HLA-DR mediated cell death is associated with, but not induced by TNF-alpha secretion in APC.

Tumor necrosis factor alpha (TNFalpha) is a pleiotropic cytokine involved in inflammatory responses which can trigger both cell apoptosis and cell activation. In antigen presenting cells (APC), TNFalpha increased antigen presentation, notably by up-regulation of HLA class II expression. In addition to their role in antigen presentation, HLA-DR molecules transduce intracellular signals which lead to cytokine up-regulation or cell death. We have previously observed that the susceptibility of APC to HLA-DR mediated apoptosis increase throughout their maturation. We therefore investigated the relationship between TNFalpha production and susceptibility to HLA-DR-mediated apoptosis of different APC. The hematopoietic progenitor cell line (KG1), monocytic cell line (THP-1), monocyte-derived dendritic cell (DC), and B-lymphoid cell line (Raji) have been studied. We report that apoptosis susceptibility and spontaneous TNFalpha release are correlated in these different cells. However, while autocrine TNFalpha production was critical for DC maturation, upregulation of TNFalpha release after HLA-DR crosslinking was not observed and neutralization of endogenous TNFalpha did not modify HLA-DR-mediated apoptosis. These data reveal that HLA-DR mediated apoptosis susceptibility and spontaneous TNFalpha release are regulated in a parallel manner and that while TNFalpha may induce maturation of APC to an "apoptosis sensitive" stage, there is no direct role for TNFalpha in HLA-DR-mediated apoptosis of APC.

Differential expression of major histocompatibility complex class Ia, Ib, and II molecules on monocytes-derived dendritic and macrophagic cells.

Blood monocyte derived antigen presenting cells (APC) such as dendritic cells and macrophages are considered as major promising tools for antitumoral immunotherapy. In order to contribute to their phenotype characterization, we have precisely investigated their levels of expression of MHC class Ia, Ib (HLA-G) and II molecules using mainly flow cytometry quantification assays. APC were generated from monocytes cultured for 7 days in the presence of GM-CSF and IL-4 or M-CSF. These cells, which exhibited known morphological and immunological features of dendritic cells and macrophages respectively, were evidenced to display high expression of MHC class Ia and class II antigens in comparison to that found in monocytes. Dendritic cells and macrophages thus expressed 2-fold more and 4-fold more MHC class Ia molecules and 5-fold and 3-fold more MHC class II DR molecules than parental monocytes. In addition, expression of MHC class II DP and DQ molecules, not or only barely detected in monocytes, was clearly demonstrated in the two kinds of APC. In contrast, monocytes, dendritic cells and macrophages failed to express MHC class Ib HLA-G antigen. The up-regulation in monocyte-derived APC of MHC class Ia and II molecules mediating the presentation of antigen peptides to lymphocytes fully supports the interest of such APC in antitumoral immunotherapy.

Differential expression of the efflux pumps P-glycoprotein and multidrug resistance-associated protein in human monocyte-derived dendritic cells.

P-glycoprotein (P-gp), an ATP-binding cassette (ABC) drug efflux pump, has been recently shown to play an important role in the physiology of Langherans cells, a subtype of dendritic cells (DC) found in the skin. The present study was designed to investigate expression and activity of P-gp and of multidrug resistance-associated protein (MRP), another ABC efflux pump sharing numerous substrates with P-gp, in human monocyte-derived DC. Immunolabeling experiments and dye efflux assays indicated that such cells displayed elevated levels of MRP activity and expression when compared to those present in parental monocytes. Generation of DC from monocytes in the presence of the MRP inhibitor indomethacin did not, however, alter the capacity of DC to stimulate allogeneic T cells proliferation in mixed lymphocyte reaction. In addition, indomethacin did not inhibit the up-regulation of the CD1a, a marker occurring during the differentiation of monocytes into DC. In contrast to that of MRP, functional expression of P-gp was not detected in monocyte-derived DC. Such antigen presenting cells that constitute a promising tool for antitumor vaccinal therapy therefore display differential expression of the efflux pumps P-gp and MRP.

Use of the anionic dye carboxy-2',7'-dichlorofluorescein for sensitive flow cytometric detection of multidrug resistance-associated protein activity.

