Cited by 108Neuroinflammation and Neurodegeneration MechanismsMultiple Sclerosis Research StudiesImmune cells in cancer

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Classically and alternatively activated bone marrow derived macrophages differ in cytoskeletal functions and migration towards specific CNS cell types

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Vereyken, Elly JF, Heijnen, Priscilla DAM, Baron, Wia, de Vries, Elga HE, Dijkstra, Christine D, Teunissen, Charlotte E·2011·Journal of Neuroinflammation

DOI 10.1186/1742-2094-8-58PMC PMC3123187

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Abstract

Background: Macrophages play an important role in neuroinflammatory diseases such as multiple sclerosis (MS) and spinal cord injury (SCI), being involved in both damage and repair. The divergent effects of macrophages might be explained by their different activation status: classically activated (CA/M1), pro-inflammatory, macrophages and alternatively activated (AA/M2), growth promoting, macrophages. Little is known about the effect of macrophages with these phenotypes in the central nervous system (CNS) and how they influence pathogenesis. The aim of this study was therefore to determine the characteristics of these phenotypically different macrophages in the context of the CNS in an in vitro setting. Results: Here we show that bone marrow derived CA and AA macrophages have a distinct migratory capacity towards medium conditioned by various cell types of the CNS. AA macrophages were preferentially attracted by the low weight (< 10 kD) fraction of neuronal conditioned medium, while CA macrophages were attracted in higher numbers by astrocyte- and oligodendrocyte conditioned medium. Intrinsic motility was twice as high in AA macrophages compared to CA macrophages. The adhesion to extracellular matrix molecules (ECM) was significantly enhanced in CA macrophages compared to control and AA macrophages. The actin cytoskeleton was differentially organized between CA and AA macrophages, possibly due to greater activity of the GTPases RhoA and Rac in CA macrophages. Phagocytosis of myelin and neuronal fragments was increased in CA macrophages compared to AA macrophages. The increase in myelin phagocytosis was associated with higher expression of CR3/MAC-1 in CA macrophages. Conclusion: In conclusion, since AA macrophages are more motile and are attracted by NCM, they are prone to migrate towards neurons in the CNS. CA macrophages have a lower motility and a stronger adhesion to ECM. In neuroinflammatory diseases the restricted migration and motility of CA macrophages might limit lesion size due to bystander damage.

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Figure 1 Analysis of macrophage phenotype. A) Using FACS analysis the expression of markers for the differently activated phenotype was determined. CA macrophages expressed significantly more MAC-1 and F4/80 compared to AA and control macrophages. MR expression was significantly higher on AA macrophages compared to control and CA macrophages. B) NO production was undetectable in both control and AA macrophages. CA macrophages did produce NO.CC BY · PubMed Central

Figure 2 Migration of macrophages towards conditioned medium from several CNS cell types. Migration towards conditioned medium from various CNS cell types and chemokines by in vitro stimulated bone marrow derived macrophages was studied using a 48-wells micro chemotaxis chamber. The data are expressed as the mean of 4 separate experiments (n = 4) ± SEM. A) AA macrophages migrate in significantly higher numbers towards NCM compared to control medium, 191 and 77 respectively and also compared to CA macrophages, 57. * = p < 0.05. B) AA macrophages migrated in significantly higher numbers towards the fraction of the NCM containing proteins smaller than 10 kD compared to CA macrophages. Both the fractions containing proteins smaller than 10 kD and between 10 and 50 kD significantly enhanced migration of AA macrophages compared to control medium. * = p < 0.05. C) CA macrophages migrate in significantly higher numbers towards ACM compared to control medium, 92 and 59 macrophages respectively. AA macrophages did not specifically migrate towards ACM. No difference was seen in the number of migrated macrophages towards ACM between CA and AA macrophages. * = p < 0.05. D) The fraction of the ACM containing proteins smaller than 10 kD significantly enhanced the migration of CA macrophages compared to control medium and AA macrophages. * = p < 0.05. E) The different stages of oligodendrocyte development conditioned medium significantly enhanced migration of both CA and AA macrophages compared to oligodendrocyte control medium, however CA macrophages migrated in significantly higher numbers compared to AA and control macrophages. * = p < 0.05 is significantly different from control medium; # is p < 0.05 different from AA and control macrophages.CC BY · PubMed Central

