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Corticosteroids reverse cytokine-induced block of survival and differentiation of oligodendrocyte progenitor cells from rats.

Mann SA, Versmold B, Marx R, Stahlhofen S, Dietzel ID, Heumann R, Berger R - J Neuroinflammation (2008)

Bottom Line: Corticosterone, dihydrocorticosterone and, most prominently dexamethasone, counteracted the deleterious effects of IFN-gamma and TNF-alpha on cell survival, A2B5-immunostaining and expression of myelin basic protein.The most potent corticosteroid tested, dexamethasone, was shown to counteract cytokine effects on membrane surface extension and capacitance.Our results show that treatment of oligodendrocyte precursors with the inflammatory cytokines TNF-alpha and IFN-gamma block the differentiation of oligodendrocyte precursors at the level of the differentiation of the voltage-gated ion currents.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Neurobiochemistry, Ruhr University Bochum 44780, Germany. s.mann@victorchang.edu.au

ABSTRACT

Background: Periventricular leukomalacia (PVL) is a frequent complication of preterm delivery. Proinflammatory cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) released from astrocytes and microglia activated by infection or ischemia have previously been shown to impair survival and maturation of oligodendrocyte progenitors and could thus be considered as potential factors contributing to the generation of this disease. The first goal of the present study was to investigate whether exposure of oligodendrocyte precursors to these cytokines arrests the maturation of ion currents in parallel to its effects on myelin proteins and morphological maturation. Secondly, in the search for agents, that can protect differentiating oligodendrocyte precursor cells from cytokine-induced damage we investigated effects of coapplications of corticosteroids with proinflammatory cytokines on the subsequent survival and differentiation of oligodendrocyte progenitor cells.

Methods: To exclude influences from factors released from other cell types purified cultures of oligodendrocyte precursors were exposed to cytokines and/or steroids and allowed to differentiate for further 6 days in culture. Changes in membrane surface were investigated with capacitance recordings and Scanning Ion Conductance Microscopy. Na+- and K+- currents were investigated using whole cell patch clamp recordings. The expression of myelin specific proteins was investigated using western blots and the precursor cells were identified using immunostaining with A2B5 antibodies.

Results: Surviving IFN-gamma and TNF-alpha treated cells continued to maintain voltage-activated Na+- and K+ currents characteristic for the immature cells after 6 days in differentiation medium. Corticosterone, dihydrocorticosterone and, most prominently dexamethasone, counteracted the deleterious effects of IFN-gamma and TNF-alpha on cell survival, A2B5-immunostaining and expression of myelin basic protein. The most potent corticosteroid tested, dexamethasone, was shown to counteract cytokine effects on membrane surface extension and capacitance. Furthermore, coapplication of dexamethasone blocked the cytokine-induced downregulation of the inwardly rectifying potassium current in 80% of the precursor cells and restored the cytokine-blocked down-regulation of the voltage activated Na+- and K+ currents during subsequent differentiation.

Conclusion: Our results show that treatment of oligodendrocyte precursors with the inflammatory cytokines TNF-alpha and IFN-gamma block the differentiation of oligodendrocyte precursors at the level of the differentiation of the voltage-gated ion currents. Co-treatment with corticosteroids at the time of cytokine application restores to a considerable extent survival and differentiation of oligodendrocytes at the level of morphological, myelin protein as well as ion current maturation suggesting the option for a functional restoration of cytokine-damaged immature oligodendrocytes.

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survival and A2B5 staining of oligodendrocytes following treatments with cytokines and corticosteroids. Phase contrast microphotographs as well as anti-A2B5 staining after three days in proliferation medium followed by 6 days in differentiation medium. Note, that after treatment of oligodendrocyte precursors with TNFα (10 ng/ml) and IFNγ (10 U/ml) for 48 h on 2–3 days in vitro (C) cells display mostly bipolar morphologies typical for progenitor cells, maintaining their A2B5 immunopositivity. Coapplication of dexamethasone during cytokine treatment (D) in the proliferation medium restored the extension of multipolar arborizations and the downregulation of A2B5 staining. (E) Average number of cells per field of view at culture day 9 (for every bar 25 fields of view from 12 coverslips of the control cultures and 4 coverslips of the corticosteroid treated cultures were counted). In both, control (left bars) and in cytokine-treated cells (right bars) corticosteroids caused a significant increase in the total number of cells surviving in culture. (F) Percentage of A2B5 positive cells at culture day 9. Note, that in control cells not treated with cytokines (left four bars) no significant effect of corticosteroids on the number of A2B5-positive cells was detected, whereas in cytokine-treated cultures the percentage of A2B5-positive cells was decreased by cotreatment with corticosteroids (CS: corticosterone, DC: deoxycorticosterone, D: dexamethasone, no corticosteroid treatment (-) * p < 0.05, ** p < 0.01, *** p < 0.001. For reasons of comparison the black bars in E and F are based on data published in [10], Figure 3B. Since cell counting was performed directly at the fluorescence microscope, fields of view were slightly larger than the photomicrographic frames shown. (G) Flow chart of the experimental design: After day 1 in proliferation medium, test cultures of oligodendrocyte precursors were treated for 48 hours with control proliferation medium or 10 U/ml IFN-γ and 10 ng/ml TNF-α in the presence or absence of glucocorticoids. The treatment was stopped by transferring the cells into differentiation medium. To judge long-term effects of treatments of precursor cells on subsequent differentiation most investigations were performed following 6 days in differentiation medium.
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Figure 1: survival and A2B5 staining of oligodendrocytes following treatments with cytokines and corticosteroids. Phase contrast microphotographs as well as anti-A2B5 staining after three days in proliferation medium followed by 6 days in differentiation medium. Note, that after treatment of oligodendrocyte precursors with TNFα (10 ng/ml) and IFNγ (10 U/ml) for 48 h on 2–3 days in vitro (C) cells display mostly bipolar morphologies typical for progenitor cells, maintaining their A2B5 immunopositivity. Coapplication of dexamethasone during cytokine treatment (D) in the proliferation medium restored the extension of multipolar arborizations and the downregulation of A2B5 staining. (E) Average number of cells per field of view at culture day 9 (for every bar 25 fields of view from 12 coverslips of the control cultures and 4 coverslips of the corticosteroid treated cultures were counted). In both, control (left bars) and in cytokine-treated cells (right bars) corticosteroids caused a significant increase in the total number of cells surviving in culture. (F) Percentage of A2B5 positive cells at culture day 9. Note, that in control cells not treated with cytokines (left four bars) no significant effect of corticosteroids on the number of A2B5-positive cells was detected, whereas in cytokine-treated cultures the percentage of A2B5-positive cells was decreased by cotreatment with corticosteroids (CS: corticosterone, DC: deoxycorticosterone, D: dexamethasone, no corticosteroid treatment (-) * p < 0.05, ** p < 0.01, *** p < 0.001. For reasons of comparison the black bars in E and F are based on data published in [10], Figure 3B. Since cell counting was performed directly at the fluorescence microscope, fields of view were slightly larger than the photomicrographic frames shown. (G) Flow chart of the experimental design: After day 1 in proliferation medium, test cultures of oligodendrocyte precursors were treated for 48 hours with control proliferation medium or 10 U/ml IFN-γ and 10 ng/ml TNF-α in the presence or absence of glucocorticoids. The treatment was stopped by transferring the cells into differentiation medium. To judge long-term effects of treatments of precursor cells on subsequent differentiation most investigations were performed following 6 days in differentiation medium.

Mentions: The effect of a transient treatment of progenitor cells with cytokines and the effect of a coapplication of corticosteroids on the subsequent differentiation into mature oligodendrocytes were studied using the culture protocol depicted in Figure 1G. Total cell numbers, percentages of A2B5 immunopositive cells, soma and process volumes and surface areas, membrane capacitances, the expression of myelin specific proteins and voltage activated Na+, K+ as well as inwardly rectifying K+ currents were evaluated following six days of differentiation in culture.


