Glucocorticoid-induced cell death is mediated through reduced glucose metabolism in lymphoid leukemia cells.

Malignant cells are known to have increased glucose uptake and accelerated glucose metabolism. Using liquid chromatography and mass spectrometry, we found that treatment of acute lymphoblastic leukemia (ALL) cells with the glucocorticoid (GC) dexamethasone (Dex) resulted in profound inhibition of glycolysis. We thus demonstrate that Dex reduced glucose consumption, glucose utilization and glucose uptake by leukemic cells. Furthermore, Dex treatment decreased the levels of the plasma membrane-associated glucose transporter GLUT1, thus revealing the mechanism for the inhibition of glucose uptake. Inhibition of glucose uptake correlated with induction of cell death in ALL cell lines and in leukemic blasts from ALL patients cultured ex vivo. Addition of di-methyl succinate could partially overcome cell death induced by Dex in RS4;11 cells, thereby further supporting the notion that inhibition of glycolysis contributes to the induction of apoptosis. Finally, Dex killed RS4;11 cells significantly more efficiently when cultured in lower glucose concentrations suggesting that modulation of glucose levels might influence the effectiveness of GC treatment in ALL. In summary, our data show that GC treatment blocks glucose uptake by leukemic cells leading to inhibition of glycolysis and that these effects play an important role in the induction of cell death by these drugs.

Cell death induced by dexamethasone in lymphoid leukemia is mediated through initiation of autophagy.

Glucocorticoids are fundamental drugs used in the treatment of lymphoid malignancies with apoptotic cell death as the hitherto proposed mechanism of action. Recent studies, however, showed that an alternative mode of cell death, autophagy, is involved in the response to anticancer drugs. The specific role of autophagy and its relationship to apoptosis remains, nevertheless, controversial: it can either lead to cell survival or can function in cell death. We show that dexamethasone induced autophagy upstream of apoptosis in acute lymphoblastic leukemia cells. Inhibition of autophagy by siRNA-mediated repression of Beclin 1 expression inhibited apoptosis showing an important role of autophagy in dexamethasone-induced cell death. Dexamethasone treatment caused an upregulation of promyelocytic leukemia protein, PML, its complex formation with protein kinase B or Akt and a PML-dependent Akt dephosphorylation. Initiation of autophagy and the onset of apoptosis were both dependent on these events. PML knockout thymocytes were resistant to dexamethasone-induced death and upregulation of PML correlated with the ability of dexamethasone to kill primary leukemic cells. Our data reveal key mechanisms of dexamethasone-induced cell death that may inform the development of improved treatment protocols for lymphoid malignancies.

Antimicrobial peptide LL-37 internalized by immature human dendritic cells alters their phenotype.

The human cathelicidin LL-37 has been shown to be involved in the barrier function of the innate immunity, being released from specific cells upon challenge and exerting immunomodulatory effects. We here demonstrate that LL-37 affects immature dendritic cells, derived from human peripheral blood monocytes (MDDC). LL-37 is internalized by MDDC with subsequent localization primarily in the cytoplasmic compartment. However, LL-37 could also be detected in the nuclei of MDDC, suggesting that LL-37 may be transported into the nucleus. The uptake of LL-37 is dose, time and energy dependent, indicating that the observed internalization process involves an endocytic pathway. Incubation of immature MDDC with LL-37 caused phenotypic changes, characterized by an increased expression of the antigen-presenting molecule HLA-DR, and the costimulatory molecule CD86. Taken together, these findings suggest that LL-37 released upon triggering of the innate immunity, may affect cellular adaptive immunity through an interaction with immature dendritic cells.

Malassezia sympodialis differently affects the expression of LL-37 in dendritic cells from atopic eczema patients and healthy individuals.

