Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers

Main methods Caco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-α, IFN-γ, IL-1), or Cytomix + PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin. Key findings Cytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix + PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization. Significance Treatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown. Keywords Tight junction ZO-1 Occludin Phosphodiesterase inhibition Pentoxifylline Caco-2 cells Intestinal permeability Shock Cytokines Inflammation Tight junction Introduction Multiple organ failure (MOF) remains a significant cause of morbidity and mortality after shock despite numerous advances in critical care. Exaggeration of the immune response can result in neutrophil activation, generation of pro-inflammatory cytokines, and distant organ injury. It is clear that the gut plays a key role in this process. Recent research has suggested the “gut-lymph” hypothesis for MOF, with studies indicating that gut derived factors carried in the intestinal lymph are key mediators of systemic inflammation ( Senthil et al., 2006; Deitch et al., 2004; Magnotti et al., 1998 ). Intestinal epithelial barrier integrity, maintained by the tight junctions, is important in preventing the egress of luminal bacteria and endotoxin. Therefore, preventing intestinal inflammation and tight junction breakdown may limit systemic inflammation and decrease MOF after shock. The tight junction is the rate limiting step for paracellular transit and defines the overall barrier function of the intact intestinal epithelium ( Turner, 2006 ). Disruption of the tight junction can result in increased intestinal permeability. Tight junction function is regulated by both structural and functional elements. Actin and myosin form a ring encircling the cell at the level of the tight junction. Activation of myosin light chain kinase (MLCK) regulates contraction of this actomyosin ring, and has been shown to increase tight junction permeability in response to pro-inflammatory stimuli ( Ma et al., 2005; Blair et al., 2006 ). The structural proteins occludin and zonula occludens protein-1 (ZO-1) have also been shown to play an important role in the maintenance of epithelial barrier integrity. Previous studies have shown decreased expression of occludin and ZO-1 in response to pro-inflammatory cytokines ( Bruewer et al., 2003; Poritz et al., 2007 ). In addition, these proteins are redistributed away from the tight junction during times of increased permeability ( Musch et al., 2006 ). These inflammatory changes to the epithelial tight junction have been shown to be mediated, in part, by nuclear factor kappa-B (NF-κB) ( Ma et al., 2004; Al-Sadi and Ma, 2007 ). Pentoxifylline (PTX, 1-[5-oxohexyl]-3,7-dimethylxanthine), a methylxanthine derivative and non-specific phosphodiesterase inhibitor, has been used extensively in the clinical setting for the treatment of peripheral vascular disease due to its ability to improve microcirculation. In our lab we have studied PTX as an immunomodulatory adjunct used during fluid resuscitation in shock and sepsis. We have previously shown that PTX decreases neutrophil degranulation, pro-inflammatory mediator synthesis, and lung injury in animal models of shock ( Coimbra et al., 2006a,b, 2005a,b ). We have also shown that PTX attenuates histologic gut injury and decreases intestinal cytokine production, including the NF-κB signaling pathway ( Deree et al., 2007a,b ). The effects of phosphodiesterase inhibition on tight junction proteins in immunostimulated intestinal epithelial cells have yet to be investigated. Modulation of occludin and ZO-1 has been shown to involve NF-κB signaling. PTX is known to decrease inflammation, at least in part, through the modulation of the NF-κB pathway. In this series of experiments, we hypothesized that the addition of PTX to immunostimulated intestinal epithelial cells will decrease the breakdown and reorganization of the tight junction proteins occludin and ZO-1. Materials and methods Cell culture Caco-2 human intestinal epithelial cells were obtained from American Type Culture Collection (ATCC, Manasas, VA). Caco-2 cells were grown at 37 °C in a 5% CO 2 humidified atmosphere. Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with high glucose (Gibco, Carlsbad, CA) supplemented with 10% FBS (ATCC), Sodium Pyruvate (2 mM, Gibco), penicillin G (10,000 U/ml, Gibco), streptomycin (10,000 μg/ml, Gibco), and 1% nonessential amino acids (Gibco). Culture media was changed every 3–4 days. Immunostimulation of Caco-2 cell monolayers Caco-2 cells (1 × 10 6  cells/well) were seeded on permeable filters with 8 μm pore size in 12-well Transwell bicameral chambers (BD Bioscience, San Jose, CA). Cells were allowed to grow for 3 days in DMEM. On day 3, DMEM was removed and Basal Seeding media (BD Bioscience, San Jose, CA) was added to the chamber for 1 day. After removal of the Basal Seeding media, cells were grown for 2 days in the presence of Enterocyte Differentiation media (BD Bioscience) to induce differentiation of the Caco-2 cells. Cells were then serum deprived overnight prior to beginning the experiment. Cells were treated with HBSS or Cytomix (IFN-γ (1000 U/ml), TNF-α (10 ng/ml), and IL-1β (10 ng/ml)) alone or in combination with PTX (2 mM, Sigma, St. Louis, MO) for 24 h at 37 °C in a 5% CO 2 . At the end of the experimental period the media was removed and cells were washed with cold HBSS. Monolayers were scraped into Eppendorf tubes and the Caco-2 cells were lysed in 500 μL lysis buffer (50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.5% deoxycholic acid, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1× protease inhibitor). Cell lysates were stored at − 70 °C for later analysis. Immunoblotting Total protein concentration of each extract was determined using the bicinchoninic acid (BCA) protein assay using the microplate procedure (Pierce, Rockford, IL). Samples containing 7 μg of protein were suspended in Sodium dodecyl sulfate (SDS) sample buffer (Invitrogen, Carlsbad, CA) and collected by boiling the samples at 100 °C for 5 min. Proteins were separated by SDS-Polyacrylamide gel electrophoresis using 8–16% tris-glycine polyacrylamide gradient gels and subsequently transferred to nitrocellulose membranes (Invitrogen). The membranes were blocked with 5% BSA in tris-buffered saline (TBS)/Tween 20 for 1 h. Primary antibodies specific for ZO-1 (1:500, Zymed), Occludin (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), or Beta Actin (1:500 Cell Signaling, Danvers, MA) were incubated with the membranes overnight at 4 °C in 5% BSA with TBS/Tween 20. Membranes were washed and incubated for 1 h at room temperature with horseradish peroxidase-linked anti-rabbit IgG (1:2000, Cell Signaling) prepared in blocking solution. After thorough washing, the Pierce Supersignal West Pico Chemiluminescent Kit was applied for antibody detection with X-ray film (Amersham Biosciences, Piscataway, NJ). Mean pixel density was estimated using UN-SCAN-IT Gel Digitizing software (Silk Scientific, Orem, UT). Western blot band density was compared to Beta actin in each lane as a loading control. Data is expressed as the relative band density of each experimental group compared to control ( n ≥ 3 per group). Confocal microscopy Glass cover slips (Fisher Scientific, Pittsburgh, PA) were incubated with 0.01% poly- l -lysine (Sigma) and sterilized under ultraviolet (UV) light under a laminar flow hood. Confluent monolayers of Caco-2 cells from each experimental group were placed on dried glass cover slips. Cells were fixed with 3.7% paraformaldehyde (Electron Microscopy Series, Hatfield, PA) for 10 min. Cover slips were washed with PBS, then cells were permeabilized with 0.01% Triton X-100 (Sigma) in PBS for 1 min. After washing with PBS, the cells were blocked for 1 h using 1% BSA. Cells were incubated overnight with the primary antibody (ZO-1, occludin, 1:100) in 1% BSA. After washing with PBS, the cells were treated with the secondary antibody, Alexa Fluor 488 (1:100, Invitrogen), in 1% BSA for 1 h. Cover slips were inverted onto glass slides after the addition of Prolong Fade (Invitrogen). Glass slides were allowed to cure overnight, in the dark. Images were viewed using an Olympus Fluoview laser scanning confocal microscope (Olympus, Melville, NY) at 60× magnification using exposure matched settings (Advanced Software V1.6, Olympus, Center Valley, PA). Statistical analysis All values are expressed as the mean ± the SEM. Each assay was performed in duplicate where appropriate. Statistical significance of differences among groups was determined by analysis of