Limits...
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.

Show MeSH

Related in: MedlinePlus

Voltage-activated sodium currents after three days in proliferation medium and further six days in differentiation medium. (A) Original current traces from cell cultured for three days under control conditions recorded in Ba2+ containing extracellular solution to avoid distortion of voltage-activated currents by inwardly rectifying potassium currents. (B) Current to voltage relationship of peak sodium currents shown in A. (C) Fraction of cells showing a INa current larger than 50 pA for each tested culture 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). The number of cells tested under each condition is indicated within the corresponding column. (D) Average inward current densities of the cells listed posititve for this current in (C). Note, that d9TI cells show a similar percentage of cells with inward currents of similar amplitudes as proliferating progenitor cells. The single cell showing an inward current under condition d9CD was ignored for the analysis of the average current density. The sodium current densities were not statistically significantly different from each other. Error bars denote mean ± SE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2562366&req=5

Figure 4: Voltage-activated sodium currents after three days in proliferation medium and further six days in differentiation medium. (A) Original current traces from cell cultured for three days under control conditions recorded in Ba2+ containing extracellular solution to avoid distortion of voltage-activated currents by inwardly rectifying potassium currents. (B) Current to voltage relationship of peak sodium currents shown in A. (C) Fraction of cells showing a INa current larger than 50 pA for each tested culture 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). The number of cells tested under each condition is indicated within the corresponding column. (D) Average inward current densities of the cells listed posititve for this current in (C). Note, that d9TI cells show a similar percentage of cells with inward currents of similar amplitudes as proliferating progenitor cells. The single cell showing an inward current under condition d9CD was ignored for the analysis of the average current density. The sodium current densities were not statistically significantly different from each other. Error bars denote mean ± SE.

Mentions: In accordance with previous publications reporting voltage-activated Na+ currents in oligodendrocyte precursor cells 70% of the bipolar cells investigated after 3 days in proliferation medium showed voltage-activated sodium currents of more than 50 pA (Figure 4C, d3C), displaying an average Na+-current density of 12.8 ± 1.5 pA/pF (n = 34, mean ± SE, Figure 4D). In control cultures treated with dexamethasone we observed Na+ currents larger than 50 pA in a slightly larger population of 75% of the cells showing a slightly larger current density of 14.0 ± 1.7 pA/pF (n = 34). Oligodendrocyte precursor cell cultures treated for two days with IFN-γ + TNF-α showed a reduced percentage of 58% of cells expressing Na+-currents with insignificantly reduced (p > 0.05) amplitudes of 8.8 ± 0.8 pA/pF (n = 30, Figure 4C, D d3TI). Again, cotreatment with dexamethasone led to a slight increase in the number of cells expressing Na+ currents to 66% showing a slight, but insignificantly increased Na+ current amplitude of 9.2 ± 1.0 pA/pF (n = 37, Figure 4C, D d3TID). Following further six days in differentiation medium the cells had changed their morphological appearance to multipolar cells. In none of the 12 cells from control cultures investigated a voltage-activated Na+ current was found (Figure 4C, d9C). Likewise, in the dexamthasone-treated control cultures only one cell out of 13 cells investigated showed a Na+-current. In contrast, 77% of the cytokine treated cells that had been cultured in differentiation medium for 6 days and which had maintained a bipolar morphology still showed voltage – activated Na+ currents with an average amplitude of 8.6 ± 1.5 pA/pF (n = 21, Figure 4C and 4D, d9TI). To investigate whether dexamethasone affects the arrest of sodium current downregulation by TNF-α and INF-γ, Na+-currents were studied in cytokine and dexamethasone co-treated cells followed by further differentiation for 6 days. As depicted in Figure 4C, D following co-treatment with cytokines and dexamethasone none of the 11 cells investigated continued to show voltage-gated Na+-currents (Figure 4C – d9TID).


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)

Voltage-activated sodium currents after three days in proliferation medium and further six days in differentiation medium. (A) Original current traces from cell cultured for three days under control conditions recorded in Ba2+ containing extracellular solution to avoid distortion of voltage-activated currents by inwardly rectifying potassium currents. (B) Current to voltage relationship of peak sodium currents shown in A. (C) Fraction of cells showing a INa current larger than 50 pA for each tested culture 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). The number of cells tested under each condition is indicated within the corresponding column. (D) Average inward current densities of the cells listed posititve for this current in (C). Note, that d9TI cells show a similar percentage of cells with inward currents of similar amplitudes as proliferating progenitor cells. The single cell showing an inward current under condition d9CD was ignored for the analysis of the average current density. The sodium current densities were not statistically significantly different from each other. Error bars denote mean ± SE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Voltage-activated sodium currents after three days in proliferation medium and further six days in differentiation medium. (A) Original current traces from cell cultured for three days under control conditions recorded in Ba2+ containing extracellular solution to avoid distortion of voltage-activated currents by inwardly rectifying potassium currents. (B) Current to voltage relationship of peak sodium currents shown in A. (C) Fraction of cells showing a INa current larger than 50 pA for each tested culture 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). The number of cells tested under each condition is indicated within the corresponding column. (D) Average inward current densities of the cells listed posititve for this current in (C). Note, that d9TI cells show a similar percentage of cells with inward currents of similar amplitudes as proliferating progenitor cells. The single cell showing an inward current under condition d9CD was ignored for the analysis of the average current density. The sodium current densities were not statistically significantly different from each other. Error bars denote mean ± SE.
Mentions: In accordance with previous publications reporting voltage-activated Na+ currents in oligodendrocyte precursor cells 70% of the bipolar cells investigated after 3 days in proliferation medium showed voltage-activated sodium currents of more than 50 pA (Figure 4C, d3C), displaying an average Na+-current density of 12.8 ± 1.5 pA/pF (n = 34, mean ± SE, Figure 4D). In control cultures treated with dexamethasone we observed Na+ currents larger than 50 pA in a slightly larger population of 75% of the cells showing a slightly larger current density of 14.0 ± 1.7 pA/pF (n = 34). Oligodendrocyte precursor cell cultures treated for two days with IFN-γ + TNF-α showed a reduced percentage of 58% of cells expressing Na+-currents with insignificantly reduced (p > 0.05) amplitudes of 8.8 ± 0.8 pA/pF (n = 30, Figure 4C, D d3TI). Again, cotreatment with dexamethasone led to a slight increase in the number of cells expressing Na+ currents to 66% showing a slight, but insignificantly increased Na+ current amplitude of 9.2 ± 1.0 pA/pF (n = 37, Figure 4C, D d3TID). Following further six days in differentiation medium the cells had changed their morphological appearance to multipolar cells. In none of the 12 cells from control cultures investigated a voltage-activated Na+ current was found (Figure 4C, d9C). Likewise, in the dexamthasone-treated control cultures only one cell out of 13 cells investigated showed a Na+-current. In contrast, 77% of the cytokine treated cells that had been cultured in differentiation medium for 6 days and which had maintained a bipolar morphology still showed voltage – activated Na+ currents with an average amplitude of 8.6 ± 1.5 pA/pF (n = 21, Figure 4C and 4D, d9TI). To investigate whether dexamethasone affects the arrest of sodium current downregulation by TNF-α and INF-γ, Na+-currents were studied in cytokine and dexamethasone co-treated cells followed by further differentiation for 6 days. As depicted in Figure 4C, D following co-treatment with cytokines and dexamethasone none of the 11 cells investigated continued to show voltage-gated Na+-currents (Figure 4C – d9TID).

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