Polybrene and interleukin-4: two opposing factors for retroviral transduction of bone-marrow-derived dendritic cells.

BACKGROUND: Gene transfer using retroviral transduction offers the advantage of long-term transgene expression in developing strategies that use dendritic cells (DCs) for immunotherapy. The goal of this study was to infect DCs in an immature state in order to take advantage of their proliferating and tolerogenic potential. METHODS: Immature DCs were generated from murine bone marrow (BM) using either GM-CSF alone or GM-CSF plus IL-4. The cells were transduced directly with retroviral supernatants or by co-culture with the GP + E-86 retroviral packaging cell line in the presence of two different cationic polymers: polybrene and protamine sulfate. Phenotypic and functional characterization of the transduced cells were then performed. RESULTS: Our results show a low efficiency of retroviral infection of DCs in the presence of polybrene. This cationic polymer was found to be directly cytotoxic to murine DCs and thus favored the growth of contaminating macrophages. This effect was not observed using protamine sulfate. Furthermore, stimulation by IL-4 early in the culture increased DC differentiation, proliferation and transduction. However, we found that DCs generated in GM-CSF plus IL-4 presented a more mature phenotype with an enhanced allogeneic stimulating activity. Finally, we showed that DCs themselves down-regulated transgene expression in the co-cultured packaging cell line in a promoter-dependent manner. CONCLUSIONS: We have defined optimal conditions to generate and transduce murine BM-derived DCs. This included: the use of protamine sulfate during exposure to retroviral infectious supernatant and the addition of IL-4 at an early stage of the culture. Nevertheless, this cytokine also induced DC maturation. These findings have potential implications in experimental gene therapy.

A self-defence mechanism of astrocytes against Fas-mediated death involving interleukin-8 and CXCR2.

Astrocytes play multiple roles from passive support to the regulation of inflammation during brain injury. This latter function is in part achieved by responses induced by the triggering of Fas expressed on astrocytes both and. It was previously shown that astrocytes are resistant to Fas-mediated death, responding to Fas triggering by interleukin-8 production. However, the cellular mechanisms by which astrocytes protect themselves from Fas-mediated death are unclear. Here, we show that survival of cultured astrocytes after Fas triggering is governed by the interaction of interleukin-8 with one of its receptors, CXCR2. Furthermore, interleukin-8 secretion and CXCR2 expression are both induced in human astrocytes after Fas stimulation, suggesting a new mechanism of self-defence against Fas-mediated death.

Exposure to exogenous DNA can modify the sensitivity of the Fas apoptotic pathway.

BACKGROUND: Gene-transfer techniques are commonly employed for both in vitro and in vivo studies. However, modifications of the target cell following the introduction of the gene of interest are not often examined. These modifications can alter the immunogenicity and/or the susceptibility of the target cell to apoptosis and may produce unwanted consequences in vivo. METHODS: Gene transfer into the murine fibroblastic Psi-CRIP packaging cell line was performed using calcium phosphate precipitation, cationic liposome-DNA complexes or a retroviral RNA-mediated method. After gene transfer, Fas expression, cytokine production, and sensitivity to Fas ligand (FasL)-mediated death were assessed. RESULTS: Following transfection of a FasL expression vector by calcium phosphate precipitation, an unexpected increase was observed in apoptotic cell death in previously Fas-resistant Psi-CRIP cells. This apoptosis was due to Fas upregulation and an increase of sensitivity to FasL-mediated death. Other plasmids coding non-cytotoxic factors also modulated this apoptotic pathway. The co-stimulatory molecule CD80 was also upregulated. Exposure to naked DNA alone elicited the same response. The effect was not dependent on the methylation status of exogenous DNA, but was found to be dependent on the target cell type and might be avoided by the use of an RNA-mediated retroviral system. CONCLUSIONS: Plasmid transfection or simple exposure to naked DNA can increase sensitivity to apoptosis. The generation of FasL packaging cell lines is therefore limited by an increase in FasL/Fas-mediated apoptosis. These findings should be considered when using genetically modified transplantable cells in order to prevent elimination by host cytotoxic cells and in particular when cells are engineered using FasL.