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Cited by 134Multiple Sclerosis Research StudiesPhagocytosis and Immune RegulationNeuroinflammation and Neurodegeneration Mechanisms

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Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during Experimental Autoimmune Encephalomyelitis

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Weinger, Jason G, Brosnan, Celia F, Loudig, Olivier, Goldberg, Michael F, Macian, Fernando, Arnett, Heather A, Prieto, Anne L, Tsiperson, Vladislav, Shafit-Zagardo, Bridget·2011·Journal of Neuroinflammation
DOI 10.1186/1742-2094-8-49PMC PMC3121615
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Abstract

BACKGROUND: Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. METHODS: WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays. RESULTS: Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals. CONCLUSIONS: These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.

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Figure 1 Axl-/- mice have a more severe clinical course than WT mice during MOG-induced EAE. a) EAE clinical course for Axl-/- and WT female mice. Three experiments were combined: WT (n = 15) and Axl-/- (n = 21) mice. All mice were housed in Barrier. b) Western blot analysis was performed using an Axl pAb on 80 μg of WT naïve (untreated), WT EAE, Tyro3-/- naïve (untreated), and Tyro3-/- EAE lumbar spinal cord homogenates. The mature Axl band migrates at 140 kDa, and the immature band (lacking post translational modifications) migrates at approximately 110 kDa. N = 3 for all groups except WT EAE where n = 4. c) The relative densitometric intensity (rdi) was determined for each band and normalized to β-actin. The average values for 140 kDa Axl are shown. * = p < 0.05, # = p < 0.01.
Figure 1 Axl-/- mice have a more severe clinical course than WT mice during MOG-induced EAE. a) EAE clinical course for Axl-/- and WT female mice. Three experiments were combined: WT (n = 15) and Axl-/- (n = 21) mice. All mice were housed in Barrier. b) Western blot analysis was performed using an Axl pAb on 80 μg of WT naïve (untreated), WT EAE, Tyro3-/- naïve (untreated), and Tyro3-/- EAE lumbar spinal cord homogenates. The mature Axl band migrates at 140 kDa, and the immature band (lacking post translational modifications) migrates at approximately 110 kDa. N = 3 for all groups except WT EAE where n = 4. c) The relative densitometric intensity (rdi) was determined for each band and normalized to β-actin. The average values for 140 kDa Axl are shown. * = p < 0.05, # = p < 0.01.CC BY · PubMed Central
Figure 2 No difference in the number of leukocytes or cytokine expression from CD4+ T cells in the CNS. Mononuclear cell preparations from brain and spinal cord were stained for flow cytometric analysis to detect differences in the phenotype of cellular infiltrates (a). The gating strategy allowed us to discriminate populations of microglia, macrophages, neutrophils, NK cells, CD4 and CD8 T cells (a) as well as CD4 T cell subsets (b). CD4 T cells were restimulated with MOG35-55 peptide and analyzed for cytokine production (c). Multi-parametric flow cytometric analysis was performed on polyfunctional CD4 T cell subsets capable of producing 3, 2, or 1 cytokine(s) simultaneously. Student's t-test revealed no significant differences between WT and Axl-/- mice. These data are representative of two experiments each performed in triplicate, with 4 mice/group/experiment at day 19.
Figure 2 No difference in the number of leukocytes or cytokine expression from CD4+ T cells in the CNS. Mononuclear cell preparations from brain and spinal cord were stained for flow cytometric analysis to detect differences in the phenotype of cellular infiltrates (a). The gating strategy allowed us to discriminate populations of microglia, macrophages, neutrophils, NK cells, CD4 and CD8 T cells (a) as well as CD4 T cell subsets (b). CD4 T cells were restimulated with MOG35-55 peptide and analyzed for cytokine production (c). Multi-parametric flow cytometric analysis was performed on polyfunctional CD4 T cell subsets capable of producing 3, 2, or 1 cytokine(s) simultaneously. Student's t-test revealed no significant differences between WT and Axl-/- mice. These data are representative of two experiments each performed in triplicate, with 4 mice/group/experiment at day 19.CC BY · PubMed Central
Figure 3 Transcripts for pro-inflammatory cytokine/chemokines are increased in the lumbar spinal cord of Axl-/- mice relative to WT during the acute phase of EAE. a) 45.7-fold increase of TNFα transcript in Axl-/- and WT lumbar spinal cord (combined) during the acute phase of EAE relative to naïve Axl-/- and WT mice (combined); 68.8-fold increase in Axl-/- EAE mice relative to untreated Axl-/-; 18.7-fold increase in WT EAE mice relative to WT untreated mice. b) Increased TNFα, MCP1 and RANTES transcripts were found in Axl-/- lumbar spinal cord compared to WT. Samples of RNA from lumbar spinal cord were grouped as untreated (non-MOG EAE-induced), mice with CI's ≤ 2, or mice with CI's > 2. The fold increase of pro-inflammatory cytokine/chemokines in Axl-/- lumbar spinal cord relative to WT lumbar spinal cord was determined. Fold increase for CI ≤ 2; TNFα = 8.1, MCP1 = 7.2, RANTES = 11.9. * p < 0.05, # p < 0.001.
