J. F. J. G. Koninkx

The heat shock response and cytoprotection of the intestinal epithelium

Abstract Following heat stress, the mammalian intestinal epithelial cells respond by producing heat shock proteins that confer protection under stressful conditions, which would otherwise lead to cell damage or death. Some of the noxious processes against which the heat shock response protects cells include heat stress, infection, and inflammation. The mechanisms of heat shock response–induced cytoprotection involve inhibition of proinflammatory cytokine production and induction of cellular proliferation for restitution of the damaged epithelium. This can mean selective interference of pathways, such as nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK), that mediate cytokine production and growth responses. Insight into elucidating the exact protective mechanisms could have therapeutic significance in treating intestinal inflammations and in aiding maintenance of intestinal integrity. Herein we review findings on heat shock response–induced intestinal epithelial protection involving regulation of NF-κB and MAPK cytokine production.

Binding of soybean agglutinin to small intestinal brush border membranes and brush border membrane enzyme activities in Atlantic salmon (Salmo salar)

To test for possible toxicity of soybean lectin for salmon, the location of soybean agglutinin binding sites in the small intestinal brush border membranes (BBM) was determined quantitatively by an enzyme-linked lectin sorbent assay (ELLSA). The assay revealed substantial binding in both the proximal (Bmax 1764±170 nM per mg BBM protein; Kd 2.16±0.23·10−5) and distal (Bmax 2096±79 nM per mg BBM protein; Kd 1.58±0.11·10−5) small intestine. The higher maximum binding and the lower dissociation constant for the distal small intestine might indicate a greater sensitivity to the toxic effect of soybean lectin in this region. Enzyme activities for alkaline phosphatase (EC 3.1.3.1) and sucrase isomaltase (EC 3.2.1.48) in brush border membrane preparations were very low in both the proximal and distal small intestine. A high enzyme activity was found for aminopeptidase (EC 3.4.11.2). Activities of sucrase isomaltase and aminopeptidase were lower in the distal than in the proximal small intestine.

Anti-inflammatory properties of heat shock protein 70 and butyrate on Salmonella-induced interleukin-8 secretion in enterocyte-like Caco-2 cells.

Intestinal epithelial cells secrete the chemokine interleukin (IL)‐8 in the course of inflammation. Because heat shock proteins (Hsps) and butyrate confer protection to enterocytes, we investigated whether they modulate Salmonella enterica serovar Enteritidis (S. serovar Enteritidis)‐induced secretion of IL‐8 in enterocyte‐like Caco‐2 cells. Caco‐2 cells incubated with or without butyrate (0–20 m M, 48 h) were infected with S. serovar Enteritidis after (1 h at 42°C, 6 h at 37°C) or without prior heat shock (37°C). Levels of Hsp70 production and IL‐8 secretion were analysed using immunostaining of Western blots and enzyme‐linked immunosorbent assay (ELISA), respectively. The cells secreted IL‐8 in response to S. serovar Enteritidis and produced Hsp70 after heat shock or incubation with butyrate. The IL‐8 secretion was inhibited by heat shock and butyrate concentrations as low as 0·2 m M for crypt‐like and 1 m M for villous‐like cells. In a dose‐dependent manner, higher butyrate concentrations enhanced IL‐8 secretion to maximal levels followed by a gradual but stable decline. This decline was associated with increasing production of Hsp70 and was more vivid in crypt‐like cells. In addition, the higher concentrations abolished the heat shock inhibitory effect. Instead, they promoted the IL‐8 production in heat‐shocked cells even in the absence of S. serovar Enteritidis. We conclude that heat shock and low concentrations of butyrate inhibit IL‐8 production by Caco‐2 cells exposed to S. serovar Enteritidis. Higher butyrate concentrations stimulate the chemokine production and override the inhibitory effect of the heat shock. The IL‐8 down‐regulation could in part be mediated via production of Hsp70.

Nippostrongylus brasiliensis Histochemical changes in the composition of mucins in goblet cells during infection in rats

Changes in the quality of mucins in jejunal goblet cells were investigated during an infection with Nippostrongylus brasiliensis in rats. At 10 days after infection, when proliferative activity in the crypts is excessive and both crypts and villi are characterized by hyperplasia of goblet cells, the histochemical composition of the population of goblet cells in comparison with controls shows a marked increase in crypt and villous goblet cells containing neutral mucins. At 15 days after infection both crypts and villi display a significant increase in goblet cells containing acid mucin and decrease in goblet cells containing neutral mucin. The acid mucins in crypt and villous goblet cells on day 15 appear to be sulphomucins predominantly, whereas in controls sialomucin-containing goblet cells dominate both in the crypts and on the villi. These experiments establish that the explusion of N. brasiliensis from the intestine of the rat coincides not only with quantitative, but also with remarkable qualitative changes in the histochemical composition of mucins in goblet cells.

