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Lymphocytes accelerate epithelial tight junction assembly: role of AMP-activated protein kinase (AMPK).

Tang XX, Chen H, Yu S, Zhang L, Caplan MJ, Chan HC - PLoS ONE (2010)

Bottom Line: In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone.This accelaration was found to be mediated by AMP-activated protein kinase (AMPK).AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Cell Biology Research Center, School of Biomedial Sciences, Faculty of Medicine, the Chinese University of Hong Kong, Shatin, Hong Kong.

ABSTRACT
The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK.

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Lymphocyte co-culture facilitates MDCK tight junction assembly.(a) Representative pictures of ZO-1 relocation to cell–cell junctions in MDCK cells cultured alone and co-cultured with lymphocytes at various time points (0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h) after calcium switch. (Scale bar: 30 µm). (b) Quantification of ZO-1 relocation to cell–cell junctions (length of ZO-1/cell (µm)) in MDCK cells cultured alone and co-cultured with lymphocytes at various time points (0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h) after calcium switch. The asterisks denote significant differences detected in the presence vs. the absence of lymphocytes by Student's t test (*, p<0.05; **, p<0.01).
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pone-0012343-g001: Lymphocyte co-culture facilitates MDCK tight junction assembly.(a) Representative pictures of ZO-1 relocation to cell–cell junctions in MDCK cells cultured alone and co-cultured with lymphocytes at various time points (0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h) after calcium switch. (Scale bar: 30 µm). (b) Quantification of ZO-1 relocation to cell–cell junctions (length of ZO-1/cell (µm)) in MDCK cells cultured alone and co-cultured with lymphocytes at various time points (0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h) after calcium switch. The asterisks denote significant differences detected in the presence vs. the absence of lymphocytes by Student's t test (*, p<0.05; **, p<0.01).

Mentions: The relocation of ZO-1 to cell–cell junctions represents an important step in the initiation of TJ assembly [9]. To evaluate the rate of calcium-induced TJ assembly, therefore, we monitored the time course of the ZO-1 relocation to the cell–cell junctions after the readdition of calcium in the MDCK cells with or without lymphocytes. At various time points measured, we found that ZO-1 relocation is accelerated significantly in the MDCK cells co-cultured with lymphocytes during calcium switch (Fig. 1a, b). Significant increase in the length of linear membrane-associated patches of ZO-1 per cell was observed in lymphocyte co-cultured MDCK cells compared to that observed in MDCK cells alone (Fig. 1a, b). This result indicates that lymphocytes can influence aspects of the TJ assembly in MDCK cells.


Lymphocytes accelerate epithelial tight junction assembly: role of AMP-activated protein kinase (AMPK).

Tang XX, Chen H, Yu S, Zhang L, Caplan MJ, Chan HC - PLoS ONE (2010)

Lymphocyte co-culture facilitates MDCK tight junction assembly.(a) Representative pictures of ZO-1 relocation to cell–cell junctions in MDCK cells cultured alone and co-cultured with lymphocytes at various time points (0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h) after calcium switch. (Scale bar: 30 µm). (b) Quantification of ZO-1 relocation to cell–cell junctions (length of ZO-1/cell (µm)) in MDCK cells cultured alone and co-cultured with lymphocytes at various time points (0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h) after calcium switch. The asterisks denote significant differences detected in the presence vs. the absence of lymphocytes by Student's t test (*, p<0.05; **, p<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2925955&req=5

pone-0012343-g001: Lymphocyte co-culture facilitates MDCK tight junction assembly.(a) Representative pictures of ZO-1 relocation to cell–cell junctions in MDCK cells cultured alone and co-cultured with lymphocytes at various time points (0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h) after calcium switch. (Scale bar: 30 µm). (b) Quantification of ZO-1 relocation to cell–cell junctions (length of ZO-1/cell (µm)) in MDCK cells cultured alone and co-cultured with lymphocytes at various time points (0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h) after calcium switch. The asterisks denote significant differences detected in the presence vs. the absence of lymphocytes by Student's t test (*, p<0.05; **, p<0.01).
Mentions: The relocation of ZO-1 to cell–cell junctions represents an important step in the initiation of TJ assembly [9]. To evaluate the rate of calcium-induced TJ assembly, therefore, we monitored the time course of the ZO-1 relocation to the cell–cell junctions after the readdition of calcium in the MDCK cells with or without lymphocytes. At various time points measured, we found that ZO-1 relocation is accelerated significantly in the MDCK cells co-cultured with lymphocytes during calcium switch (Fig. 1a, b). Significant increase in the length of linear membrane-associated patches of ZO-1 per cell was observed in lymphocyte co-cultured MDCK cells compared to that observed in MDCK cells alone (Fig. 1a, b). This result indicates that lymphocytes can influence aspects of the TJ assembly in MDCK cells.

Bottom Line: In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone.This accelaration was found to be mediated by AMP-activated protein kinase (AMPK).AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Cell Biology Research Center, School of Biomedial Sciences, Faculty of Medicine, the Chinese University of Hong Kong, Shatin, Hong Kong.

ABSTRACT
The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK.

Show MeSH
Related in: MedlinePlus