Dominik Faust

Effects of deoxycholate on human colon cancer cells: apoptosis or proliferation

Abstract Background  Deoxycholic acid has long been attributed as a tumour promoter in the colon. It exerts its growth‐related actions in a phorbol ester‐like manner, by stimulating protein kinase C. The aim of this study was to investigate the effect of deoxycholic acid on proliferation and apoptosis in the colon, by exposing colon cancer cells to it in increasing concentrations.

Regulation of α1-proteinase inhibitor release by proinflammatory cytokines in human intestinal epithelial cells

α1‐Proteinase inhibitor (α1‐PI) is the main serine proteinase inhibitor in human plasma. Apart from its synthesis in the liver, this anti‐inflammatory protein is also synthesized by and excreted from human intestinal epithelial cells. Antiinflammatory actions of α1‐PI are thought to be of relevance in the pathogenesis of inflammatory bowel disease. To investigate the role of macrophage‐derived cytokines on α1‐PI secretion from intestinal epithelial cells, we cultured Caco‐2 cells until differentiation (14 days in culture) on permeable filter supports. Monolayers of differentiated Caco‐2 cells were then co‐cultured with human peritoneal macrophages, grown on plastic in the basolateral chamber. Under these conditions, α1‐PI secretion from Caco‐2 cells was enhanced by 45%, probably by a direct action of macrophage‐derived cytokines on Caco‐2 cells. To extend this observation further, we treated differentiated Caco‐2 cells with macrophage‐derived proinflammatory cytokines (IL‐1β, IL‐8, TNF‐α), as well as with lymphocyte‐derived cytokines IL‐2, IL‐6 and IFN‐γ. As early as after 24h treatment, IL‐2 and IL‐8 induced a significant and dose‐dependent increase of α‐1‐PI secretion into cell culture medium; this effect was completely reversed after immunoneutralization by the antibodies against IL‐2 and IL‐8 α1‐PI secretion was only slightly decreased after treatment with IFN‐γ, while IL‐1β, IL‐6 and TNF‐α had no effect. α1‐PI secretion correlated well with the expression of this protein in differentiated Caco‐2 cells after cytokine treatment, as confirmed by Western blot. Our data imply that, in vitro, α1‐PI secretion in enterocyte‐like Caco‐2 cells is up‐regulated by IL‐2 and IL‐8. Our results suggest that both lymphocyte‐ and macrophage‐derived cytokines regulate secretion of the anti‐inflammatory protein α1‐PI in intestinal epithelial cells.

Permeability characteristics of polyamines across intestinal epithelium using the Caco-2 monolayer system: comparison between transepithelial flux and mitogen-stimulated uptake into epithelial cells

The polyamines putrescine, spermidine, and spermine are present in foods in high amounts, and are used for cell growth throughout the body. Surprisingly little is known about the mechanisms of polyamine absorption in the gut. To elucidate the mechanisms, transepithelial transport of polyamines was studied in human enterocytelike Caco-2 cells, grown on permeable filter supports. Transport of all three polyamines across Caco-2 cell monolayers was linear; intraepithelial accumulation of polyamines was higher in confluent than in differentiated Caco-2 cells, but still negligible in comparison with the overall transport across the monolayers. Epidermal growth factor (EGF) enhanced polyamine accumulation in Caco-2 cells four-fold, and basolateral uptake was higher than apical uptake if the cells were stimulated to grow. The amounts of polyamines taken up by the cells were nevertheless negligible in comparison with the net polyamine flux across the monolayers. Basolateral excretion of polyamines was in the picomolar range, whereas their transepithelial transport, occurring presumably by passive diffusion through the paracellular pathway, contributed hundreds of micromoles of polyamines to the basolateral chamber. We conclude that transepithelial transport of polyamines occurs by passive diffusion, and that it is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

Rejection quantity in kidney transplant recipients is associated with increasing intracellular interleukin-2 in CD8+ T-cells.

