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Cited by 117Cellular transport and secretionNeuroinflammation and Neurodegeneration MechanismsSkin and Cellular Biology Research

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IFN-γ-induced increase in the mobility of MHC class II compartments in astrocytes depends on intermediate filaments

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Vardjan, Nina, Gabrijel, Mateja, Potokar, Maja, Švajger, Urban, Kreft, Marko, Jeras, Matjaž, de Pablo, Yolanda, Faiz, Maryam, Pekny, Milos, Zorec, Robert·2012·Journal of Neuroinflammation
DOI 10.1186/1742-2094-9-144PMC PMC3423045
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Abstract

Background: In immune-mediated diseases of the central nervous system, astrocytes exposed to interferon-γ (IFN-γ) can express major histocompatibility complex (MHC) class II molecules and antigens on their surface. MHC class II molecules are thought to be delivered to the cell surface by membrane-bound vesicles. However, the characteristics and dynamics of this vesicular traffic are unclear, particularly in reactive astrocytes, which overexpress intermediate filament (IF) proteins that may affect trafficking. The aim of this study was to determine the mobility of MHC class II vesicles in wild-type (WT) astrocytes and in astrocytes devoid of IFs. Methods: The identity of MHC class II compartments in WT and IF-deficient astrocytes 48 h after IFN-γ activation was determined immunocytochemically by using confocal microscopy. Time-lapse confocal imaging and Alexa Fluor 546-dextran labeling of late endosomes/lysosomes in IFN-γ treated cells was used to characterize the motion of MHC class II vesicles. The mobility of vesicles was analyzed using ParticleTR software. Results: Confocal imaging of primary cultures of WT and IF-deficient astrocytes revealed IFN-γ induced MHC class II expression in late endosomes/lysosomes, which were specifically labeled with Alexa Fluor 546-conjugated dextran. Live imaging revealed faster movement of dextran-positive vesicles in IFN-γ-treated than in untreated astrocytes. Vesicle mobility was lower in IFN-γ-treated IF-deficient astrocytes than in WT astrocytes. Thus, the IFN-γ-induced increase in the mobility of MHC class II compartments is IF-dependent. Conclusions: Since reactivity of astrocytes is a hallmark of many CNS pathologies, it is likely that the up-regulation of IFs under such conditions allows a faster and therefore a more efficient delivery of MHC class II molecules to the cell surface. In vivo, such regulatory mechanisms may enable antigen-presenting reactive astrocytes to respond rapidly and in a controlled manner to CNS inflammation.

