Effect of Hydrogen Peroxide on Interleukin-8 Synthesis and Death of Caco-2 Cells

Abstract

Intestinal epithelial cells can secrete interleukin-8 (IL-8), among other substances in response to different stimuli, which plays an important role in mucosal immune response. Above a certain concentration range, hydrogen peroxide causes cell death by necrosis or apoptosis. We investigated the time- and dose-dependent induction of IL-8 by hydrogen peroxide in the human colon adenocarcinoma cell line Caco-2. In addition, the changes of transepithelial electrical resistance and cell death induction in response to hydrogen peroxide were studied. Nonfilter-grown and filter-grown Caco-2 cells were employed in our experiments. Interleukin-8 synthesis was measured by ELISA. Necrosis was determined by DAPI staining of cells, apoptosis by measuring caspase-3 enzyme activity or annexin V staining. In nonfilter-grown Caco-2 cells, 1 mM of hydrogen peroxide induced the highest level of IL-8 production 24 hr after treatment. In filter-grown Caco-2 cells, IL-8 was produced only on the apical side in response to 1 mM of hydrogen peroxide. This level was 10-fold lower than that measured in nonfilter-grown Caco-2 cells 24 hr after the treatment. In filter-grown Caco-2 cells 10 mM hydrogen peroxide induced the highest IL-8 level on the apical as well as basolateral side. Transepithelial electrical resistance decreased markedly upon application of 40 mM hydrogen peroxide. Late effect of hydrogen peroxide was observed in nonfilter-grown Caco-2 cells, as 1 mM hydrogen peroxide caused necrosis after 24 hr while early-necrosis induction occurred in filter-grown cells exposed to 40 mM of hydrogen peroxide after 1 hr. Filter-grown Caco-2 cells were less sensitive to hydrogen peroxide than the nonfilter-grown ones.

Measurement of Barrier Tissue Integrity with an Organic Electrochemical Transistor

“…It should be noted that we observe a continuous cell monolayer after H 2 O 2 exposure, up to concentrations of 50 mM (immunofluorescence staining, data not shown). This is in agreement with reports in the literature that observe TER disruption on addition of modest amounts of H 2 O 2 (10–40 mM), without gross disruption of monolayer integrity 25, 26. Permeability assays after exposure to ethanol indicate that barrier properties are disrupted for concentrations over 20%.…”

“…Experiments discussed here were performed using complete cell culture media as the delivery vehicle. The fact that the OECT operates so well in such a complex environment is an advantage, and we believe that we are sensing a more subtle disruption of barrier tissue than previously demonstrated 25. Figure 3d shows apparent permeability data of Caco‐2 layers exposed to H 2 O 2 and ethanol, in complete cell culture media.…”

Acute Oxidative Stress Affects IL-8 and TNF-α Expression in IPEC-J2 Porcine Epithelial Cells

“…Studying the early effects of H 2 O 2 on filter-grown Caco-2 cells, it was found that 1-4 mM H 2 O 2 had no impact on cell viability, whilst 20% of nuclei were stained with DAPI after 10 mM H 2 O 2 exposure, indicating cell necrosis. IPEC-J2 cells seem to be more sensitive since 10% and 15% of the IPEC-J2 cells became necrotic after 2 and 4 mM H 2 O 2 treatment, respectively [26]. The decrease in TER may reflect an increase in the movement of solutes and ions across the epithelium by transcellular pathways.…”

“…They secrete cytokines and chemokines due to adverse stimuli such as oxidative stress. IEC were proven to produce IL-8 in response to H 2 O 2 in vitro [4][5][6]26]. All these studies were performed with human carcinoma-derived cell lines.…”

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

“…The apical junction is responsible for the regulation of ions and molecules passing across the barrier 5,6 via a specific opening and closing function. A dysfunction or disruption of these protein complexes is often related to disease [7][8][9][10][11] . In addition, many enteric pathogens/ toxins are known to specifically target this complex, thereby entering the body and leading to diarrhea, most likely as a consequence of massive dysregulation of ion/water flow across the barrier [12][13][14] .…”