Multidrug resistance-associated protein (MRP) and P-glycoprotein are drug efflux pumps conferring multidrug resistance to tumor cells and sharing numerous substrates. In order to determine a flow cytometric assay allowing to analyse MRP activity in cancerous cells in a sensitive and specific manner, cellular accumulation and efflux of the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CDF) were studied by flow cytometry using mainly MRP-overexpressing lung GLC4/Sb30 cells and parental GLC4 cells. GLC4/Sb30 cells were found to display reduced accumulation and enhanced efflux of the dye when compared to their parental counterparts. Probenecid, a well known blocker of MRP, strongly enhanced CDF accumulation in GLC4/Sb30 cells through inhibiting efflux of the dye; it also increased CDF levels in GLC4 cells, although to a lesser extent, which may likely be linked to the low, but detectable, expression of MRP in these cells. Comparison of CDF retention with that of calcein demonstrated that the former dye was the most efficiently effluxed by GLC4/Sb30 cells. In contrast to MRP overexpression, that of P-glycoprotein was not found to alter cellular CDF labelling whereas it strongly impaired calcein staining. These results indicate that CDF is a substrate for MRP, but not for P-gp, which may likely be useful for sensitive and specific flow cytometric determination of MRP activity in clinical samples.

Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from peripheral blood.

Multidrug resistance proteins (MRPs) such as MRP1, MRP2 and MRP3 are membrane efflux pumps involved in multidrug resistance and handling organic anions. In the present study, MRP activity was investigated in normal mature leucocytes and CD34-positive hematopoietic cells from peripheral blood using the flow cytometric carboxy-2',7'-dichlorofluorescein (CF) efflux assay. Basal and similar cellular exports of CF, an anionic fluorescent dye substrate for MRP1 and MRP2 transporters, were evidenced in lymphocytes whatever their subsets (CD3, CD4, CD8, CD20 and CD56 cells), in CD14 monocytes and in CD15 granulocytes whereas higher CF efflux was found in CD34 cells. Such outwardly-directed transports of CF were inhibited by known blockers of MRP function such as probenecid whereas the P-glycoprotein modulator verapamil did not alter the retention of the dye in the blood leukocytes. Peripheral mature blood leukocytes were moreover found to express MRP1 mRNAs and MRP1 protein as assessed by Northern-blot and Western-blot analyses, whereas MRP2 and MRP3 transcripts were not present or only at very low levels. Mature leukocytes therefore display basal constitutive MRP-related transport activity regardless of cell lineage and likely related to MRP1 expression whereas higher MRP-related efflux can be detected in peripheral CD34 hematopoietic cells.

High multidrug resistance protein activity in acute myeloid leukaemias is associated with poor response to chemotherapy and reduced patient survival.

Multidrug resistance protein (MRP) activity was investigated in 44 newly diagnosed acute myeloid leukaemia (AML) patients using a functional assay based on efflux of carboxy-2',7'-dichlorofluorescein, an anionic dye handled by both MRP1 and MRP2. Elevated MRP transport was detected in 29% of cases, but was not significantly correlated with sex, age, white blood cell count at diagnosis or karyotype. In contrast, it was associated with secondary AML (P = 0.002), CD34 positivity (P = 0.041) and P-glycoprotein activity (P = 0.01). There was a lower rate of complete remission in MRP-positive patients versus MRP-negative patients (23% versus 81%; P = 0.001); overall survival was also better for MRP-negative patients (P = 0.004). These data indicate a probable role for MRP activity in the clinical outcome of AML.

HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC.

Professional APC are characterized by their ability to present peptide via HLA class II in the presence of costimulatory molecules (CD40, CD80, and CD86). The efficiency of Ag presentation can be classed as follows: mature dendritic cells (DC) are most efficient, immature DC and macrophages are intermediate, and monocytes are considered poor APC. There is a large body of evidence demonstrating that HLA-DR transmits signals in the APC. In this study, we have addressed the question of the outcome of HLA-DR signals on APC of the monocyte/DC lineages throughout their differentiation from immature to mature APC. DC were generated from both monocytes and CD34+ cells of the same individual, macrophages were differentiated from monocytes. Immunophenotypical analysis clearly distinguished these populations. HLA-DR-mediated signals led to marked apoptosis in mature DC of either CD34 or monocytic origin. Significantly less apoptosis was observed in immature DC of either origin. Nonetheless, even immature DC were more susceptible to HLA-DR-mediated apoptosis than macrophages, whereas monocytes were resistant to HLA-DR-mediated apoptosis. The mechanism of HLA-DR-mediated apoptosis was independent of caspase activation. Taken together, these data lead to the notion that signals generated via HLA-DR lead to the demise of mature professional APC, thereby providing a means of limiting the immune response.