Hyperosmotic stress induces nuclear factor-kappaB activation and interleukin-8 production in human intestinal epithelial cells - PubMed (original) (raw)

Hyperosmotic stress induces nuclear factor-kappaB activation and interleukin-8 production in human intestinal epithelial cells

Zoltán H Németh et al. Am J Pathol. 2002 Sep.

Abstract

Inflammatory bowel disease of the colon is associated with a high osmolarity of colonic contents. We hypothesized that this hyperosmolarity may contribute to colonic inflammation by stimulating the proinflammatory activity of intestinal epithelial cells (IECs). The human IEC lines HT-29 and Caco-2 were used to study the effect of hyperosmolarity on the IEC inflammatory response. Exposure of IECs to hyperosmolarity triggered expression of the proinflammatory chemokine interleukin (IL)-8 both at the secreted protein and mRNA levels. In addition, hyperosmotic stimulation induced the release of another chemokine, GRO-alpha. These effects were because of activation of the transcription factor, nuclear factor (NF)-kappaB, because hyperosmolarity stimulated both NF-kappaB DNA binding and NF-kappaB-dependent transcriptional activity. Hyperosmolarity activated both p38 and p42/44 mitogen-activated protein kinases, which effect contributed to hyperosmolarity-stimulated IL-8 production, because p38 and p42/44 inhibition prevented the hyperosmolarity-induced increase in IL-8 production. In addition, the proinflammatory effects of hyperosmolarity were, in a large part, mediated by activation of Na(+)/H(+) exchangers, because selective blockade of Na(+)/H(+) exchangers prevented the hyperosmolarity-induced IEC inflammatory response. In summary, hyperosmolarity stimulates IEC IL-8 production, which effect may contribute to the maintenance of inflammation in inflammatory bowel disease.

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Figures

Figure 1.

Figure 1.

Hyperosmolarity stimulates IL-8 production by both HT-29 (A and B) and Caco-2 (C) cells. Cells were incubated with hyperosmotic medium prepared by the addition of mannitol (A and C) or NaCl (B) to isosmolar growth medium. NaCl concentrations depicted on the x axis represent NaCl amounts added to isosmolar growth medium that already contains ∼140 mmol/L of NaCl (B). In control wells, cells were incubated with isosmolar growth medium. D: Hyperosmotic medium containing either mannitol or NaCl also stimulates GRO-α production by HT-29 cells. Supernatants for IL-8 and GRO-α measurement were taken 18 hours after the hyperosmolar challenge. Data are mean ± SEM of n = 6 to 12 wells from two different experiments. *, P < 0.05; **, P < 0.01.

Figure 2.

Figure 2.

Treatment of HT-29 cells with the selective NHE inhibitors amiloride (A), MIA (B), or EIPA (C) suppresses mannitol-induced IL-8 production. Treatment of HT-29 cells with the nonamiloride NHE inhibitors cimetidine (D), clonidine (E), or harmaline (F), reproduces the suppressive effect of selective NHE inhibitors on the production of IL-8 by mannitol-induced HT-29 cells. When harmaline was used, supernatants for IL-8 measurement were taken 4 hours after the hyperosmotic challenge. In the case of amiloride, MIA, EIPA, cimetidine, and clonidine, IL-8 levels were measured from supernatants obtained 18 hours after the hyperosmotic challenge. Hyperosmotic medium prepared by the addition of 100 mmol/L of mannitol to isosmolar growth medium was used for hyperosmotic stimulation. Data are mean ± SEM of n = 6 to 12 wells from two different experiments. *, P < 0.05; **, P < 0.01. Dotted bar, no mannitol; cross-hatched bars, mannitol.

Figure 3.

Figure 3.

A: DMSO (0.5%) inhibits both basal and mannitol (100 mmol/L)-stimulated IL-8 production by HT-29 cells. Growth medium on cells was switched with fresh growth medium with or without mannitol, both in the presence or absence of 0.5% DMSO. IL-8 production was measured from supernatants taken 18 hours after the addition of fresh medium. B: Increasing medium pH fails to augment the production of IL-8 by HT-29 cells. Growth medium on cells was switched with fresh growth medium with pH values ranging from 7.3 to 7.8. IL-8 production was measured from supernatants taken 18 hours after the addition of fresh medium. Data are mean ± SEM of n = 6 to 12 wells from two different experiments. **, P < 0.01.

Figure 4.

Figure 4.

Hyperosmolarity induces p38 and p42/44 activation in HT-29 cells (A). Cells were incubated with hyperosmotic medium prepared by the addition of mannitol (for a final concentration of 100 mmol/L) to isosmolar medium for the indicated time periods. p38 and p42/44 activation was determined using Western blotting using antibodies raised against the active, double-phosphorylated form of p38 and p42/44. This figure is representative of two separate experiments. Treatment of HT-29 cells with the selective p38 inhibitor SB203580 or selective p42/44 inhibitor PD98059 suppresses the hyperosmolarity-induced IL-8 response (B). Hyperosmolarity was achieved by the addition of mannitol (for a final concentration of 100 mmol/L) to isosmolar medium. In control wells, cells were incubated with isosmolar medium. Data are mean ± SEM of n = 12 wells from two separate experiments. *, P < 0.05; **, P < 0.01. Dotted bar, no mannitol; cross-hatched bars, mannitol.

Figure 5.

Figure 5.

Hyperosmolarity induces up-regulation of IL-8 mRNA levels in HT-29 cells. Amiloride pretreatment (300 μmol/L) inhibits hyperosmolarity-induced IL-8 mRNA accumulation. Lanes 1 and 2: Control (isosmolar medium); lanes 3 and 4: hyperosmolarity (100 mmol/L of mannitol); lanes 5 and 6: amiloride and hyperosmolarity. GAPDH levels were not affected by both hyperosmolar and amiloride treatment. IL-8 and GAPDH mRNA levels were quantitated using reverse transcriptase-PCR. This figure is representative of three separate experiments.

Figure 6.

Figure 6.

Hyperosmolarity induces NF-κB DNA binding in HT-29 cells. Cells were incubated with hyperosmotic medium prepared by the addition of mannitol (for a final concentration of 100 mmol/L) to isosmolar medium for the indicated time periods. NF-κB DNA binding was assessed using EMSA. The figure is representative of two separate experiments.

Figure 7.

Figure 7.

Amiloride pretreatment (300 μmol/L) inhibits hyperosmolarity-induced NF-κB DNA binding in HT-29 cells. NF-κB-specific complexes are indicated by arrows as determined by antibody supershifting. Hyperosmolarity was produced by the addition of mannitol (for a final concentration of 100 mmol/L) to isosmolar medium for 45 minutes. Control cells were treated with isosmolar medium. This figure is representative of three separate experiments.

Figure 8.

Figure 8.

Hyperosmolar stimulation increases NF-κB-dependent transcriptional activity as compared to isosmolar stimulation. HT-29 cells were transiently transfected with a NF-κB-luciferase promoter construct, after which the cells were incubated for 16 hours with either hyperosmolar (100 mmol/L of mannitol) or isosmolar (control) medium. NF-κB-dependent transcriptional activity was determined using the luciferase assay (pNF-κB-Luc). This figure also shows that hyperosmolarity does not influence luciferase activity in cells transfected with an enhancerless construct (pTAL-Luc). Data are mean ± SEM of n = 14 to 16 wells from two separate experiments. **, P < 0.01.

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