Figure 3 Motility and migratory capacity. Motility of in vitro stimulated bone marrow derived macrophages was determined by timelapse video microscopy. Speed and distance covered were determined using TrackIt software. The migration towards fMLP and MCP-1 was used as a measure for migratory capacity of in vitro stimulated bone marrow derived macrophages. Adhesion was determined by plating fluorescently labeled macrophages on (un)coated 96 wells plates, washing the plates after 2 h and measuring fluorescence. Data are the mean of 3 separate experiments (n = 3) ± SEM. *= p < 0.05. A) During the recorded time the average distance AA macrophages covered was significantly enhanced twofold compared to CA macrophages. The distance was recorded in μm. B) AA macrophages had a twofold higher speed compared to CA macrophages. Speed was recorded in μm/sec. C) No difference in migration was seen between the different macrophage phenotypes when comparing migration towards either fMLP or MCP-1. D) CA macrophages displayed a trend towards higher adherence on plastic compared to control and AA macrophages. On fibronectin and collagen adherence of CA macrophages was twofold higher compared to both control and AA macrophages. E) The expression of adhesion receptors was higher in CA macrophages compared to both AA and control macrophages.CC BY · PubMed Central

Figure 4 Cytoskeleton of differently activated macrophages. A-C) Visualization of actin cytoskeleton, red: actin (rhodamine phalloidin); blue: nuclear staining (Hoechst). Representative pictures are shown. CA macrophages appear flattened and rounded. AA macrophages are more stretched and elongated. Control macrophages appear to possess morphology in between the two extremes, with some cells being more elongated while others are more rounded. The scale bar is 50 μm and a 40 times magnification was used. D) Rac and Rho activity. In CA macrophages small GTPases are more activated compared to both control and AA macrophages.CC BY · PubMed Central

Figure 5 Phagocytosis of myelin by differently activated macrophages. Phagocytosis of fluorescently labeled myelin by activated bone marrow derived macrophages and expression of CR3 was determined using flow cytometry. MFI was measured and data are the mean of 4 separate experiments (n = 4) ± SEM. *= p < 0.05 and #= p < 0.05. A) CA macrophages phagocytosed significantly more myelin compared to control and AA macrophages. B-D) Visualization of myelin internalization by phagocytosis, red: DiI labeled myelin; green: Mac-1 on macrophage membrane. Representative images, taken on a confocal microscope, are shown. Red myelin is seen inside the green labeled membrane of the differently activated macrophages. All macrophage phenotypes appear to have taken up myelin. A 20 times magnification was used. E) Using anti-MAC-1 antibody, phagocytosis of myelin by CA and control macrophages could be blocked significantly, as indicated by #. Anti-MAC-1 antibody did not affect myelin phagocytosis by AA macrophages.CC BY · PubMed Central

Figure 6 Influence of DPI and flavonoids on phagocytosis of myelin. To determine the influence of DPI, a NADPH oxidase blocker, and flavonoids on myelin phagocytosis, these substances were added together with fluorescently labeled myelin. Phagocytosis is determined as MFI of the macrophages. Data are the mean of 4 separate experiments (n = 4) ± SEM. *= p < 0.05. Both luteolin and quercetin significantly blocked phagocytosis of myelin by control, CA and AA macrophages. DPI only significantly reduced phagocytosis of myelin by control and AA macrophages. DPI did not significantly decrease phagocytosis of myelin by CA macrophages.CC BY · PubMed Central