Effect of the insecticidal Galanthus nivalis agglutinin on metabolism and the activities of brush border enzymes in the rat small intestine

Abstract With increasing use of lectin genes in crop plants to improve insect resistance, the dietary exposure of humans to lectins will rise and it is necessary to assess whether the presently most favored insecticidal lectin, Galanthus nivalis agglutinin, would be harmful for mammals. Effects of Galanthus nivalis agglutinin on gut and brush border enzymes were studied in rats over a 10-day dietary exposure and compared with those of a known antinutrient, phytohaemagglutinin. At a level that provides insecticidal protection for plants but did not reduce the growth of young rats, Galanthus nivalis agglutinin had negligible effects on the weight and length of the small intestine even though there was a slight, but significant hypertrophy of this tissue. However, the activities of brush border enzymes were affected; sucrase-isomaltase activity was nearly halved and those of alkaline phosphatase and aminopeptidase were significantly increased. Although most of the changes in gut metabolism caused by the incorporation of Galanthus nivalis agglutinin in the diet were less extensive than those found with toxic phytohaemagglutinin, some of them may be potentially deleterious. Thus, further and longer animal studies are needed to establish whether it is safe to use Galanthus nivalis agglutinin in transgenic plants destined for human consumption.

Decreased levels of heat shock proteins in gut epithelial cells after exposure to plant lectins

BACKGROUND The enterocytes of the intestinal epithelium are regularly exposed to potentially harmful substances of dietary origin, such as lectins. Expression of heat shock proteins (HSPs) by this epithelium may be part of a protective mechanism developed by intestinal epithelial cells to deal with noxious components in the intestinal lumen. AIM To investigate if the lectins PHA, a lectin from kidney beans (Phaseolus vulgaris) and WGA, a lectin from wheat germ (Triticum aestivum) could modify the heat shock response in gut epithelial cells and to establish the extent of this effect. METHODS Jejunal tissue sections from PHA and WGA fed rats were screened for expression of HSP70, HSP72, and HSP90 using monoclonal antibodies. Differentiated Caco-2 cells, the in vitro counterpart of villus enterocytes, were exposed to 100 μg/ml of PHA-E4 or WGA for 48 hours and investigated for changes in DNA and protein synthesis by double labelling with [2-14C]thymidine and L-[methyl-3H]methionine. The relative concentrations of HSP60, HSP70, HSP72, and HSP90 and binding protein (BiP) in these cells exposed to lectins were analysed by polyacrylamide gel electrophoresis and immunoblotting. To establish if lectin exposed differentiated Caco-2 cells were still capable of producing a heat shock response, these cells received a heat shock (40°C, 41°C, and 42°C) for one hour and were allowed to recover for six hours at 37°C. During heat shock and recovery periods, lectin exposure was continued. RESULTS Constitutive levels of HSPs were measured in the intestinal cells of lactalbumin fed (control) rats, as may be expected from the function of this tissue. However, in PHA and WGA fed rats a marked decline in the binding of antibodies against several HSPs to the intestinal epithelium was found. These results were confirmed by in vitro experiments using differentiated Caco-2 cells exposed to PHA-E4 and WGA. However, after exposure to lectins, these cells were still capable of heat induced heat shock protein synthesis, and total protein synthesis was not impaired indicating specific inhibition of HSP synthesis in non-stressed cells. CONCLUSIONS We conclude that PHA and WGA decrease levels of stress proteins in rat gut and enterocyte-like Caco-2 cells, leaving these cells less well protected against the potentially harmful content of the gut lumen.

Mutual paracrine effects of colorectal tumour cells and stromal cells: modulation of tumour and stromal cell differentiation and extracellular matrix component production in culture

