- Meeting abstract
Pro-inflammatory cytokines decrease the expression of tight junction proteins in Caco-2 intestinal epithelial cells through a process that is dependent on peroxynitrite formation and PARP activation
Critical Care volume 6, Article number: P102 (2002)
We sought to test the hypotheses that cytomix-induced hyperpermeability is mediated by changes in the expression of the tight junction proteins, ZO-1 and occludin, secondary to ONOO- formation and/or activation of the enzyme, poly (ADP-ribosyl) polymerase (PARP).
Caco-2 monolayers grown on permeable filters for 21 days in bicameral Transwell chambers were incubated with control medium, medium containing cytomix (CM), a cocktail containing the pro-inflammatory cytokines, IL-1β (1 μg/ml), TNF (10 ng/ml) and IFN-γ (1000 U/ml), or medium containing CM and one of these other pharmacologic agents: ethyl pyruvate (EP; H2O2 scavenger; 10 mM): PJ34 (PARP inhibitor; 5 μM): 3-aminobenzamide (3-AB; PARP inhibition; 3 μM): FeTPPS (ONOO- decomposition catalyst; 50 μM): C-PTIO (NO• scavenger; 100 μM): PDTC (NF-κB inhibitor; 100 μM) or L-NIL (selective iNOS inhibitor; 20 μM). Permeability was expressed as the apical-to-basolateral clearance (nLcm-1 h-1) of fluorescein-labelled dextran (FD4) during the last 48 hours of incubation. Expression of occludin and ZO-1 were assessed by Western blot and immunohistochemistry.
Western blot showed that after 48 hours of incubation with cytomix the intensity of the bands were significantly decreased as compared with the control group. After incubation with the various pharmacological agents with cytomix the intensity of the bands have significantly increased. Immunohistochemistry showed that ZO-1 and occludin were localized to the cell boundaries in control Caco-2 monolayers. Staining was continuous and 48 hours after treatment with CM, ZO-1 and occludin immunostaining was more diffuse. Coincubating cells with CM and agents that interrupted the NF-κB → iNOS → NO• → ONOO- → PARP pathway prevented and these alterations in the immunostaining of ZO-1 and occludin.
Taken together, our data support the view that CM increases the permeability of Caco-2 monolayers by activating NF-κB and initiating a chain of events that ultimately leads to PARP activation and decreased expression of the tight junction proteins, ZO-1 and occludin.
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Sappington, P., Han, X., Delude, R. et al. Pro-inflammatory cytokines decrease the expression of tight junction proteins in Caco-2 intestinal epithelial cells through a process that is dependent on peroxynitrite formation and PARP activation. Crit Care 6, P102 (2002). https://doi.org/10.1186/cc1555
- Western Blot