Irradiation leads to apoptosis of Kupffer cells by a Hsp27-dependant pathway followed by release of TNF-α

In a previous publication, we were able to show that irradiation of Kupffer cells, the liver resident macrophages, leads to an increased TNF-α concentration in the culture medium. The pathomechanisms underlying this phenomenon, however, remained to be elucidated. Here, we show that following irradiation of Kupffer cells, the apoptosis rate increased drastically within 48 h. At the same time, the total TNF-α concentration in cell lysates of Kupffer cells attached to the culture plate decreased. However, normalization of the TNF-α concentration with respect to cell number revealed that TNF-α concentration per attached cell remained constant during the observation period. Western blot analysis showed that heat shock protein 27 (Hsp27) is strongly downregulated and bax is upregulated in irradiated Kupffer cells as compared to sham-irradiated cells. Overexpression of Hsp27 in Kupffer cells was shown to prevent the effect of irradiation on bax expression, apoptosis and, at the same time, on increase of TNF-α concentration in the Kupffer cell medium. We conclude that irradiation of Kupffer cells leads to apoptosis because of downregulation of Hsp27 and consecutive upregulation of bax expression. Furthermore, we suggest that apoptosis of Kupffer cells leads to an increase of TNF-α concentration in the culture medium which may be due to cell death rather than active release or synthesis

An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion