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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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Inwardly rectifying K+ currents after three days in proliferation medium and further six days in differentiation medium. (A) Original voltage clamp recordings of current traces from cell cultured after 9 days in control solution. No leak and capacitance subtraction used to visualize current components at a holding potential of -85 mV. (Aa) Current traces in physiological extracellular solution, (Ab) in the same solution containing in addition 1 mM Ba2+. (Ac) plot of the difference between traces shown in (Aa) and (Ab) corresponding to the inwardly rectifying K+ current (KIR). (B) Current versus voltage relationship for recordings shown in Ac (mean current between the dashed lines in Ac). (C) Fraction of cells showing a KIR current larger than 50 pA for each tested culture condition. The number of cells tested under each condition (control: C, pretreatment with TNF-α and IFN-γ for 48 hours:TI, control cultures treated with dexamethasone: CD, cultures co-treated with dexamethasone and TNF-α and IFN-γ for 48 hours: TID) is indicated within the corresponding column. (D) Average current densities of KIR for the cells listed positive for this current in (C). Note, that after 3 days in proliferation medium containing TNF-α and IFN-γ for 48 hrs the number of cells showing a KIR was reduced to only 20%. Error bars indicate ± SE.
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Figure 6: Inwardly rectifying K+ currents after three days in proliferation medium and further six days in differentiation medium. (A) Original voltage clamp recordings of current traces from cell cultured after 9 days in control solution. No leak and capacitance subtraction used to visualize current components at a holding potential of -85 mV. (Aa) Current traces in physiological extracellular solution, (Ab) in the same solution containing in addition 1 mM Ba2+. (Ac) plot of the difference between traces shown in (Aa) and (Ab) corresponding to the inwardly rectifying K+ current (KIR). (B) Current versus voltage relationship for recordings shown in Ac (mean current between the dashed lines in Ac). (C) Fraction of cells showing a KIR current larger than 50 pA for each tested culture condition. The number of cells tested under each condition (control: C, pretreatment with TNF-α and IFN-γ for 48 hours:TI, control cultures treated with dexamethasone: CD, cultures co-treated with dexamethasone and TNF-α and IFN-γ for 48 hours: TID) is indicated within the corresponding column. (D) Average current densities of KIR for the cells listed positive for this current in (C). Note, that after 3 days in proliferation medium containing TNF-α and IFN-γ for 48 hrs the number of cells showing a KIR was reduced to only 20%. Error bars indicate ± SE.

Mentions: After three days in proliferation medium 70% of the control cells investigated showed an inwardly rectifying K+ current exceeding 50 pA (mean value 12.3 ± 2.1 nA/pF – Figure 6C, D – d3C). Most of the inwardly rectifying K+ currents showed an inactivation at negative membrane potentials reflected in the negative slope of the I/V relationship (Figure 6Aa, B). We did not further examine whether this inactivation was voltage-dependent [32] or due to the presence of Na+ in the recording solution [30]. However, we only observed the inactivation in about 60% of the cells recorded from using identical solutions and voltage protocols, suggesting an inhomogeneous expression pattern in the oligodendrocytes. Following treatment with dexamethasone a similar fraction of 72% of the cells recorded from showed inwardly rectifying K+-currents, with an insignificantly reduced current density of 7.9 ± 0.5 pA/pF (n = 20, Figure 6C, D – d3CD). Remarkably, in oligodendrocyte precursor cells treated for two days with IFN-γ + TNF-α only 20% of the cells investigated showed an inwardly rectifying K+ current exceeding 50 pA (Figure 6 – d3TI). The average current density of the 9 cells out of 45 cells recorded, that showed a current exceeding 50 pA displayed a slightly, but insignificantly (p > 0.05) smaller amplitude as recorded in the control cells (9.1 ± 2.1 pA/pF). Most interestingly, co-treatment with dexamethasone restored the percentage of cells expressing inwardly rectifying potassium currents to 72%, showing almost identical current densities as observed in only dexamethasone treated control cultures (7.2 ± 0.7 pA/pF, n = 16).