BACKGROUND: Atopic eczema (AE) is a multifactorial disease, which has increased in prevalence. The skin-colonizing yeast Malasezzia sympodialis can induce IgE- and T-cell reactivity in patients with AE. LL-37 is an endogenous peptide antibiotic belonging to the cathelicidin family. The aim of this study was to examine whether exposure to M. sympodialis would affect the expression of LL-37 in dendritic cells. METHODS: The presence of LL-37 was analyzed in monocyte-derived dendritic cells (MDDCs) generated from healthy individuals and patients with AE by Western blotting and the corresponding cDNA by real-time quantitative RT-PCR. Antibacterial activity was measured with an inhibition zone assay in fractions after reverse phase chromatography. RESULTS: For the first time we here present data, showing that LL-37 is produced by MDDCs. Notably, the secretion of LL-37 was substantially enhanced in M. sympodialis-exposed MDDCs generated from patients with a high degree of eczema, as measured by SCORAD, compared to healthy controls and patients with a low SCORAD. The relative expression of LL-37 transcript in MDDCs generated from patients was up-regulated after 1 h of exposure to M. sympodialis and declined gradually at the time points analyzed, whereas the transcription was unaffected in the MDDCs of healthy controls. CONCLUSIONS: Our results suggest that M. sympodialis can trigger the innate immune response differently in patients with AE and healthy individuals. The enhanced LL-37 secretion from the MDDCs in the patients with AE may reflect the severity of their inflammatory response to M. sympodialis.

Malassezia enhances natural killer cell-induced dendritic cell maturation.

Human natural killer (NK) cells can induce cell death in autologous dendritic cells (DCs), though an interaction between these two cell types can also lead to a reciprocal activation. We have recently shown cell contact between NK cells and DCs in vivo, in Malassezia-induced lesional skin of patients with atopic eczema, where the yeast acts as an allergen although it is part of the normal skin microflora. Here we characterize the interaction of human NK cells and monocyte-derived DCs (MDDCs) by using an in vitro system where short-term activated polyclonal NK cells are cocultured with autologous, immature, Malassezia-stimulated or lipopolysaccharide-matured MDDCs. We found that the number of CD83(+) MDDCs increased in the immature and Malassezia-stimulated MDDCs upon coculture with NK cells, while an increased number of CD86(+) cells was detected in the Malassezia-stimulated MDDCs. NK cell-MDDC interaction induced the production of interleukin-8 (IL-8). In conclusion, our results imply that NK cells provide maturation signals and may play a role in inducing IL-8 production in DCs. Furthermore, the increased expression of CD86 on Malassezia-stimulated MDDCs might have a function in subsequent T-cell activation by DCs, and indicate a role for NK cell-DC interaction in modulating the immune responses to microbial stimuli.

The allergenic yeast Malassezia furfur induces maturation of human dendritic cells.

BACKGROUND: The yeast Malassezia furfur (M. furfur), present in the normal microflora of human skin, can act as an allergen that incites specific IgE reactivity and T cell proliferation in atopic dermatitis (AD) patients. The role of antigen presenting dendritic cells (DCs) in the onset and maintenance of AD is not well established. OBJECTIVE: The objective of the present study was to assess whether the interaction of M. furfur with human DCs will result in DC maturation, cytokine production and lymphocyte proliferation. METHODS: Monocyte-derived dendritic cells (MDDCs) were generated from human peripheral blood. Immature MDDCs were cultured with or without M. furfur or plastic beads, and with or without CD40L stimulation. Interaction of yeast cells by MDDCs was studied by time-lapse photography and cytokines were detected in culture supernatants with ELISA. The ability of MDDCs pre-incubated with M. furfur to induce proliferation in autologous lymphocytes was measured by [(3)H]-thymidine incorporation. RESULTS: Time-lapse photography showed that the majority of immature MDDCs internalized whole M. furfur yeast cells within 1 h. The presence of M. furfur induced maturation (CD83 expression) of MDDCs, and up-regulation of the costimulatory molecules CD80 and CD86. Production of TNF-alpha, IL-1 beta and IL-18 by MDDCs increased significantly (P < 0.05 for TNF-alpha and IL-1 beta, and P < 0.01 for IL-18) after the addition of M. furfur, while IL-10 and IL-12p70 levels remained unaltered. The CD40L-stimulated IL12p70 production by MDDCs was decreased in the presence of M. furfur (P < 0.05). Finally, immature MDDCs pre-incubated with M. furfur induced a proliferative response in autologous CD14-depleted peripheral blood mononuclear cells, in a dose-dependent manner. CONCLUSION: The data indicate that immature MDDCs can internalize the opportunistic yeast M. furfur. This process was associated with MDDC maturation, production of pro-inflammatory and immunoregulatory cytokines, which might favour induction of a Th2-type immune response, and a capacity to stimulate lymphocyte proliferation. This chain of events most likely contributes to the inflammatory reaction in AD.