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Figure 1 IFN-γ induces MHC class II expression on WT andGFAP-/-Vim-/- deficient mouse astrocytes. Cell-surface expression of MHC class II molecules on WT (left) and GFAP-/-Vim-/- (right) astrocytes was analyzed by flow cytometry in the absence (Ctrl.) and 48 h after the addition of 600 U/ml IFN-γ (+IFN-γ). Note that in WT cells, the fraction of low fluorescence intensity MHC class II positive cells upon IFN-γ treatment appears to be higher than in IFN-γ treated GFAP-/-Vim-/- astrocytes. Dotted lines indicate isotype controls; solid lines indicate anti-Alexa Fluor488-MHC class II antibodies. The results are representatives of four independent experiments.
Figure 1 IFN-γ induces MHC class II expression on WT andGFAP-/-Vim-/- deficient mouse astrocytes. Cell-surface expression of MHC class II molecules on WT (left) and GFAP-/-Vim-/- (right) astrocytes was analyzed by flow cytometry in the absence (Ctrl.) and 48 h after the addition of 600 U/ml IFN-γ (+IFN-γ). Note that in WT cells, the fraction of low fluorescence intensity MHC class II positive cells upon IFN-γ treatment appears to be higher than in IFN-γ treated GFAP-/-Vim-/- astrocytes. Dotted lines indicate isotype controls; solid lines indicate anti-Alexa Fluor488-MHC class II antibodies. The results are representatives of four independent experiments.CC BY · PubMed Central
Figure 2 Alexa Fluor546-dextran labels late endosomes/lysosomes in WT andGFAP-/-Vim-/- mouse astrocytes. Fluorescence images of WT (a) and GFAP-/-Vim-/- (b) astrocytes labeled with dextran and immunostained with antibodies against LAMP1, a marker of late endosomes/lysosomes. Merged images (overlay) show that the majority of dextran puncta are colocalized with LAMP1 puncta. Lower panels (insets) show boxed regions at higher magnification. Scale bars: 10 μm, 2 μm (insets). Arrowheads point to typical structures expressing both signals. Note that the green signal corresponding to the membrane bound LAMP-1 signal encircles the luminal signal of red dextran.
Figure 2 Alexa Fluor546-dextran labels late endosomes/lysosomes in WT andGFAP-/-Vim-/- mouse astrocytes. Fluorescence images of WT (a) and GFAP-/-Vim-/- (b) astrocytes labeled with dextran and immunostained with antibodies against LAMP1, a marker of late endosomes/lysosomes. Merged images (overlay) show that the majority of dextran puncta are colocalized with LAMP1 puncta. Lower panels (insets) show boxed regions at higher magnification. Scale bars: 10 μm, 2 μm (insets). Arrowheads point to typical structures expressing both signals. Note that the green signal corresponding to the membrane bound LAMP-1 signal encircles the luminal signal of red dextran.CC BY · PubMed Central
Figure 3 Alexa Fluor546-dextran labels MHC class II–positive compartments in IFN-γ-treated WT andGFAP-/-Vim-/- mouse astrocytes. Fluorescence images of control (Ctrl.) and IFN-γ-treated (+IFN-γ) WT (a) and GFAP-/-Vim-/- (b) astrocytes labeled with dextran, fixed, and immunostained with antibodies against MHC class II molecules. IFN-γ induces punctuate expression of MHC class II molecules. White pixels (mask) represent the colocalization mask of green (MHC II) and red fluorescence pixels (dextran). Note that the threshold value of 51 arbitrary units of 255 intensity levels, which corresponds to 20% of the maximum intensity level, was selected to separate the background intensity levels from the signal of single red and green pixels. Scale bars: 10 μm.
Figure 3 Alexa Fluor546-dextran labels MHC class II–positive compartments in IFN-γ-treated WT andGFAP-/-Vim-/- mouse astrocytes. Fluorescence images of control (Ctrl.) and IFN-γ-treated (+IFN-γ) WT (a) and GFAP-/-Vim-/- (b) astrocytes labeled with dextran, fixed, and immunostained with antibodies against MHC class II molecules. IFN-γ induces punctuate expression of MHC class II molecules. White pixels (mask) represent the colocalization mask of green (MHC II) and red fluorescence pixels (dextran). Note that the threshold value of 51 arbitrary units of 255 intensity levels, which corresponds to 20% of the maximum intensity level, was selected to separate the background intensity levels from the signal of single red and green pixels. Scale bars: 10 μm.CC BY · PubMed Central
Figure 4 MHC class II molecules are expressed in late endosomes/lysosomes in IFN-γ-activated WT andGFAP-/-Vim-/-mouse astrocytes. Fluorescence images of IFN-γ-activated WT (a) and GFAP-/-Vim-/- (b) astrocytes after immunostaining with antibodies against LAMP1 and MHC class II molecules. The two signals overlap to a great extent (overlay), indicating that IFN-γ induces expression and localization of MHC class II molecules in late endosomes/lysosomes in WT and GFAP-/-Vim-/- astrocytes. Lower panels (insets) show boxed regions at higher magnification. Scale bars: 10 μm; 2 μm (insets). Arrowheads point to typical structures expressing both signals.
Figure 4 MHC class II molecules are expressed in late endosomes/lysosomes in IFN-γ-activated WT andGFAP-/-Vim-/-mouse astrocytes. Fluorescence images of IFN-γ-activated WT (a) and GFAP-/-Vim-/- (b) astrocytes after immunostaining with antibodies against LAMP1 and MHC class II molecules. The two signals overlap to a great extent (overlay), indicating that IFN-γ induces expression and localization of MHC class II molecules in late endosomes/lysosomes in WT and GFAP-/-Vim-/- astrocytes. Lower panels (insets) show boxed regions at higher magnification. Scale bars: 10 μm; 2 μm (insets). Arrowheads point to typical structures expressing both signals.CC BY · PubMed Central
Figure 5 IFN-γ induces a larger increase in the mobility of dextran labeled vesicles in WT than inGFAP-/-Vim-/-mouse astrocytes. (a, b) Top, representative fluorescence images of a control (Ctrl.) and an IFN-γ-treated (+IFN-γ) WT astrocyte (a) and a GFAP-/-Vim-/- astrocyte (b) labeled with dextran. Bottom, trajectories of dextran-loaded vesicles in an exemplary cell before (Spon.) and after treatment with 1 mM ATP (ATP). The tracks of individual vesicles were monitored for 30 s (see also the movies, Additional files 1, 2, 3, 4, 5, 6, 7, 8). Vesicle tracks were longer in WT and GFAP-/-Vim-/- cells treated with IFN-γ than in untreated cells. ATP decreased vesicle mobility. Scale bars: 10 μm. (c) Histogram of average vesicle TLs in control (Ctrl.) and IFN-γ-treated (+IFN-γ) WT and GFAP-/-Vim-/- cells. (d) Histogram of mean MDs of vesicles in control (Ctrl.) and IFN-γ-treated (+IFN-γ) WT and GFAP-/-Vim-/- cells. Numbers on graphs are numbers of analyzed vesicles. Values are mean ± s.e.m. *P <0.05.
Figure 5 IFN-γ induces a larger increase in the mobility of dextran labeled vesicles in WT than inGFAP-/-Vim-/-mouse astrocytes. (a, b) Top, representative fluorescence images of a control (Ctrl.) and an IFN-γ-treated (+IFN-γ) WT astrocyte (a) and a GFAP-/-Vim-/- astrocyte (b) labeled with dextran. Bottom, trajectories of dextran-loaded vesicles in an exemplary cell before (Spon.) and after treatment with 1 mM ATP (ATP). The tracks of individual vesicles were monitored for 30 s (see also the movies, Additional files 1, 2, 3, 4, 5, 6, 7, 8). Vesicle tracks were longer in WT and GFAP-/-Vim-/- cells treated with IFN-γ than in untreated cells. ATP decreased vesicle mobility. Scale bars: 10 μm. (c) Histogram of average vesicle TLs in control (Ctrl.) and IFN-γ-treated (+IFN-γ) WT and GFAP-/-Vim-/- cells. (d) Histogram of mean MDs of vesicles in control (Ctrl.) and IFN-γ-treated (+IFN-γ) WT and GFAP-/-Vim-/- cells. Numbers on graphs are numbers of analyzed vesicles. Values are mean ± s.e.m. *P <0.05.CC BY · PubMed Central
Figure 6 IFN-γ increases the proportion of directional dextran labeled vesicles in live WT mouse astrocytes. Graph shows MD as a function of total TL over 30 s. Vesicles with MD >1 μm (dashed line) were characterized as directional vesicles (black circles; nd, number of directional vesicles). Other vesicles were characterized as non-directional vesicles (white circles; nn, number of nondirectional vesicles). (a) The percentage of directional vesicles in IFN-γ-treated WT cells was greater than in untreated control cells (20.3% vs. 8%; P <0.001). After ATP treatment, the percentage of directional vesicles decreased from 8% to 5.2% (P <0.01) in control cells and from 20.3% to 5.9% in IFN-γ-treated WT cells (P <0.001). (b) The percentage of directional vesicles in IFN-γ-treated GFAP-/-Vim-/- astrocytes did not change significantly compared to untreated control cells (4.4% vs. 5.7%, P = 0.081). After ATP treatment, the percentage of directional vesicles was not affected in control cells (4.4% vs. 3.9%; P = 0.602), but was reduced from 5.7% to 2.5% in IFN-γ-treated GFAP-/-Vim-/- cells (P <0.001). Note that the slopes of the lines fitted for directional and non-directional vesicles are presented in Table 2.
Figure 6 IFN-γ increases the proportion of directional dextran labeled vesicles in live WT mouse astrocytes. Graph shows MD as a function of total TL over 30 s. Vesicles with MD >1 μm (dashed line) were characterized as directional vesicles (black circles; nd, number of directional vesicles). Other vesicles were characterized as non-directional vesicles (white circles; nn, number of nondirectional vesicles). (a) The percentage of directional vesicles in IFN-γ-treated WT cells was greater than in untreated control cells (20.3% vs. 8%; P <0.001). After ATP treatment, the percentage of directional vesicles decreased from 8% to 5.2% (P <0.01) in control cells and from 20.3% to 5.9% in IFN-γ-treated WT cells (P <0.001). (b) The percentage of directional vesicles in IFN-γ-treated GFAP-/-Vim-/- astrocytes did not change significantly compared to untreated control cells (4.4% vs. 5.7%, P = 0.081). After ATP treatment, the percentage of directional vesicles was not affected in control cells (4.4% vs. 3.9%; P = 0.602), but was reduced from 5.7% to 2.5% in IFN-γ-treated GFAP-/-Vim-/- cells (P <0.001). Note that the slopes of the lines fitted for directional and non-directional vesicles are presented in Table 2.CC BY · PubMed Central

