Search

In colonic epithelium, one of the pathways of lipopolysaccharide (LPS) activation of NF-[kappa]B and IL-8 is via Toll-like receptor (TLR)4, MyD88, IRAK1/4, and B-cell CLL/ lymphoma 10 (Bcl10). However, this innate immune pathway accounts for only ~50% of the NF-[kappa]B activation, so additional mechanisms to explain the LPS-induced effects are required. In this report, we identify a second pathway of LPS-induced stimulation, mediated by reactive oxygen species (ROS), in human colonic epithelial tissue cells in tissue culture and in ex vivo mouse colonic tissue. Measurements of IL-8, KC, Bcl10, phospho-I[kappa]B[alpha], nuclear NF-[kappa]B, and phosphorylated Hsp27 were performed by ELISA. The TLR4-Bcl10 pathway was inhibited by Bcl10 siRNA and in studies with colonic tissue from the TLR4-deficient mouse. The ROS pathway was inhibited by Tempol, a free radical scavenger, or by okadaic acid, an inhibitor of Hsp27 dephosphorylation by protein phosphatase 2A (PP2A). The ROS pathway was unaffected in the TLR4-deficient tissue or by silencing of Bcl10. The combination of exposure to the free radical scavenger Tempol and of TLR4 or Bcl10 suppression was required to completely inhibit the LPS-induced activation. The ROS pathway was associated with dephosphorylation of Hsp27. LPS appears to activate both the regulatory component of the I[kappa]B[alpha]-kinase (IKK) signalosome through Bcl10 interaction with Nemo (IKK[gamma]) and the catalytic component through Hsp27 interaction with IKK[beta]. Since LPS exposure is associated with septic shock and the systemic inflammatory response syndrome, distinguishing between these two pathways of LPS activation may facilitate new approaches to prevention and treatment. IKK signalosome; Bcl10; lipopolysaccharide; Hsp27; TLR4; reactive oxygen species

Multiple studies in animal models have shown that the commonly used food additive carrageenan (CGN) induces inflammation and intestinal neoplasia. We performed the first studies to determine the effects of CGN exposure on human intestinal epithelial cells (IEC)in tissue culture and tested the effect of very low concentrations (1-10 mg/L) of undegraded, high-molecular weight CGN. These concentrations of CGN are less than the anticipated exposure of the human colon to CGN from the average Western diet. In the human colonic epithelial cell line NCM460 and in primary human colonic epithelial cells that were exposed to CGN for 1-8 d, we found increased cell death, reduced cell proliferation, and cell cycle arrest compared with unexposed control cells. After 6-8 d of CGN exposure, the percentage of cells reentering G0-G1 significantly decreased and the percentages of cells in S and G2-M phases significantly increased. Increases in activated p53, p21, and p15 followed CGN exposure, consistent with CGN-induced cell cycle arrest. Additional data, including DNA ladder, poly ADP ribose polymerase Western blot, nuclear DNA staining, and activities of caspases 3 and 7, indicated no evidence of increased apoptosis following CGN exposure and were consistent with CGN-induced necrotic cell death. These data document for the first time, to our knowledge, marked adverse effects of low concentrations of CGN on survival of normal human IEC and suggest that CGN exposure may have a role in development of human intestinal pathology.

Lipopolysaccharide (LPS) is recognized as an inducer of the inflammatory response associated with gram-negative sepsis and systemic inflammatory response syndrome. LPS induction proceeds through Toll-like receptor (TLR) in immune cells and intestinal epithelial cells (IEC). This report presents the first identification of Bc110 (B-cell CLL/lymphoma 10) as a mediator of the LPS-induced activation of IL-8 in human IEC. Be110 is a caspase-recruitment domain-containing protein, associated with constitutive activation of NF-[kappa]B in MALT (mucosa-associated lymphoid tissue) lymphomas. The normal human IEC line NCM460, normal primary human colonocytes, and ex vivo human colonic tissue were exposed to 10 ng/ml of LPS for 2-6 h. Effects on Bc110, phospho-I[kappa]B[alpha], NF-[kappa]B, and IL-8 were determined by Western blot, ELISA, immunohistochemistry, and confocal microscopy. Effects of Bc110 silencing by small-interfering RNA (siRNA), TLR4 blocking antibody, TLR4 silencing by siRNA, and an IL-1 receptor-associated kinase (IRAK)-1/4 inhibitor on LPS-induced activation were examined. Following Bc110 silencing, LPS-induced increases in NF-[kappa]B, I[kappa]B[alpha], and IL-8 were significantly reduced (P < 0.001). Increasing concentrations of LPS were associated with higher concentrations of Bc110 protein when quantified by ELISA, and the association between LPS exposure and increased Bc110 was also demonstrated by Western blot, immunohistochemistry, and confocal microscopy. Exposure to TLR4 antibody, TLR4 siRNA, or an IRAK-1/4 inhibitor eliminated the LPS-induced increases in Bc100, NF-[kappa]B, and IL-8. Identification of Bc110 as a mediator of LPS-induced activation of NF-[kappa]B and IL-8 in normal human IEC provides new insight into mechanisms of epithelial inflammation and new opportunities for therapeutic intervention. lipopolysaccharide; inflammation; colonocytes; Toll-like receptor 4