Interactions of tumour and stromal cells influence tumour cell proliferation and differentiation, stromal cell phenotypic transdifferentiation and secretion of extracellular matrix (ECM) components. In this study, we established a monolayer and a three‐dimensional cell‐to‐cell interaction model between canine mammary stromal cells and human colonic carcinoma cell lines (Caco‐2 and HT‐29) to investigate mutual paracrine effects of tumour cells and stromal cells on (i) tumour cell differentiation, (ii) production of ECM components and (iii) phenotypic transdifferentiation of stromal cells. We showed that when Caco‐2 or HT‐29 cells are cultured in collagen gels, they form a few small solid cell clusters with no lumina, but when cocultured with stromal cells, the tumour cells formed glandular structures with central lumina. This fibroblast‐induced organization and differentiation of Caco‐2 cells (not HT‐29 cells) appeared to be mediated by transforming growth factor‐β (TGF‐β). Culturing of stromal cells, Caco‐2 cells or HT‐29 cells alone in both monolayers and gels resulted in weak tenascin‐C expression in stromal cells and HT‐29 cells and no expression in the Caco‐2 cells. Coculturing of stromal cells with tumour cells resulted in increased tenascin‐C expression in the stromal cells and HT‐29 cells and induced expression of tenascin‐C in the Caco‐2 cells. This induction and increased expression of tenascin‐C appeared to be mediated by TGF‐β. Culturing of stromal cells, Caco‐2 cells or HT‐29 cells alone on monolayers and in gels resulted in a weak expression of chondroitin sulfate (CS), chondroitin‐6‐sulfate (C‐6‐S) and versican in stromal cells and no expression in Caco‐2 and HT‐29 cells. Coculturing of stromal cells with tumour cells on monolayers and in gels resulted in increased CS, C‐6‐S and versican expression in stromal cells. This tumour cell‐induced expression of CS, C‐6‐S and versican appeared to be mediated by TGF‐β and platelet‐derived growth factor (PDGF). Coculturing of Caco‐2 and HT‐29 and stromal cells promoted the transdifferentiation of stromal cells into myofibroblasts, and this appeared to be mediated by TGF‐β. These results suggest that TGF‐β and PDGF are part of a paracrine system involved in stromal–epithelial cell interaction important in stromal cell differentiation and ECM component production.

Biological and pathobiological aspects of the glycocalyx of the small intestinal epithelium. A review

The literature on the glycocalyx of small intestinal epithelium is reviewed. The structure, general and barrier functions, synthesis, and degradation of the glycocalyx, and pathobiological aspects of the glycocalyx in relation to its barrier function are mentioned. Topics for future research are indicated.

Interaction of legume lectins with the cellular metabolism of differentiated Caco-2 cells

The binding of the legume lectins Phaseolus vulgaris E4 and L4, Glycine max agglutinin, Vicia faba agglutinin, and Pisum sativum agglutinin to intact differentiated Caco-2 cells and to brush border membranes of differentiated Caco-2 cells was investigated, and their impact on the cellular metabolism and the microvilli of these cells was assessed. P. vulgaris isolectin E4 showed the most intense staining after binding of fluorescein isothiocyanate-labeled lectin to intact Caco-2 cells. P. sativum agglutinin showed the weakest staining intensity. The dissociation constant for P. vulgaris isolectin E4 and P. sativum agglutinin binding was 0.11 x 10(-5) and 1.69 x 10(-5) mol/L, respectively. The values of the dissociation constants for P. vulgaris isolectin L4, G. max agglutinin, and V. faba agglutinin were situated in between these extremes. Stimulation of thymidine, glucosamine, and fucose incorporation was observed after exposure to P. vulgaris isolectins and soybean agglutinin. V. faba agglutinin had an inhibitory effect, whereas P. sativum agglutinin showed little or no effect. Compared with control cells and P. vulgaris isolectin L4- and P. sativum agglutinin-incubated cells, the microvilli of P. vulgaris isolectin E4-, soybean agglutinin-, and V. faba agglutinin-incubated cells were shortened significantly. The data provide evidence that a correlation exists, not only between the dissociation constants of the lectins and the fluorescent staining intensity, but also between the dissociation constants of the lectins and the extent of the legume lectin-induced changes in the cellular metabolism.

Expression levels of heat shock proteins in enterocyte-like Caco-2 cells after exposure to Salmonella enteritidis

Abstract The enterocytes of the small intestine are occasionally exposed to pathogenic bacteria, such as Salmonella enteritidis 857, an etiologic agent of intestinal infections in humans. The expression of the heat shock response by enterocytes may be part of a protective mechanism developed against pathogenic bacteria in the intestinal lumen. We aimed at investigating whether S enteritidis 857 is able to induce a heat shock response in crypt- and villus-like Caco-2 cells and at establishing the extent of the induction. To establish whether S enteritidis 857 interfered with the integrity of the cell monolayer, the transepithelial electrical resistance (TEER) of filter-grown, differentiated (villus-like) Caco-2 cells was measured. We clearly observed damage to the integrity of the cell monolayer by measuring the TEER. The stress response was screened in both crypt- and villus-like Caco-2 cells exposed to heat (40–43°C) or to graded numbers (101–108) of bacteria and in villus-like cells exposed to S enteritidis 857 endotoxin. Expression of the heat shock proteins Hsp70 and Hsp90 was analyzed by polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies. Exposure to heat or Salmonella resulted in increased levels of Hsp70 and Hsp90 in a temperature-effect or Salmonella-dose relationship, respectively. Incubation of Caco-2 cells with S enteritidis 857 endotoxin did not induce heat shock gene expression. We conclude that S enteritidis 857 significantly increases the levels of stress proteins in enterocyte-like Caco-2 cells. However, our data on TEER clearly indicate that this increase is insufficient to protect the cells.