Corticosteroids reverse cytokine-induced block of survival and differentiation of oligodendrocyte progenitor cells from rats.

Mann SA, Versmold B, Marx R, Stahlhofen S, Dietzel ID, Heumann R, Berger R - J Neuroinflammation (2008)

survival and A2B5 staining of oligodendrocytes following treatments with cytokines and corticosteroids. Phase contrast microphotographs as well as anti-A2B5 staining after three days in proliferation medium followed by 6 days in differentiation medium. Note, that after treatment of oligodendrocyte precursors with TNFα (10 ng/ml) and IFNγ (10 U/ml) for 48 h on 2–3 days in vitro (C) cells display mostly bipolar morphologies typical for progenitor cells, maintaining their A2B5 immunopositivity. Coapplication of dexamethasone during cytokine treatment (D) in the proliferation medium restored the extension of multipolar arborizations and the downregulation of A2B5 staining. (E) Average number of cells per field of view at culture day 9 (for every bar 25 fields of view from 12 coverslips of the control cultures and 4 coverslips of the corticosteroid treated cultures were counted). In both, control (left bars) and in cytokine-treated cells (right bars) corticosteroids caused a significant increase in the total number of cells surviving in culture. (F) Percentage of A2B5 positive cells at culture day 9. Note, that in control cells not treated with cytokines (left four bars) no significant effect of corticosteroids on the number of A2B5-positive cells was detected, whereas in cytokine-treated cultures the percentage of A2B5-positive cells was decreased by cotreatment with corticosteroids (CS: corticosterone, DC: deoxycorticosterone, D: dexamethasone, no corticosteroid treatment (-) * p < 0.05, ** p < 0.01, *** p < 0.001. For reasons of comparison the black bars in E and F are based on data published in [10], Figure 3B. Since cell counting was performed directly at the fluorescence microscope, fields of view were slightly larger than the photomicrographic frames shown. (G) Flow chart of the experimental design: After day 1 in proliferation medium, test cultures of oligodendrocyte precursors were treated for 48 hours with control proliferation medium or 10 U/ml IFN-γ and 10 ng/ml TNF-α in the presence or absence of glucocorticoids. The treatment was stopped by transferring the cells into differentiation medium. To judge long-term effects of treatments of precursor cells on subsequent differentiation most investigations were performed following 6 days in differentiation medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562366&req=5

Figure 1: survival and A2B5 staining of oligodendrocytes following treatments with cytokines and corticosteroids. Phase contrast microphotographs as well as anti-A2B5 staining after three days in proliferation medium followed by 6 days in differentiation medium. Note, that after treatment of oligodendrocyte precursors with TNFα (10 ng/ml) and IFNγ (10 U/ml) for 48 h on 2–3 days in vitro (C) cells display mostly bipolar morphologies typical for progenitor cells, maintaining their A2B5 immunopositivity. Coapplication of dexamethasone during cytokine treatment (D) in the proliferation medium restored the extension of multipolar arborizations and the downregulation of A2B5 staining. (E) Average number of cells per field of view at culture day 9 (for every bar 25 fields of view from 12 coverslips of the control cultures and 4 coverslips of the corticosteroid treated cultures were counted). In both, control (left bars) and in cytokine-treated cells (right bars) corticosteroids caused a significant increase in the total number of cells surviving in culture. (F) Percentage of A2B5 positive cells at culture day 9. Note, that in control cells not treated with cytokines (left four bars) no significant effect of corticosteroids on the number of A2B5-positive cells was detected, whereas in cytokine-treated cultures the percentage of A2B5-positive cells was decreased by cotreatment with corticosteroids (CS: corticosterone, DC: deoxycorticosterone, D: dexamethasone, no corticosteroid treatment (-) * p < 0.05, ** p < 0.01, *** p < 0.001. For reasons of comparison the black bars in E and F are based on data published in [10], Figure 3B. Since cell counting was performed directly at the fluorescence microscope, fields of view were slightly larger than the photomicrographic frames shown. (G) Flow chart of the experimental design: After day 1 in proliferation medium, test cultures of oligodendrocyte precursors were treated for 48 hours with control proliferation medium or 10 U/ml IFN-γ and 10 ng/ml TNF-α in the presence or absence of glucocorticoids. The treatment was stopped by transferring the cells into differentiation medium. To judge long-term effects of treatments of precursor cells on subsequent differentiation most investigations were performed following 6 days in differentiation medium.
Mentions: The effect of a transient treatment of progenitor cells with cytokines and the effect of a coapplication of corticosteroids on the subsequent differentiation into mature oligodendrocytes were studied using the culture protocol depicted in Figure 1G. Total cell numbers, percentages of A2B5 immunopositive cells, soma and process volumes and surface areas, membrane capacitances, the expression of myelin specific proteins and voltage activated Na+, K+ as well as inwardly rectifying K+ currents were evaluated following six days of differentiation in culture.

