing experimental autoimmune encephalomyelitis by selecting antigen-specific regulatory T cells.

Meiron M, Zohar Y, Anunu R, Wildbaum G, Karin N - J. Exp. Med. (2008)

CXCL12-Ig preserves the biological activities of native CXCL12. (A) Purified CXCL12-Ig was separated on 12% SDS-PAGE and subjected to Western blot analysis under reducing and nonreducing conditions (with or without β-mercaptoethanol) using anti-CXCL12 mAb (clone 79014) as a primary antibody (molecular masses are shown). (B) THP-1 cells (human monocytic cell line) were subjected to a migration assay using a Transwell system. Lower chambers were supplemented with culture medium, rCXCL12, CXCL12-Ig, CXCL12-Ig plus anti-CXCR4 mAb, or β-actin–Ig. The number of cells migrating to the lower chamber was counted by FACS 3 h later. Results shown represent three independent experiments and are the mean of the migration percentage (number of cells that migrated to the lower chamber divided by the number of cells originally plated in the upper chamber) ± SE. (C) Freshly isolated peritoneal macrophages were supplemented with PBS, rCXCL12, or CXCL12-Ig. Supernatants were collected 48 h later, and the IL-10 concentration was determined by ELISA. The results shown represent three experiments done in triplicates, and are the mean IL-10 concentration ± SE. (D) Primary spleen cell cultures responding to their target MOGp35-55 antigen were supplemented with PBS, rCXCL12, CXCL12-Ig, or β-actin–Ig. Supernatants were collected 48 h later, and IL-10 levels were determined by standard ELISA. The results represent three experiments done in triplicates, and are the mean IL-10 concentration ± SE. (E) Dose-dependent inhibition of CXCL12-induced migration of anti-CD3/anti-CD28–activated spleen T cells from naive C57BL/6 mice. Results are shown as the mean ± SE of three independent experiments with similar results. (F and G) IL-10 and IL-2 production of anti-CD3/anti-CD28–activated spleen T cells in the presence of 100 ng/ml CXCL12, 100 nM AMD3100, or CXCL12 plus 100 nM AMD3100. The results represent three experiments done in triplicates and are shown as the mean cytokine concentration ± SE.
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Related In: Results  -  Collection

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fig3: CXCL12-Ig preserves the biological activities of native CXCL12. (A) Purified CXCL12-Ig was separated on 12% SDS-PAGE and subjected to Western blot analysis under reducing and nonreducing conditions (with or without β-mercaptoethanol) using anti-CXCL12 mAb (clone 79014) as a primary antibody (molecular masses are shown). (B) THP-1 cells (human monocytic cell line) were subjected to a migration assay using a Transwell system. Lower chambers were supplemented with culture medium, rCXCL12, CXCL12-Ig, CXCL12-Ig plus anti-CXCR4 mAb, or β-actin–Ig. The number of cells migrating to the lower chamber was counted by FACS 3 h later. Results shown represent three independent experiments and are the mean of the migration percentage (number of cells that migrated to the lower chamber divided by the number of cells originally plated in the upper chamber) ± SE. (C) Freshly isolated peritoneal macrophages were supplemented with PBS, rCXCL12, or CXCL12-Ig. Supernatants were collected 48 h later, and the IL-10 concentration was determined by ELISA. The results shown represent three experiments done in triplicates, and are the mean IL-10 concentration ± SE. (D) Primary spleen cell cultures responding to their target MOGp35-55 antigen were supplemented with PBS, rCXCL12, CXCL12-Ig, or β-actin–Ig. Supernatants were collected 48 h later, and IL-10 levels were determined by standard ELISA. The results represent three experiments done in triplicates, and are the mean IL-10 concentration ± SE. (E) Dose-dependent inhibition of CXCL12-induced migration of anti-CD3/anti-CD28–activated spleen T cells from naive C57BL/6 mice. Results are shown as the mean ± SE of three independent experiments with similar results. (F and G) IL-10 and IL-2 production of anti-CD3/anti-CD28–activated spleen T cells in the presence of 100 ng/ml CXCL12, 100 nM AMD3100, or CXCL12 plus 100 nM AMD3100. The results represent three experiments done in triplicates and are shown as the mean cytokine concentration ± SE.
Mentions: Chemokines possess a very short half-life time in vivo, and therefore, their potential use as drugs is limited. To overcome this, we adopted the strategy of generating Ig-based fusion proteins and constructed a chimeric protein composed of CXCL12 fused to IgG1 (Fc). The fusion protein was expressed as a disulphide-linked homodimer, similar to IgG1, and it had a molecular mass of ∼72 kD, consisting of two identical 36-kD subunits (Fig. 3 A). Next, we determined whether our CXCL12-Ig maintains the functional properties of the chemokine, including its ability to attract human THP-1 monocytic cell line cells (P < 0.001; Fig. 3 B), as well as Jurkat cells (not depicted), in a Transwell system. CXCL12-Ig was also tested for its ability to elicit IL-10 production in LPS-activated peritoneal macrophages (Fig. 3 C), and in primary T cells undergoing antigen-specific in vitro activation (Fig. 3 D). Both the commercially available rCXCL12 and our fusion protein could significantly (P < 0.01) induce IL-10 production in these cells.

Bottom Line: The beneficial effect included selection of antigen-specific T cells that were CD4(+)CD25(-)Foxp3(-)IL-10(high), which could adoptively transfer disease resistance, and suppression of Th17 selection.However, in vitro functional analysis of these cells suggested that, even though CXCL12-Ig-induced tolerance is IL-10 dependent, IL-10-independent mechanisms may also contribute to their regulatory function.Collectively, our results not only demonstrate, for the first time, that a chemokine functions as a regulatory mediator, but also suggest a novel way for treating multiple sclerosis and possibly other inflammatory autoimmune diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel.

ABSTRACT
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system induced by antigen-specific effector Th17 and Th1 cells. We show that a key chemokine, CXCL12 (stromal cell-derived factor 1alpha), redirects the polarization of effector Th1 cells into CD4(+)CD25(-)Foxp3(-)interleukin (IL) 10(high) antigen-specific regulatory T cells in a CXCR4-dependent manner, and by doing so acts as a regulatory mediator restraining the autoimmune inflammatory process. In an attempt to explore the therapeutic implication of these findings, we have generated a CXCL12-immunoglobulin (Ig) fusion protein that, when administered during ongoing EAE, rapidly suppresses the disease in wild-type but not IL-10-deficient mice. Anti-IL-10 neutralizing antibodies could reverse this suppression. The beneficial effect included selection of antigen-specific T cells that were CD4(+)CD25(-)Foxp3(-)IL-10(high), which could adoptively transfer disease resistance, and suppression of Th17 selection. However, in vitro functional analysis of these cells suggested that, even though CXCL12-Ig-induced tolerance is IL-10 dependent, IL-10-independent mechanisms may also contribute to their regulatory function. Collectively, our results not only demonstrate, for the first time, that a chemokine functions as a regulatory mediator, but also suggest a novel way for treating multiple sclerosis and possibly other inflammatory autoimmune diseases.

Show MeSH
Related in: MedlinePlus