Interaction with colon cancer cells hyperactivates TGF-ß signaling in cancer-associated fibroblasts.

The interaction between epithelial cancer cells and cancer-associated fibroblasts (CAFs) has a major role in cancer progression and eventually in metastasis. In colorectal cancer (CRC), CAFs are present in high abundance, but their origin and functional interaction with epithelial tumor cells has not been elucidated. In this study we observed strong activation of the transforming growth factor-ß (TGF-ß)/Smad signaling pathway in CRC CAFs, accompanied by decreased signaling in epithelial tumor cells. We evaluated the TGF-ß1 response and the expression of target genes including matrix metalloproteinases (MMPs) and plasminogen activator inhibitor (PAI)-1 of various epithelial CRC cell lines and primary CAFs in vitro. TGF-ß1 stimulation caused high upregulation of MMPs, PAI-1 and TGF-ß1 itself. Next we showed that incubation of CAFs with conditioned medium (CM) from epithelial cancer cells led to hyperactivation of the TGF-ß signaling pathway, enhanced expression of target genes like PAI-1, and the expression of α-smooth muscle actin (α-SMA). We propose that the interaction of tumor cells with resident fibroblasts results in hyperactivated TGF-ß1 signaling and subsequent transdifferentiation of the fibroblasts into α-SMA-positive CAFs. In turn this leads to cumulative production of TGF-ß and proteinases within the tumor microenvironment, creating a cancer-promoting feedback loop.

Tissue level, activation and cellular localisation of TGF-beta1 and association with survival in gastric cancer patients.

Transforming growth factor-beta1 (TGF-beta1), a tumour suppressing as well as tumour-promoting cytokine, is stored as an extracellular matrix-bound latent complex. We examined TGF-beta1 activation and localisation of TGF-beta1 activity in gastric cancer. Gastric tumours showed increased stromal and epithelial total TGF-beta1 staining by immunohistochemistry. Active TGF-beta1 was present in malignant epithelial cells, but most strongly in smooth muscle actin expressing fibroblasts. Normal gastric mucosa from the same patient showed some staining for total, and little for active TGF-beta1. Active TGF-beta1 levels were determined by ELISA on tissue homogenates, confirming a strong increase in active TGF-beta1 in tumours compared to corresponding normal mucosa. Moreover, high tumour TGF-beta1 activity levels were significantly associated with clinical parameters, including worse survival of the patients. Total and active TGF-beta1 levels were not correlated, suggesting a specific activation process. Of the different proteases tested, active TGF-beta1 levels were only correlated with urokinase activity levels. The correlation with urokinase activity suggests a role for plasmin in TGF-beta1 activation in the tumour microenvironment, resulting in transformation of resident fibroblasts to tumour promoting myofibroblasts. In conclusion we have shown localisation and clinical relevance of TGF-beta1 activity levels in gastric cancer.

Suppression of intestinal mucosal natural killer cells by corticosteroids.

BACKGROUND: Corticosteroid therapy of patients with inflammatory bowel disease can give rise to systemic side-effects. Budesonide is a topically acting corticosteroid with low systemic bioavailability and is efficacious in the treatment of inflammatory bowel disease. Natural killer cells were previously found to be altered, both systemically and locally, in patients with inflammatory bowel disease. Modulatory effects of budesonide, prednisolone, dexamethasone, and cortisol on peripheral blood NK cells have already been described, but have never been assessed on mucosal NK cells from the intestine. METHODS: The effect of the synthetic corticosteroids prednisolone and budesonide, the endogenous corticosteroid cortisol, and adrenocorticotropic hormone was analysed on NK cells isolated from the lamina propria of human intestinal resection specimens. RESULTS: The three corticosteroids suppressed intestinal NK cell activity, not only during the cytotoxicity assay, but also after pre-incubation of the lamina propria mononuclear cells. ACTH, however, did not affect the activity of intestinal NK cells. We previously showed that corticosteroid-suppressed peripheral blood NK cell activity could be restored in vivo, but not in vitro, by the administration of ACTH. In the present study, the in vitro incubation of budesonide- or prednisolone-suppressed mucosal NK cells with cortisol, alone or combined with ACTH, did not revert the suppressed NK cell activity. These findings are similar to our previous observations with peripheral blood NK cells. CONCLUSIONS: Intestinal mucosal NK cells can be suppressed by systemically as well as locally acting corticosteroids. This suppression in NK cell activity is not reversed by incubation with cortisol and/or ACTH.

In vitro cellular cytotoxicity in Crohn's disease and ulcerative colitis: relation with disease activity and treatment, and the effect of recombinant gamma-interferon.

In a previous study using total mononuclear cells and lymphocytes, enriched by elutriation centrifugation, of patients with Crohn's disease and ulcerative colitis were found to have a decreased NK cell activity. In the present study the relation with disease activity and treatment, and the effect of recombinant gamma-interferon (gamma-IFN) on NK cell and monocyte cytotoxicity has been studied in 19 patients with Crohn's disease, 11 with ulcerative colitis, two with indeterminate colitis and 12 healthy controls. Patients with active Crohn's disease and active ulcerative colitis were shown to have an impaired NK cell activity compared to the control group. However, no difference was found in the percentage of CD16 (Leu 11+) cells, as determined by fluorocytometry, between patients with active or inactive disease. Moreover, the NK cell impairment was not related to corticosteroid treatment. Recombinant gamma-interferon (gamma-IFN) stimulated significantly the cytotoxic activity of the total mononuclear cells and the monocyte-enriched fraction against all target cell lines, both in patients and controls. No relation was found between the increase in cytotoxicity by gamma-IFN and disease activity in the patients. Stimulation with gamma-IFN demonstrated that the monocyte cytotoxic response of inflammatory bowel disease patients is normal. The present study reveals that the impairment in NK cell activity in patients with inflammatory bowel disease is related to disease activity and therefore suggests to be secondary to the inflammatory process.

Natural, lectin- and phorbol ester-induced cellular cytotoxicity in Crohn's disease and ulcerative colitis.

Cellular cytotoxicity of peripheral blood cells was studied in patients with Crohn's disease or ulcerative colitis and healthy controls. The spontaneous cytotoxicity or natural killer (NK) cell activity, evaluated against the erythroleukemia K-562 and the colon cancer CaCo-2 and HT-29 cell lines, of total mononuclear cells and enriched lymphocytes was depressed in Crohn's disease and ulcerative colitis patients compared to the controls. Phytohaemagglutinin (PHA) increased the cytotoxicity in the patients, to a similar maximal level as the stimulated controls. In contrast, the phorbol ester, phorbol-myristate-acetate (PMA), enhanced the cytotoxicity in patients and in controls, but in the patients not to the levels of the controls. No cytotoxicity was observed in the monocyte-enriched fraction both in patients and controls using the same assay system. A similar small but significant stimulation of monocyte cytotoxicity was obtained by PHA and PMA in patients and in controls. In conclusion, inflammatory bowel disease is associated with a depressed NK cell activity in peripheral blood which is not target specific. PHA but not PMA could restore the deficient NK cell activity. Monocytes seem not to be involved in the decreased NK cell activity in patients with inflammatory bowel disease.