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GDNF protects enteric glia from apoptosis: evidence for an autocrine loop.

Steinkamp M, Gundel H, Schulte N, Spaniol U, Pflueger C, Zizer E, von Boyen GB - BMC Gastroenterol (2012)

Bottom Line: Increased GDNF expression and Caspase 3/7 activities were detected in in specimen of CD patients but not in healthy controls.Moreover, inactivation of GDNF sensitized in EGC cell to IFN-γ/TNF-α induced apoptosis.Alterations in this novel EGC self-protecting mechanism could lead to a higher susceptibility towards apoptosis and thus contribute to disruption of the mucosal integrity and severity of inflammation in CD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, Endocrinology and Metabolism, University of Giessen und Marburg GmbH, Site Marburg, Baldingerstrasse, 35037 Marburg, Germany.

ABSTRACT

Background: Enteric glia cells (EGC) play an important role in the maintenance of intestinal mucosa integrity. During the course of acute Crohn's disease (CD), mucosal EGC progressively undergo apoptosis, though the mechanisms are largely unknown. We investigated the role of Glial-derived neurotrophic factor (GDNF) in the regulation of EGC apoptosis.

Methods: GDNF expression and EGC apoptosis were determined by immunofluorescence using specimen from CD patients. In primary rat EGC cultures, GDNF receptors were assessed by western blot and indirect immunofluorescence microscopy. Apoptosis in cultured EGC was induced by TNF-α and IFN-γ, and the influence of GDNF on apoptosis was measured upon addition of GDNF or neutralizing anti-GDNF antibody.

Results: Increased GDNF expression and Caspase 3/7 activities were detected in in specimen of CD patients but not in healthy controls. Moreover, inactivation of GDNF sensitized in EGC cell to IFN-γ/TNF-α induced apoptosis.

Conclusions: This study proposes the existence of an autocrine anti-apoptotic loop in EGC cells which is operative in Crohn's disease and dependent of GDNF. Alterations in this novel EGC self-protecting mechanism could lead to a higher susceptibility towards apoptosis and thus contribute to disruption of the mucosal integrity and severity of inflammation in CD.

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Activation of caspase 3/7 activity in primary EGC cultures. Fluorometric detection of caspase 3/7 activity after incubation of the EGC cultures with TNF-α (100 ng/ml), IFN-γ (100 ng/ml), neutralizing anti-GDNF-antibody (0.5 μg/ml), alone or in combination as described above. The bars indicate the mean values ± SD, normalized to controls, three independent experiments were performed in duplicates (N = 3). Asterisk indicates a significant difference between both values (p < 0.05).
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Figure 5: Activation of caspase 3/7 activity in primary EGC cultures. Fluorometric detection of caspase 3/7 activity after incubation of the EGC cultures with TNF-α (100 ng/ml), IFN-γ (100 ng/ml), neutralizing anti-GDNF-antibody (0.5 μg/ml), alone or in combination as described above. The bars indicate the mean values ± SD, normalized to controls, three independent experiments were performed in duplicates (N = 3). Asterisk indicates a significant difference between both values (p < 0.05).

Mentions: Since glial cells are assumed as highly apoptosis resistant cells, we aimed to expose these cells to an inflammatory cytokine environment using TNF-α and IFN-γ. Neither of these factors alone was able to induce apoptosis in primary rat EGC cultures, but in combination they significantly increased apoptosis rates in the cultures by nearly 2 fold, as indicated by a caspase 3/7 activation in these cells. Noteworthy, we have also tested various other combinations of important cytokines with implication in Crohn's disease (e.g. IL-1ß, IL-6) but failed to induce apoptosis (data not shown). The addition of GDNF had no effect on the apoptosis rate in the cell cultures, but it was also not able to alter the apoptosis rate in the cultures after induction of apoptosis by the combination of TNF-α and IFN-γ (Figure 4). However, if a neutralizing antibody against GDNF was added to neutralize endogenous secreted GDNF, the apoptosis rates in the cultures that were stimulated with TNF-α and IFN-γ raised significantly (Figure 5). Therefore, it is likely that an autocrine antiapoptotic loop is activated in EGC when exposed to inflammatory conditions.