Carrageenan is a high molecular weight sulfated polygalactan used to improve the texture of commercial food products. Its use increased markedly during the last half century, although carrageenan is known to induce inflammation in rheumatological models and in intestinal models of colitis. We performed studies to determine its direct effects on human intestinal cells, including normal human intestinal epithelial cells from colonic surgeries, the normal intestinal epithelial cell line NCM460, and normal rat ileal epithelial cells. Cells were treated with high molecular weight [lambda]-carrageenan at a concentration of 1 [micro]g/ml for 1-96 h. IL-8, IL-8 promoter activity, total and nuclear NF-[kappa]B, I[kappa]B[alpha], phospho-I[kappa]B[alpha], and Bc110 were assessed by immunohistochemistry, Western blot, ELISA, and cDNA microarray. Increased Bc110, nuclear and cytoplasmic NF-[kappa]B, IL-8 promoter activation, and IL-8 secretion were detected following carrageenan exposure. Knockdown of Bc110 by siRNA markedly reduced the increase in IL-8 that followed carrageenan exposure in the NCM460 cells. These results show, for the first time, that exposure of human intestinal epithelial cells to carrageenan triggers a distinct inflammatory pathway via activation of Bc110 with NF-[kappa]B activation and upregulation of IL-8 secretion. Since Bc110 contains a caspase-recruitment domain, similar to that found in NOD2/CARD15 and associated with genetic predisposition to Crohn's disease, the study findings may represent a link between genetic and environmental etiologies of inflammatory bowel disease. Because of the high use of carrageenan as a food additive in the diet, the findings may have clinical significance. inflammation; colon; NF-[kappa]B; I[kappa]B[alpha]

Abstract Background The receptors for adhesion of Plasmodium falciparum-infected red blood cells (RBC) in the placenta have been identified as chondroitin-4-sulphate (C4S) proteoglycans, and the more sulphate-rich chondroitin oligosaccharides have been reported to inhibit adhesion. Since the anti-malarial drug chloroquine accumulates in lysosomes and alters normal lysosomal processes, the effects of chloroquine on the lysosomal enzyme arylsulphatase B (ASB, N-acetylgalactosamine-4-sulphatase), which removes 4-sulphate groups from chondroitin-4-sulphate, were addressed. The underlying hypothesis derived from the recognized impairment of attachment of parasite-infected erythrocytes in the placenta, when chondroitin-4-sulphation was increased. If chloroquine reduced ASB activity, leading to increased chondroitin-4-sulphation, it was hypothesized that the anti-malarial mechanism of chloroquine might derive, at least in part, from suppression of ASB. Methods Experimental methods involved cell culture of human placental, bronchial epithelial, and cerebrovascular cells, and the in vitro exposure of the cells to chloroquine at increasing concentrations and durations. Measurements of arylsulphatase B enzymatic activity, total sulphated glycosaminoglycans (sGAG), and chondroitin-4-sulphate (C4S) were performed using in vitro assays, following exposure to chloroquine and in untreated cell preparations. Fluorescent immunostaining of ASB was performed to determine the effect of chloroquine on cellular ASB content and localization. Mass spectrometry and high performance liquid chromatography were performed to document and to quantify the changes in chondroitin disaccharides following chloroquine exposure. Results In the human placental, bronchial epithelial, and cerebrovascular cells, exposure to increasing concentrations of chloroquine was associated with reduced ASB activity and with increased concentrations of sGAG, largely attributable to increased C4S. The study data demonstrated: 1) decline in ASB activity following chloroquine exposure; 2) inverse correlation between ASB activity and C4S content; 3) increased content of chondroitin-4-sulphate disaccharides following chloroquine exposure; and 4) decline in extent of chloroquine-induced ASB reduction with lower baseline ASB activity. Confocal microscopy demonstrated the presence of ASB along the cell periphery, indicating extra-lysosomal localization. Conclusions The study data indicate that the therapeutic mechanism of chloroquine action may be attributable, at least in part, to reduction of ASB activity, leading to increased chondroitin-4-sulphation in human placental, bronchial epithelial, and cerebrovascular cells. In vivo, increased chondroitin-4-sulphation may reduce the attachment of P. falciparum-infected erythrocytes to human cells. Extra-lysosomal localization of ASB and reduced impact of chloroquine when baseline ASB activity is less suggest possible mechanisms of resistance to the effects of chloroquine.