Figure 7 Phagocytosis of neuronal fragments by differently activated macrophages. Phagocytosis of fluorescently labeled neuronal fragments by activated bone marrow derived macrophages was determined using flow cytometry. Phagocytosis was blocked using anti-MAC-1 antibodies, luteolin, quercetin and DPI. Data are the mean of 4 separate experiments (n = 4) ± SEM. *= p < 0.05 and #= p < 0.05. A) CA macrophages phagocytosed significantly more neuronal fragments compared to control and AA macrophages. B-D) Visualization of neuronal fragment internalization by phagocytosis, red: DiI labeled neuronal fragments; green: Mac-1 on macrophage membrane. Representative images, taken on a confocal microscope (Leica TCS SP2), are shown. Red labelled neuronal fragments are clearly present within the green labelled membrane of the differently activated macrophages. Some binding is also present, with the neuronal fragments being present outside the membrane. A 20 times magnification was used. E) To determine whether phagocytosis could be blocked macrophages were exposed to luteolin, quercetin, MAC-1 (anti-CR3 antibody) or DPI (NADPH oxidase blocker). Phagocytosis of neuronal fragments by all types of macrophages was significantly reduced by exposure to luteolin and quercetin. Using MAC-1 antibody did not reduce neuronal fragment phagocytosis. DPI led to a significant reduction in control macrophages, however, CA and AA macrophages did not reduce phagocytosis significantly.CC BY · PubMed Central

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Authors: Vereyken, E. J., Heijnen, P. D., Baron, W., de Vries, E. H., Dijkstra, C. D., & Teunissen, C. E.
Affiliations: Amsterdam UMC Location Vrije Universiteit Amsterdam (NL)
University Medical Center Groningen (NL)
Corresponding author: Dijkstra, Christine D
ORCID: Baron, Wia
Teunissen, Charlotte E
Venue: Journal of Neuroinflammation
Publisher: Springer Science and Business Media LLC
Published: 2011-05-26
Volume: 8
Issue: 1
Pages: 58-58
ISSN: 1742-2094 (ISSN-L)
DOI: 10.1186/1742-2094-8-58
PMC: PMC3123187
OpenAlex: W2165155335
Cited by: 108
References: 85
Citations by year: 2026: 6 · 2025: 5 · 2024: 5 · 2023: 11 · 2022: 6 · 2021: 11 · 2020: 8 · 2019: 5 · 2018: 8 · 2017: 7 · 2016: 5 · 2015: 7 · 2014: 9 · 2013: 6 · 2012: 6
FWCI: 4.06
Type: journal article
Language: en
Open access: gold
doi.org
Licence: cc-by
Locations: Journal of Neuroinflammation (publishedVersion, cc-by)
PubMed Central (publishedVersion, cc-by)
PubMed (publishedVersion)
Amsterdam UMC (VU Amsterdam) - Institutional Repository (publishedVersion, other-oa)
University of Groningen research database (University of Groningen / Centre for Information Technology) (publishedVersion, cc-by)
Digital Academic REpository of VU University Amsterdam (Vrije Universiteit Amsterdam) (submittedVersion)
DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Pure Amsterdam UMC (submittedVersion, other-oa)
University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion, cc-by)
Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
Funders: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (016.056.024)
Topics: Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Health Sciences › Medicine › Clinical Neurology › Multiple Sclerosis Research Studies
Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
Keywords: macrophage polarization, M1 macrophages, M2 macrophages, bone marrow-derived macrophages, macrophage migration, conditioned medium, actin cytoskeleton, RhoA, Rac GTPase, extracellular matrix adhesion, phagocytosis, myelin phagocytosis, astrocytes, oligodendrocytes, neurons, neuroinflammation, multiple sclerosis, spinal cord injury
MeSH terms: Actins, Animals, Cell Movement, Cells, Cultured, Central Nervous System, Cytoskeleton, Flavonoids, Macrophages, Mice, Inbred C57BL, Myelin Sheath, Neuroglia, Neurons, Phagocytosis, Phenotype, Culture Media, Conditioned, Mice

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Classically and alternatively activated bone marrow derived macrophages differ in cytoskeletal functions and migration towards specific CNS cell types

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