GDNF protects enteric glia from apoptosis: evidence for an autocrine loop.

Steinkamp M, Gundel H, Schulte N, Spaniol U, Pflueger C, Zizer E, von Boyen GB - BMC Gastroenterol (2012)

Activation of caspase 3/7 activity in primary EGC cultures. Fluorometric detection of caspase 3/7 activity after incubation of the EGC cultures with TNF-α (100 ng/ml), IFN-γ (100 ng/ml), neutralizing anti-GDNF-antibody (0.5 μg/ml), alone or in combination as described above. The bars indicate the mean values ± SD, normalized to controls, three independent experiments were performed in duplicates (N = 3). Asterisk indicates a significant difference between both values (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298702&req=5

Figure 5: Activation of caspase 3/7 activity in primary EGC cultures. Fluorometric detection of caspase 3/7 activity after incubation of the EGC cultures with TNF-α (100 ng/ml), IFN-γ (100 ng/ml), neutralizing anti-GDNF-antibody (0.5 μg/ml), alone or in combination as described above. The bars indicate the mean values ± SD, normalized to controls, three independent experiments were performed in duplicates (N = 3). Asterisk indicates a significant difference between both values (p < 0.05).
Mentions: Since glial cells are assumed as highly apoptosis resistant cells, we aimed to expose these cells to an inflammatory cytokine environment using TNF-α and IFN-γ. Neither of these factors alone was able to induce apoptosis in primary rat EGC cultures, but in combination they significantly increased apoptosis rates in the cultures by nearly 2 fold, as indicated by a caspase 3/7 activation in these cells. Noteworthy, we have also tested various other combinations of important cytokines with implication in Crohn's disease (e.g. IL-1ß, IL-6) but failed to induce apoptosis (data not shown). The addition of GDNF had no effect on the apoptosis rate in the cell cultures, but it was also not able to alter the apoptosis rate in the cultures after induction of apoptosis by the combination of TNF-α and IFN-γ (Figure 4). However, if a neutralizing antibody against GDNF was added to neutralize endogenous secreted GDNF, the apoptosis rates in the cultures that were stimulated with TNF-α and IFN-γ raised significantly (Figure 5). Therefore, it is likely that an autocrine antiapoptotic loop is activated in EGC when exposed to inflammatory conditions.

Bottom Line: Increased GDNF expression and Caspase 3/7 activities were detected in in specimen of CD patients but not in healthy controls.Moreover, inactivation of GDNF sensitized in EGC cell to IFN-γ/TNF-α induced apoptosis.Alterations in this novel EGC self-protecting mechanism could lead to a higher susceptibility towards apoptosis and thus contribute to disruption of the mucosal integrity and severity of inflammation in CD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, Endocrinology and Metabolism, University of Giessen und Marburg GmbH, Site Marburg, Baldingerstrasse, 35037 Marburg, Germany.

ABSTRACT

Background: Enteric glia cells (EGC) play an important role in the maintenance of intestinal mucosa integrity. During the course of acute Crohn's disease (CD), mucosal EGC progressively undergo apoptosis, though the mechanisms are largely unknown. We investigated the role of Glial-derived neurotrophic factor (GDNF) in the regulation of EGC apoptosis.

Methods: GDNF expression and EGC apoptosis were determined by immunofluorescence using specimen from CD patients. In primary rat EGC cultures, GDNF receptors were assessed by western blot and indirect immunofluorescence microscopy. Apoptosis in cultured EGC was induced by TNF-α and IFN-γ, and the influence of GDNF on apoptosis was measured upon addition of GDNF or neutralizing anti-GDNF antibody.

Results: Increased GDNF expression and Caspase 3/7 activities were detected in in specimen of CD patients but not in healthy controls. Moreover, inactivation of GDNF sensitized in EGC cell to IFN-γ/TNF-α induced apoptosis.

Conclusions: This study proposes the existence of an autocrine anti-apoptotic loop in EGC cells which is operative in Crohn's disease and dependent of GDNF. Alterations in this novel EGC self-protecting mechanism could lead to a higher susceptibility towards apoptosis and thus contribute to disruption of the mucosal integrity and severity of inflammation in CD.

Show MeSH
Related in: MedlinePlus