Figure 3 Transcripts for pro-inflammatory cytokine/chemokines are increased in the lumbar spinal cord of Axl-/- mice relative to WT during the acute phase of EAE. a) 45.7-fold increase of TNFα transcript in Axl-/- and WT lumbar spinal cord (combined) during the acute phase of EAE relative to naïve Axl-/- and WT mice (combined); 68.8-fold increase in Axl-/- EAE mice relative to untreated Axl-/-; 18.7-fold increase in WT EAE mice relative to WT untreated mice. b) Increased TNFα, MCP1 and RANTES transcripts were found in Axl-/- lumbar spinal cord compared to WT. Samples of RNA from lumbar spinal cord were grouped as untreated (non-MOG EAE-induced), mice with CI's ≤ 2, or mice with CI's > 2. The fold increase of pro-inflammatory cytokine/chemokines in Axl-/- lumbar spinal cord relative to WT lumbar spinal cord was determined. Fold increase for CI ≤ 2; TNFα = 8.1, MCP1 = 7.2, RANTES = 11.9. * p < 0.05, # p < 0.001.CC BY · PubMed Central
Figure 4 Axl-/- mice have larger inflammatory cuffs and increased demyelination in the spinal cord than WT mice. One micron Trump's fixed cross-sections of WT (a,c,e) and Axl-/- (b,d,f) spinal cords were stained with toluidine blue. a,b) Representative 5× sections of whole spinal cord. Two 20× sections with areas of infiltration and demyelination are shown for each 5× section (WT: c,e; Axl-/-: d,f). The 20× sections are labeled with an asterisk (dorsal funiculus; c,d) or arrow (lateral funiculus; e,f) denoting the corresponding area on the 5× section. g) quantitative data for the mean lesion size (mm2), see methods. Ratio of CD3+ to Iba1+ cells per lesion.
Figure 4 Axl-/- mice have larger inflammatory cuffs and increased demyelination in the spinal cord than WT mice. One micron Trump's fixed cross-sections of WT (a,c,e) and Axl-/- (b,d,f) spinal cords were stained with toluidine blue. a,b) Representative 5× sections of whole spinal cord. Two 20× sections with areas of infiltration and demyelination are shown for each 5× section (WT: c,e; Axl-/-: d,f). The 20× sections are labeled with an asterisk (dorsal funiculus; c,d) or arrow (lateral funiculus; e,f) denoting the corresponding area on the 5× section. g) quantitative data for the mean lesion size (mm2), see methods. Ratio of CD3+ to Iba1+ cells per lesion.CC BY · PubMed Central
Figure 5 Demyelination and infiltration of CD45+ cells in spinal cords of Axl-/- mice are enhanced relative to WT mice. Seven micron paraffin-embedded day 14 WT and Axl-/- spinal cord sections were stained with MBP or CD45 mAb's, and frozen sections were stained with an Axl pAb, and were visualized by DAB. Representative images of WT (a,b) and Axl-/- (g,h,i,j,k) spinal cord stained for MBP. Arrows in a,g,i denote areas of demyelination shown at 40× in b,h,j, respectively. Asterisks in i denote demyelinated lesions; higher magnification of one lesion at 40× is shown in k. e,f) images of a WT mouse stained for Axl; the boxed area in e is shown at 20× in f. c,l) low magnification images of WT (c) and Axl-/- (l) mice stained with CD45. d) serial section of b stained for CD45. Serial sections of j,k stained for CD45 are shown in m,n.