BACKGROUND CD8+ T-cells and interleukin-2 play an important role during organ rejection in kidney transplant recipients. Numerous studies showed increased interleukin-2 levels during acute rejection. The aim of our study is to show an association between intracellular interleukin-2 in CD8+ T-cells and the incidence of those who underwent organ rejection in kidney transplant recipients. METHODS 407 transplant recipients were included into this study. The rejection incidence was investigated from the patient records. White blood cells from recipients were separated using a ficoll gradient. The cells were double-gated (CD3+ and CD8+) for the analysis of cellular percentage for intracellular interleukin-2 with a flow cytometer. RESULTS The percentage of CD8+ cells with detectable intracellular interleukin-2 was significantly higher in renal transplant recipients with a documented rejection compared to recipients without any history of rejection (9.06±0.50, N=133 vs. 4.28±0.24, N=274, p<0.0001, t-test). Further, there was a significant increase in patients with one (8.02±0.54, N=80, p<0.0001, t-test), two (10.40±1.17, N=33, p<0.0001, t-test) or three (11.82±1.58, N=18, p<0.0001, t-test) rejection events. CONCLUSIONS Past studies showed, that during acute organ rejection intracellular interleukin-2 is increased in cytotoxic T-cells, supposed to be a marker for this event. We were able to show, that intracellular interleukin-2 in CD8+ T-cells is also increased after organ rejection. Furthermore it seems to depend on the quantity of rejection events. Further studies in recipients with increased intracellular interleukin-2 in cytotoxic CD8+ T-cells and documented history of organ rejection are needed to identify this as a possible risk factor for further rejection events.

Anti-inflammatory drugs modulate C1q secretion in human peritoneal macrophages in vitro

The complement system plays an important role in the humoral immune response. Activation of the classical complement pathway is mediated by its subcomponent, C1q, which is involved in the pathogenesis of several autoimmune disorders. Among the main C1q-synthesising tissues, macrophages have been attributed as the main source. We investigated the effects of anti-inflammatory drugs (methylprednisolone and acetylsalicylic acid (ASA)) on C1q secretion in human peritoneal macrophages in vitro. The macrophages were isolated from peritoneal lavage fluid of patients with end-stage renal disease undergoing continuous ambulatory peritoneal dialysis, and were maintained in culture for up to 6 days. ASA decreased while methylprednisolone increased C1q secretion from human peritoneal macrophages in vitro, which correlated well with the percentage of CD14 positive cells after treatment. We conclude that different response of the macrophages to treatment with methylprednisolone and ASA may point out to the importance of macrophage activation after treatment, as well as an increased abundance of membrane C1q accompanied by increased phagocytosis.

Butyrate and the cytokine-induced alpha1-proteinase inhibitor release in intestinal epithelial cells.

Background α1‐proteinase inhibitor (α1‐PI), an anti‐inflammatory protein thought to play a role in the intestinal inflammation, is synthesised by and released from the intestinal epithelial cells. IL‐1β is a key proinflammatory cytokine in the abnormal immune response that occurs in inflammatory bowel disease. Butyrate is a normal luminal constituent in the colon, known to be of benefit in preventing inflammatory bowel disease. Direct modes of action of butyrate in intestinal inflammation have been poorly studied so far. The aim of this study was to investigate the effects of butyrate on cytokine‐mediated α1‐PI release in intestinal epithelial cells.

Human peritoneal macrophages in culture: a model for studying inflammatory disorders in vitro

Abstract. In this study we characterized a model of human peritoneal macrophages maintained in culture for up to 48 h that can be used to study different functions of this cell population in vitro. The cells remained viable and functionally active over time, with well-preserved phagocytic properties. They expressed a macrophage marker, CD14. Once in culture, human peritoneal macrophages secreted C1q and nitric oxide in a pattern described in murine, guinea pig, and rat peritoneal macrophages . The described model can be used to study physiology and pathophysiology of peritoneal macrophages in vitro, offering all the advantages of the use of a human cell population.