variance (ANOVA) with Bonferroni correction. A p -value < 0.05 was considered statistically significant. Results Occludin expression Caco-2 cells treated with Cytomix resulted in a 53% ± 6 ( p < 0.01) decrease in occludin protein expression compared to control cells ( Fig. 1 ). Cells treated with Cytomix + PTX exhibited minimal decrease in occludin protein (98% ± 7 of control, p < 0.03 vs. Cytomix). Control cells treated with PTX alone showed no change in occludin protein expression. ZO-1 expression Caco-2 cell ZO-1 protein expression decreased 53% ± 2 ( p < 0.01) compared to control after treatment with Cytomix ( Fig. 2 ). The addition of PTX to Cytomix treated cells returned ZO-1 protein to control levels (97% ± 10 of control, p < 0.03). Control cells treated with PTX alone showed no significant change compared to control. Confocal microscopy ZO-1 is a cytoplasmic protein that forms a link between the transmembrane occludin and peri-junctional actin within the cytoskeleton ( Fanning et al., 1998 ). Immunofluorescence staining of control Caco-2 monolayers shows ZO-1 localizing at the cellular borders, with a smooth, continuous staining that encircles the cell ( Fig. 3 A ). In monolayers treated with Cytomix, the appearance of ZO-1 becomes more disordered and irregular ( Fig. 3 B). Several areas of punctuate staining are noted throughout the cell, away from areas of cell–cell contact. Cytomix stimulated Caco-2 monolayers treated with PTX have a pattern of ZO-1 localization similar to control ( Fig. 3 C). Images of these PTX treated cells show a smooth, continuous circle of ZO-1 staining, with clear definition of the periphery of the cell. There is a similar pattern of localization in cells stained with fluorescent occludin antibody. Control cells exhibit a smooth pattern of staining, with localization seen at the periphery of the cell ( Fig. 3 D). Cytomix treatment alters the localization of occludin exhibited by a more discontinuous pattern of staining ( Fig. 3 E). Treatment with PTX decreases the Cytomix-induced alterations in occludin localization ( Fig. 3 F). Discussion Distant organ injury after shock occurs after an exaggerated host response to the inciting injury. It is believed that the gut may be the source of pro-inflammatory mediators, and may play a central role in the systemic inflammatory response that can lead to acute respiratory distress syndrome (ARDS), MOF, and death. The importance of the gut as the “motor of MOF” has been recognized for decades, and has been the subject of extensive research. Studies by Deitch, et al. have shown that gut derived factors carried in the intestinal lymphatics after shock can cause acute lung injury and activate neutrophils ( Deitch, 2001; Davidson et al., 2004 ). This was further supported by their study showing that ligation of the mesenteric lymphatic ducts prevented distant organ injury ( Watkins et al., 2008 ). Increased intestinal permeability may allow bacteria and endotoxin from the gut to access normally protected portions of the intestinal wall, leading to a significant inflammatory response. Investigations into modulating intestinal epithelial permeability, specifically the tight junction breakdown, may be beneficial in attenuating the intestinal inflammatory response to shock, ultimately decreasing distant organ injury. PTX has been investigated for its immunomodulatory properties, with studies showing decreased mortality in neonatal sepsis and attenuation of inflammation in patients undergoing cardiac surgery ( Haque and Mohan, 2003; Heinze et al., 2007 ). Through its ability to increase the deformability of red blood cells, PTX is able to improve microcirculation and decrease ischemia-reperfusion injury ( Yada-Langui et al., 2004 ). In our lab, we have extensively researched the use of PTX as an immunomodulatory adjunct to resuscitation in models of hemorrhagic shock and sepsis ( Coimbra et al., 2004, 2005a,b, 2006a,b; Deree et al., 2007a,b,c ). We have previously shown that the use of PTX with hypertonic saline as a resuscitation strategy decreases intestinal levels of the inducible form of nitric oxide synthase (iNOS) and nitric oxide (NO) compared to standard ringers lactate in a model of hemorrhagic shock. This study also noted a marked reduction of intestinal tumor necrosis factor-alpha (TNF-α), and a decrease in both cytoplasmic