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)

Inwardly rectifying K+ currents after three days in proliferation medium and further six days in differentiation medium. (A) Original voltage clamp recordings of current traces from cell cultured after 9 days in control solution. No leak and capacitance subtraction used to visualize current components at a holding potential of -85 mV. (Aa) Current traces in physiological extracellular solution, (Ab) in the same solution containing in addition 1 mM Ba2+. (Ac) plot of the difference between traces shown in (Aa) and (Ab) corresponding to the inwardly rectifying K+ current (KIR). (B) Current versus voltage relationship for recordings shown in Ac (mean current between the dashed lines in Ac). (C) Fraction of cells showing a KIR current larger than 50 pA for each tested culture condition. The number of cells tested under each condition (control: C, pretreatment with TNF-α and IFN-γ for 48 hours:TI, control cultures treated with dexamethasone: CD, cultures co-treated with dexamethasone and TNF-α and IFN-γ for 48 hours: TID) is indicated within the corresponding column. (D) Average current densities of KIR for the cells listed positive for this current in (C). Note, that after 3 days in proliferation medium containing TNF-α and IFN-γ for 48 hrs the number of cells showing a KIR was reduced to only 20%. Error bars indicate ± SE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Inwardly rectifying K+ currents after three days in proliferation medium and further six days in differentiation medium. (A) Original voltage clamp recordings of current traces from cell cultured after 9 days in control solution. No leak and capacitance subtraction used to visualize current components at a holding potential of -85 mV. (Aa) Current traces in physiological extracellular solution, (Ab) in the same solution containing in addition 1 mM Ba2+. (Ac) plot of the difference between traces shown in (Aa) and (Ab) corresponding to the inwardly rectifying K+ current (KIR). (B) Current versus voltage relationship for recordings shown in Ac (mean current between the dashed lines in Ac). (C) Fraction of cells showing a KIR current larger than 50 pA for each tested culture condition. The number of cells tested under each condition (control: C, pretreatment with TNF-α and IFN-γ for 48 hours:TI, control cultures treated with dexamethasone: CD, cultures co-treated with dexamethasone and TNF-α and IFN-γ for 48 hours: TID) is indicated within the corresponding column. (D) Average current densities of KIR for the cells listed positive for this current in (C). Note, that after 3 days in proliferation medium containing TNF-α and IFN-γ for 48 hrs the number of cells showing a KIR was reduced to only 20%. Error bars indicate ± SE.
Mentions: After three days in proliferation medium 70% of the control cells investigated showed an inwardly rectifying K+ current exceeding 50 pA (mean value 12.3 ± 2.1 nA/pF – Figure 6C, D – d3C). Most of the inwardly rectifying K+ currents showed an inactivation at negative membrane potentials reflected in the negative slope of the I/V relationship (Figure 6Aa, B). We did not further examine whether this inactivation was voltage-dependent [32] or due to the presence of Na+ in the recording solution [30]. However, we only observed the inactivation in about 60% of the cells recorded from using identical solutions and voltage protocols, suggesting an inhomogeneous expression pattern in the oligodendrocytes. Following treatment with dexamethasone a similar fraction of 72% of the cells recorded from showed inwardly rectifying K+-currents, with an insignificantly reduced current density of 7.9 ± 0.5 pA/pF (n = 20, Figure 6C, D – d3CD). Remarkably, in oligodendrocyte precursor cells treated for two days with IFN-γ + TNF-α only 20% of the cells investigated showed an inwardly rectifying K+ current exceeding 50 pA (Figure 6 – d3TI). The average current density of the 9 cells out of 45 cells recorded, that showed a current exceeding 50 pA displayed a slightly, but insignificantly (p > 0.05) smaller amplitude as recorded in the control cells (9.1 ± 2.1 pA/pF). Most interestingly, co-treatment with dexamethasone restored the percentage of cells expressing inwardly rectifying potassium currents to 72%, showing almost identical current densities as observed in only dexamethasone treated control cultures (7.2 ± 0.7 pA/pF, n = 16).

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