Granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis

Abstract

Experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, can be induced by immunization with a number of myelin antigens. In particular, myelin oligodendrocyte glycoprotein, a central nervous system (CNS)-specific antigen expressed on the myelin surface, is able to induce a paralytic MS-like disease with extensive CNS inflammation and demyelination in several strains of animals. Although not well understood, the egress of immune cells into the CNS in EAE is governed by a complex interplay between pro and antiinflammatory cytokines and chemokines. The hematopoietic growth factor, granulocyte macrophage colony-stimulating factor (GM-CSF), is considered to play a central role in maintaining chronic inflammation. The present study was designed to investigate the previously unexplored role of GM-CSF in autoimmune-mediated demyelination. GM-CSF(-/)- mice are resistant to EAE, display decreased antigen-specific proliferation of splenocytes, and fail to sustain immune cell infiltrates in the CNS, thus revealing key activities for GM-CSF in the development of inflammatory demyelinating lesions and control of migration and/or proliferation of leukocytes within the CNS. These results hold implications for the pathogenesis of inflammatory and demyelinating diseases and may provide the basis for more effective therapies for inflammatory diseases, and more specifically for multiple sclerosis.

Figure 1

GM-CSF regulates the onset and progression of clinical EAE provoked by MOG_35–55 peptide. Mice were immunized with MOG_35–55 peptide emulsified in CFA and monitored daily for the development of clinical disease. Mean clinical disease score in GM-CSF^−/− and WT mice (n = 8 for each group). Note this is a representative of three independent experiments; in total 20/21 GM-CSF^−/− mice failed to develop any clinical symptoms. The single mouse that developed symptoms did not progress past a clinical score of 1. In contrast all WT mice developed clinical symptoms.

Figure 2

Mean clinical disease score in (A) GM-CSF^−/− and (B) WT mice treated with rGM-CSF (n = 5 for each group). rGM-CSF (10 ng/mouse) was injected subcutaneously every day, starting at day 0 until day 40 after immunization with MOG_35–55 peptide.

Figure 5

Production of cytokines, GM-CSF (A), IL-2 (B), IFN-γ (C), and IL-6 (D) by spleen cells from GM-CSF^−/− and WT mice immunized with MOG_35–55 peptide. Cytokine concentrations were measured from supernatants after 72 h of in vitro culture with MOG_35–55 peptide. Each bar represents the mean concentration ± SEM (n = 6 for both groups), each tested in triplicate.

Figure 3

Brain histopathology of paraffin sections from mice immunized with MOG_35–55. H&E staining showing typical inflammatory cuffing around blood vessels with cellular infiltration into the CNS parenchyma of WT mice killed (A) 18 and (C) 40 d after immunization (original magnification: ×10). H&E staining showing small focal inflammatory lesions in GM-CSF^−/− mice killed 18 d after immunization (B) and no inflammation in GM-CSF^−/− mice killed 40 d after immunization (D) (original magnification: ×10). 26–30 sagital sections per mouse (n = 6 for each group) were examined without knowledge of the injection regime by two independent investigators.

Figure 4

Antigen-specific proliferative responses by spleen cells from mice immunized with MOG_35–55 peptide. Cells from GM-CSF^−/− and WT mice (n = 6 for both groups) were isolated 18 d after disease induction and stimulated in vitro with (A) MOG_35–55 peptide and (B) Con A. At day 40 cells were taken from GM-CSF^−/− and WT mice, and GM-CSF^−/− and WT mice treated with rGM-CSF (n = 5 for each group) and stimulated in vitro with (C) MOG_35–55 peptide and (D) Con A. Each bar represents the mean stimulation indices ± SEM.

Figure 7

Influence of anti–GM-CSF mAb on the clinical course of MOG_35–55-induced EAE in WT mice. (A) Mean clinical score of MOG_35–55-induced EAE in WT mice treated by intraperitoneal injection with PBS (n = 4), anti–GM-CSF mAb (n = 8), or isotype control IgG (n = 8, anti-βgal) from time of sensitization with MOG_35–55 peptide until day 24. (B) Mean clinical score of MOG_35–55-induced EAE in WT mice treated by intraperitoneal injection with PBS, anti–GM-CSF mAb, or isotype control IgG (n = 6 for each group) from first signs of clinical disease (score = 2) until day 33.

Figure 6

MOG-specific Ab response in mice immunized with MOG_35–55 peptide. Serum was collected 40 d after sensitization and tested by ELISA with (A) rMOG and (B) MOG_35–55 peptide-coated plates. (C), Ig isotype response was assessed by ELISA 33 with MOG_35–55 peptide-coated plates. Each bar represents the mean specific absorbance (corrected against BSA coated plates) ± SEM (n = 5 for each group), each tested in triplicate at 1:100 dilutions. For the GM-CSF^−/− mice and WT controls this study was repeated (n = 8) with no difference in results (data not shown).

Figure 6

MOG-specific Ab response in mice immunized with MOG_35–55 peptide. Serum was collected 40 d after sensitization and tested by ELISA with (A) rMOG and (B) MOG_35–55 peptide-coated plates. (C), Ig isotype response was assessed by ELISA 33 with MOG_35–55 peptide-coated plates. Each bar represents the mean specific absorbance (corrected against BSA coated plates) ± SEM (n = 5 for each group), each tested in triplicate at 1:100 dilutions. For the GM-CSF^−/− mice and WT controls this study was repeated (n = 8) with no difference in results (data not shown).

Figure 6

MOG-specific Ab response in mice immunized with MOG_35–55 peptide. Serum was collected 40 d after sensitization and tested by ELISA with (A) rMOG and (B) MOG_35–55 peptide-coated plates. (C), Ig isotype response was assessed by ELISA 33 with MOG_35–55 peptide-coated plates. Each bar represents the mean specific absorbance (corrected against BSA coated plates) ± SEM (n = 5 for each group), each tested in triplicate at 1:100 dilutions. For the GM-CSF^−/− mice and WT controls this study was repeated (n = 8) with no difference in results (data not shown).