IκBα phosphorylation and nuclear factor kappa-B (NF-κB) activation ( Deree et al., 2007a ). Multiple prior studies have shown that immunostimulation of Caco-2 monolayers results in increased permeability ( Chavez et al., 1999; Boivin et al., 2007; Wang et al., 2006 ). Occludin is a 65 kDa transmembrane protein which is localized at tight junctions of both epithelial and endothelial cells. Occludin has 4 membrane spanning domains near its N-terminal end, which are integral in tight junction formation ( Furuse et al., 1993 ). ZO-1 is 225 kDa membrane associated protein that has been shown to co-localize with the cytoplasmic end of occludin at tight junctions ( Furuse et al., 1993; Willott et al., 1992 ). Proinflammatory cytokines have been shown to induce a decrease in occludin and ZO-1 protein levels, and change their cellular localization ( Ma et al., 2004; Al-Sadi and Ma, 2007; Han et al., 2003; Sappington et al., 2003; Cario et al., 2004 ). This study documents for the first time that treatment with PTX attenuates the breakdown and cellular redistribution of the key tight junction proteins occludin and ZO-1 in an in vitro model of immunostimulated intestinal epithelial cells. In this study, we chose the dose of PTX (2 mM) based on our extensive use of this drug in both in vitro and in vivo models. We have found this dose to be effective in attenuating inflammatory signaling with minimal cytotoxicity. The effects of PTX on intestinal occludin and ZO-1 may be related to changes in intracellular signaling. Epithelial barrier permeability is regulated by several signaling pathways involving, calcium, Ras, Rho, cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) ( Sawada et al., 2003 ). PTX is known to exert its effects through phosphodiesterase inhibition, resulting in increase intracellular levels of cAMP, and acts through both PKA dependent and independent mechanisms ( Semmler et al., 1993 ). Therefore, PTX may modulate the tight junction by activating PKA, which has been shown to improve ZO-1 recruitment to the tight junction ( Kohler et al., 2004 ). However, phosphodiesterase inhibition alone likely does not explain all of the immunomodulatory effects seen with PTX ( Windmeier and Gressner, 1997 ). The mitogen-activated protein kinase (MAPK) signaling pathway is involved in the regulation of occludin and ZO-1, and may be another mechanism by which PTX modulates these tight junction proteins. The regulation of occludin and ZO-1 occurs via phosphorylation and dephosphorylation, which involves signaling via the MAPK pathway ( Basuroy et al., 2006 ). PTX has previously been shown to attenuate this inflammatory signaling cascade. Specifically, we have shown that PTX inhibits both p38 MAPK and ERK 1/2 activation in human neutrophils ( Deree et al., 2007d ). Occludin and ZO-1 protein breakdown is induced by pro-inflammatory cytokines such as TNF-α and IL-1β. Ma, et al. have shown that the TNF-α induces an increase in tight junction permeability and ZO-1 protein downregulation, which is mediated by NF-κB activation ( Ma et al., 2004 ). They have also shown that IL-1β stimulation of Caco-2 cells resulted in decreased occludin protein expression, and distorted its normal junctional localization, both of which were prevented in the presence of an NF-κB inhibitor ( Al-Sadi and Ma, 2007 ). These findings indicate the importance of NF-κB activation in regulating occludin and ZO-1, and are a possible mechanism by which PTX is able to attenuate their breakdown. It has been shown that agents, such as PTX, that increase cAMP can lead to inhibition of NF-κB dependent pro-inflammatory gene expression ( Minguet et al., 2005; Takahashi et al., 2002 ). Accordingly, results of our previous work indicate that PTX consistently reduces the generation of TNF-α, and the activation and nuclear translocation of the NF-κB transcriptional factor ( Coimbra et al., 2005b, 2006a ). Therefore, PTX may be exerting its effects by limiting inflammatory signaling via the NF-κB pathway. Conclusion This series of experiments shows that PTX prevents the inflammatory-induced alterations of the tight junction proteins occludin and ZO-1. While the results of this in vitro work are certainly promising, further investigation into the effects of PTX on tight junction proteins is warranted. 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