Uptake of the yeast Malassezia furfur and its allergenic components by human immature CD1a+ dendritic cells.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with increasing prevalence, though still little is known of the pathomechanisms and the causes of the disease. Patients with AD often have specific IgE reactivity to the yeast Malassezia furfur (M. furfur), present in the normal microflora on human skin. To investigate the possible interaction of immature and mature antigen-presenting dendritic cells with the yeast M. furfur and its allergenic components. Monocyte-derived dendritic cells (MDDCs) generated from human peripheral blood were allowed to interact with FITC-labelled whole M. furfur yeast cells, M. furfur extract, a recombinant allergen from M. furfur designated rMal f 5 and M. furfur mannan, in the absence of IgE antibodies. Interaction and uptake were detected using flow cytometry and confocal laser scanning microscopy. Internalization of M. furfur yeast cells and yeast components by immature MDDCs was found using confocal laser scanning microscopy. Results from flow cytometric studies showed that a median of 94% (range, 65-98%) of the immature CD1a+ MDDCs were M. furfur extract positive, 81% (75-97%) rMal f 5 positive and 93% (62-98%) mannan positive. Mature CD1a+ MDDCs were significantly less efficient in this respect, with the corresponding figures only 26% (6-37%, P < 0.01), 6% (2-15%, P < 0.05) and 32% (9-50%, P < 0.01), respectively. Uptake of the non-glycosylated rMal f 5 by immature CD1a+ MDDCs was decreased to 27% (15-38%) by inhibition of pinocytosis. The binding of M. furfur extract and mannan was inhibited in a dose-dependent manner by methyl-alpha-D-mannopyranoside, suggesting uptake via the mannose receptor. Human immature CD1a+ MDDCs can efficiently take up M. furfur and allergenic components from the yeast in the absence of IgE antibodies, implying that sensitization of AD patients to M. furfur can be mediated by immature dendritic cells in the skin.

Expression of cystic fibrosis transmembrane conductance regulator in liver tissue from patients with cystic fibrosis.

The authors examined the expression of cystic fibrosis transmembrane conductance regulator (CFTR) and its relationship to histopathological changes in cystic fibrosis (CF) liver tissue. Immunohistochemistry was used to examine expression of CFTR, intercellular adhesion molecule-1 (ICAM-1) and liver cell-type markers in liver cryosections in 11 patients with CF-associated liver disease, and non-CF controls with (n = 17) and without (n = 3) liver disease. In CF patients prominent inflammatory infiltrates were not found, yet hepatic stellate cells were identified within fibrotic areas around bile ducts. Proliferating bile ducts displayed ICAM-1 immunoreactivity in 3 cases, but bile ducts were otherwise negative. In 2 patients homozygous for R764X and for 1112delT no CFTR immunoreactivity was detected. Bile-duct epithelial cells in patients carrying the DeltaF508 mutation displayed aberrant cytoplasmic immunolocalization of CFTR, as determined with confocal laser scanning microscopy, in contrast to the distinct CFTR expression at the luminal surface seen in controls. No clear relationship between CFTR expression and fibrosis or inflammation was evidenced in CF patients. In conclusion, these findings are consistent with an impairment of DeltaF508 CFTR processing in intrahepatic biliary epithelium. ICAM-1 expression on bile-duct epithelial cells and inflammatory infiltrates were rare findings in CF liver tissue, indicating that immunological mechanisms are unlikely to be involved in initiation of CF-associated liver disease.