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Metadata

Authors
Vardjan, N., Gabrijel, M., Potokar, M., Švajger, U., Kreft, M., Jeras, M., de Pablo, Y., Faiz, M., Pekny, M., & Zorec, R.
Affiliations
  • University of Ljubljana (SI)
  • Blood Transfusion Centre of Slovenia (SI)
  • University of Gothenburg (SE)
Corresponding author
Zorec, Robert
ORCID
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2012-06-26
Volume
9
Issue
1
Pages
144-144
ISSN
1742-2094 (ISSN-L)
DOI
10.1186/1742-2094-9-144
PMC
PMC3423045
OpenAlex
W2139576028
Cited by
117
References
54
Citations by year
2026: 5 · 2025: 7 · 2024: 4 · 2023: 7 · 2022: 5 · 2021: 7 · 2020: 9 · 2019: 13 · 2018: 8 · 2017: 14 · 2016: 10 · 2015: 14 · 2014: 8 · 2013: 4 · 2012: 2
FWCI
4.24
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroinflammation (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • European Commission (279017, 237956)
  • Javna Agencija za Raziskovalno Dejavnost RS (J3-0133, P3-310, L1-2402, J3-4146, J3-0031)
  • Medical Research Council
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Skin and Cellular Biology Research
Keywords
interferon-gamma, MHC class II, astrocytes, intermediate filaments, vesicle mobility, late endosomes, lysosomes, antigen presentation, reactive astrocytes, vesicle trafficking, neuroinflammation
MeSH terms
Animals, Histocompatibility Antigens Class II, Astrocytes, Cell Compartmentation, Cells, Cultured, Interferon-gamma, Intermediate Filament Proteins, Mice, Inbred C57BL, Up-Regulation, Mice, Knockout, Protein Transport, Mice, Mice, 129 Strain

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