Bottom Line: Corticosterone, dihydrocorticosterone and, most prominently dexamethasone, counteracted the deleterious effects of IFN-gamma and TNF-alpha on cell survival, A2B5-immunostaining and expression of myelin basic protein.The most potent corticosteroid tested, dexamethasone, was shown to counteract cytokine effects on membrane surface extension and capacitance.Our results show that treatment of oligodendrocyte precursors with the inflammatory cytokines TNF-alpha and IFN-gamma block the differentiation of oligodendrocyte precursors at the level of the differentiation of the voltage-gated ion currents.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Neurobiochemistry, Ruhr University Bochum 44780, Germany. s.mann@victorchang.edu.au

ABSTRACT

Background: Periventricular leukomalacia (PVL) is a frequent complication of preterm delivery. Proinflammatory cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) released from astrocytes and microglia activated by infection or ischemia have previously been shown to impair survival and maturation of oligodendrocyte progenitors and could thus be considered as potential factors contributing to the generation of this disease. The first goal of the present study was to investigate whether exposure of oligodendrocyte precursors to these cytokines arrests the maturation of ion currents in parallel to its effects on myelin proteins and morphological maturation. Secondly, in the search for agents, that can protect differentiating oligodendrocyte precursor cells from cytokine-induced damage we investigated effects of coapplications of corticosteroids with proinflammatory cytokines on the subsequent survival and differentiation of oligodendrocyte progenitor cells.

Methods: To exclude influences from factors released from other cell types purified cultures of oligodendrocyte precursors were exposed to cytokines and/or steroids and allowed to differentiate for further 6 days in culture. Changes in membrane surface were investigated with capacitance recordings and Scanning Ion Conductance Microscopy. Na+- and K+- currents were investigated using whole cell patch clamp recordings. The expression of myelin specific proteins was investigated using western blots and the precursor cells were identified using immunostaining with A2B5 antibodies.

Results: Surviving IFN-gamma and TNF-alpha treated cells continued to maintain voltage-activated Na+- and K+ currents characteristic for the immature cells after 6 days in differentiation medium. Corticosterone, dihydrocorticosterone and, most prominently dexamethasone, counteracted the deleterious effects of IFN-gamma and TNF-alpha on cell survival, A2B5-immunostaining and expression of myelin basic protein. The most potent corticosteroid tested, dexamethasone, was shown to counteract cytokine effects on membrane surface extension and capacitance. Furthermore, coapplication of dexamethasone blocked the cytokine-induced downregulation of the inwardly rectifying potassium current in 80% of the precursor cells and restored the cytokine-blocked down-regulation of the voltage activated Na+- and K+ currents during subsequent differentiation.

Conclusion: Our results show that treatment of oligodendrocyte precursors with the inflammatory cytokines TNF-alpha and IFN-gamma block the differentiation of oligodendrocyte precursors at the level of the differentiation of the voltage-gated ion currents. Co-treatment with corticosteroids at the time of cytokine application restores to a considerable extent survival and differentiation of oligodendrocytes at the level of morphological, myelin protein as well as ion current maturation suggesting the option for a functional restoration of cytokine-damaged immature oligodendrocytes.

Show MeSH
Related in: MedlinePlus