Figure 5 Demyelination and infiltration of CD45+ cells in spinal cords of Axl-/- mice are enhanced relative to WT mice. Seven micron paraffin-embedded day 14 WT and Axl-/- spinal cord sections were stained with MBP or CD45 mAb's, and frozen sections were stained with an Axl pAb, and were visualized by DAB. Representative images of WT (a,b) and Axl-/- (g,h,i,j,k) spinal cord stained for MBP. Arrows in a,g,i denote areas of demyelination shown at 40× in b,h,j, respectively. Asterisks in i denote demyelinated lesions; higher magnification of one lesion at 40× is shown in k. e,f) images of a WT mouse stained for Axl; the boxed area in e is shown at 20× in f. c,l) low magnification images of WT (c) and Axl-/- (l) mice stained with CD45. d) serial section of b stained for CD45. Serial sections of j,k stained for CD45 are shown in m,n.CC BY · PubMed Central
Figure 6 Axl-/- mice have significantly fewer Iba1+ microglia/macrophages within and surrounding lesions than WT mice. Ten micron frozen WT and Axl-/- spinal cord sections were stained with an Iba1 pAb. Representative 40× images for WT (a) and Axl-/- (b) mice are shown. The arrows point to Iba1+ microglia/macrophages and the asterisks denote lesion area. c) Iba1+ activated microglia were counted in and surrounding multiple lesions in multiple mice for WT and Axl-/- mice. Statistical analysis was performed; student's t-test confirmed significance between WT and Axl-/- mice; p < 0.01.
Figure 6 Axl-/- mice have significantly fewer Iba1+ microglia/macrophages within and surrounding lesions than WT mice. Ten micron frozen WT and Axl-/- spinal cord sections were stained with an Iba1 pAb. Representative 40× images for WT (a) and Axl-/- (b) mice are shown. The arrows point to Iba1+ microglia/macrophages and the asterisks denote lesion area. c) Iba1+ activated microglia were counted in and surrounding multiple lesions in multiple mice for WT and Axl-/- mice. Statistical analysis was performed; student's t-test confirmed significance between WT and Axl-/- mice; p < 0.01.CC BY · PubMed Central
Figure 7 Double-label immunofluorescence confirm Axl-/- (b,d) mice have fewer Iba1+ microglia/macrophages than WT (a,c) mice in ventral (a,b), lateral and dorsal funiculi (c,d). Ten micron frozen spinal cord sections were stained with an Iba1 pAb (red), a CD3 mAb (green), counterstained with Hoechst stain (blue), and visualized by immunofluorescence. a,b) Left panel shows merged triple fluorescence, middle panel shows CD3/Hoechst, and right panel shows Iba1/Hoechst for the ventral funiculus of WT (a) and Axl-/- (b) spinal cords. c,d) Merged images of lateral and dorsal funiculi for WT and Axl-/- spinal cords. All images are 40×. e) CD3+/Iba1+ cells/lesion for WT and Axl-/- mice.
Figure 7 Double-label immunofluorescence confirm Axl-/- (b,d) mice have fewer Iba1+ microglia/macrophages than WT (a,c) mice in ventral (a,b), lateral and dorsal funiculi (c,d). Ten micron frozen spinal cord sections were stained with an Iba1 pAb (red), a CD3 mAb (green), counterstained with Hoechst stain (blue), and visualized by immunofluorescence. a,b) Left panel shows merged triple fluorescence, middle panel shows CD3/Hoechst, and right panel shows Iba1/Hoechst for the ventral funiculus of WT (a) and Axl-/- (b) spinal cords. c,d) Merged images of lateral and dorsal funiculi for WT and Axl-/- spinal cords. All images are 40×. e) CD3+/Iba1+ cells/lesion for WT and Axl-/- mice.CC BY · PubMed Central
Figure 8 Axl-/- mice do not clear myelin debris as efficiently as WT mice. Ten micron frozen WT and Axl-/- spinal cord sections were stained with the lipid dye Oil-Red-O (a,b,c,d,g). Myelin debris appears as dense red clumps. Arrowheads in g show sparse Oil-Red-O+ deposits in the WT lesion. Representative 10× (a,b,c,d left panel) and corresponding 40× (a,b,c,d right panel) images are shown for the ventral (a,c) and lateral (b,d) funiculi of WT (a,b) and Axl-/- mice (c,d, e,f,g,h,i) 40× images of sections showing H+E (e), MBP (f), Iba1 (h) and MAC3 (i) for WT and Axl-/- mice.
Figure 8 Axl-/- mice do not clear myelin debris as efficiently as WT mice. Ten micron frozen WT and Axl-/- spinal cord sections were stained with the lipid dye Oil-Red-O (a,b,c,d,g). Myelin debris appears as dense red clumps. Arrowheads in g show sparse Oil-Red-O+ deposits in the WT lesion. Representative 10× (a,b,c,d left panel) and corresponding 40× (a,b,c,d right panel) images are shown for the ventral (a,c) and lateral (b,d) funiculi of WT (a,b) and Axl-/- mice (c,d, e,f,g,h,i) 40× images of sections showing H+E (e), MBP (f), Iba1 (h) and MAC3 (i) for WT and Axl-/- mice.CC BY · PubMed Central
Figure 9 The spinal cord of Axl-/- mice has more axonal damage in the ventral and dorsal funiculi than WT mice. Seven micron paraffin-embedded WT and Axl-/- spinal cord sections were stained with SMI32, a mAb for non-phosphorylated neurofilaments. Representative 20× images are shown for the ventral and dorsal funiculi of WT and Axl-/- mice. b) SMI32+ non-phosphorylated neurofilament deposits were counted in the ventral and dorsal funiculi of three WT and three Axl-/- mice. Three or four sections were counted for each mouse and averaged. A student's t-test was performed to test significance; * = p < 0.05, # = p < 0.01.
Figure 9 The spinal cord of Axl-/- mice has more axonal damage in the ventral and dorsal funiculi than WT mice. Seven micron paraffin-embedded WT and Axl-/- spinal cord sections were stained with SMI32, a mAb for non-phosphorylated neurofilaments. Representative 20× images are shown for the ventral and dorsal funiculi of WT and Axl-/- mice. b) SMI32+ non-phosphorylated neurofilament deposits were counted in the ventral and dorsal funiculi of three WT and three Axl-/- mice. Three or four sections were counted for each mouse and averaged. A student's t-test was performed to test significance; * = p < 0.05, # = p < 0.01.CC BY · PubMed Central

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Authors
Weinger, J. G., Brosnan, C. F., Loudig, O., Goldberg, M. F., Macian, F., Arnett, H. A., Prieto, A. L., Tsiperson, V., & Shafit-Zagardo, B.
Affiliations
  • Albert Einstein College of Medicine (US)
  • Amgen (United States) (US)
  • Indiana University Bloomington (US)
ORCID
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2011-05-15
Volume
8
Issue
1
Pages
49-49
ISSN
1742-2094 (ISSN-L)
DOI
10.1186/1742-2094-8-49
PMC
PMC3121615
OpenAlex
W2081982964
Cited by
134
References
57
Citations by year
2026: 9 · 2025: 14 · 2024: 10 · 2023: 9 · 2022: 9 · 2021: 10 · 2020: 15 · 2019: 10 · 2018: 13 · 2017: 5 · 2016: 10 · 2015: 6 · 2014: 9 · 2013: 3 · 2012: 2
FWCI
3.14
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Journal of Neuroinflammation (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Multiple Sclerosis Society
  • Multiple Sclerosis Society
  • National Institutes of Health
  • National Institute of General Medical Sciences (GM 07491, T32GM007491)
  • National Institute of Allergy and Infectious Diseases (R01AI059738)
  • National Institute of Neurological Disorders and Stroke (R21NS061128)
Topics
  • Health Sciences › Medicine › Clinical Neurology › Multiple Sclerosis Research Studies
  • Life Sciences › Immunology and Microbiology › Immunology › Phagocytosis and Immune Regulation
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
AXL receptor tyrosine kinase, TAM receptors, Gas6, experimental autoimmune encephalomyelitis, multiple sclerosis, demyelination, myelin debris clearance, neuroinflammation, myelin oligodendrocyte glycoprotein, axonal injury
MeSH terms
Axl Receptor Tyrosine Kinase, Animals, Central Nervous System, Encephalomyelitis, Autoimmune, Experimental, Glycoproteins, Inflammation, Macrophages, Myelin Sheath, Peptide Fragments, Proto-Oncogene Proteins, Cytokines, Microglia, Mice, Knockout, Receptor Protein-Tyrosine Kinases, Mice, Myelin-Oligodendrocyte Glycoprotein

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