Zoulikha Amraoui

Monophosphoryl Lipid A Activates Both Human Dendritic Cells and T Cells

The induction of dendritic cell (DC) maturation is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. In this study, we have investigated the effects of monophosphoryl lipid A (MPL) on human monocyte-derived DC as well as peripheral blood T cells. Calcium mobilization, mitogen-activated protein kinase activation, and the NF-κB transcription factor were induced after MPL stimulation of DC and required high doses of MPL (100 μg/ml). Maturation parameters such as production of IL-12 and increases in cell surface expression of HLA-DR, CD80, CD86, CD40, and CD83 were observed following DC treatment with MPL. However, lower levels of IL-12 were induced by MPL when compared with lipopolysaccharide. This is likely to be related to differences in the kinetics of extracellular signal-related kinase 1/2 and p-38 phosphorylation induced by both molecules. Although maturation induced by MPL was weaker when compared with lipopolysaccharide, it appeared to be sufficient to support optimal activation of allogeneic naive CD45RA+ T cell and anti-tetanus toxoid CD4 T cells. MPL at low doses (5 μg/ml) had no impact on DC maturation, while its addition to DC-T cell cocultures induced full T cell activation. The observed effect was related to the fact that MPL also acts directly on T cells, likely through their Toll-like receptors, by increasing their intracellular calcium and up-regulating their CD40 ligand expression. Together, these data support a model where MPL enhances T cell responses by having an impact on DC and T cells.

Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4.

Phosphoinositide 3‐kinases (PI3K) are known to regulate Toll‐like receptor (TLR)‐mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll‐IL‐1 receptor domain‐containing adapter‐inducing IFN‐β (TRIF)‐dependent signaling, which induces IFN‐β gene expression downstream of TLR3 and TLR4. First, treatment of monocyte‐derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN‐β expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA‐binding activity of NF‐κB, but not interferon response factor (IRF)‐3, the key transcription factors required for TLR‐mediated IFN‐β synthesis. In parallel, wortmannin‐treated DC exhibited enhanced levels of IκB kinase (IKK)‐α/β phosphorylation and IκB‐α degradation with a concomitant increase in NF‐κB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF‐κB‐dependent promoters as well as IFN‐β promoter activities without interfering with transcription at the positive regulatory domain III‐I. Furthermore, wortmannin enhanced NF‐κB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF‐mediated NF‐κB transcriptional activity. Finally, in co‐immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF‐dependent NF‐κB activity, and thereby increases IFN‐β synthesis elicited by TLR3 or TLR4 ligands.

Tumoricidal activity of monocyte-derived dendritic cells: Evidence for a caspase-8-dependent, Fas-associated death domain-independent mechanism.

Monocyte-derived dendritic cells (DC) were found to be cytotoxic for several tumor cell lines including Jurkat cells, which were killed through a calcium-independent pathway. K562 cells were resistant, excluding a NK cell-like activity. DC-mediated apoptosis did not involve classical death receptors because it was not reversed by blocking TNF/TNFR, CD95/CD95 ligand, or TNF-related apoptosis-inducing ligand/TNF-related apoptosis-inducing ligand receptor interactions. Fas-associated death domain-deficient, but not caspase-8 deficient, Jurkat cells were killed by DC. Indeed, caspase-8 cleavage was demonstrated in Jurkat cells cocultured with DC, and the use of specific caspase inhibitors confirmed that apoptosis triggered by DC was caspase-8 dependent. Furthermore, the involvement of Bcl-2 family members in the control of DC-mediated apoptosis was demonstrated by Bid cleavage in Jurkat cells cocultured with DC and resistance of Jurkat cells overexpressing Bcl-2 to DC-mediated cytotoxicity. Overall, these data indicate that monocyte-derived DC exert a caspase-8-dependent, Fas associated death domain-independent tumoricidal activity, a finding that could be relevant to their therapeutic use in cancer.

Adsorption of ß2-microglobulin on dialysis membranes: comparison of different dialyzers and effects of reuse procedures

In order to measure ß2-microglobulin adsorption on dialysis membranes, uremic plasma was passed through different dialyzers in a simulated hemodialysis circuit in which both plasma and dialysate compartments were organized as closed loops, the ultrafiltration pressure being adjusted to minimize water shifts. Under these conditions, comparison of the amounts of ß2-m in the plasma and dialysate compartments allowed us to calculate the binding of ß2-m to the membrane at different times of the procedure. Whereas cuprophane membrane (Gambro gf 180m, 1.8m2) did not bind ß2-m, AN69 (Filtral, 1.1 m2), high flux polysulfone (F60, 1.2m2) and modified polyamide (Polyflux 130, Gambro, 1.3m2) were found to adsorb 49 ± 8 mg (mean ± SEM), 17 ± 5 mg and 38 ± 4 mg of 82-m, respectively. These data were confirmed in trace labeling experiments with 125I-ß2-m. Adsorption was a saturable phenomenon occurring during the first 90 min of in vitro dialysis. After reuse with peracetic acid, the adsorption capacity of AN69 membrane was lowered to 20 ± 4 mg of ß2-m, contrasting with the unchanged adsorption after reuse with sodium hypochlorite. These data indicate that adsorption significantly contributes to ß2-m removal during hemodialysis with certain dialyzers and that reuse procedures may affect the propensity of dialysis membranes to bind 82-m.

Critical role of protein kinase C epsilon for lipopolysaccharide-induced IL-12 synthesis in monocyte-derived dendritic cells.

In the present study we have investigated the potential involvement of protein kinase C (PKC) in the maturation of human dendritic cells (DC) by bacterial lipopolysaccharide (LPS). LPS stimulation of DC derived from human monocytes resulted in PKC phosphorylation. Inhibition of PKC activation using bisindolylmaleimide (Bis), a pan‐PKC inhibitor, was associated with a dose‐dependent decrease of LPS‐induced IL‐12 production. In contrast, up‐regulation of MHC class II, CD80 and CD86 was not altered. Consistent with the diminished IL‐12 synthesis, DC stimulated with LPS in presence of Bis were deficient in the induction of IFN‐γ production by allogeneic CD4+ T cells. Furthermore, we found that PKC inhibition impaired LPS‐induced IκB‐α degradation and subsequent nuclear factor (NF)‐κB activation in DC. LPS resulted in the phosphorylation of conventional α/β and novel ϵ PKC isoforms in DC. Inhibition of LPS‐induced PKC activity using pseudosubstrate peptides specific for PKC isoforms established that PKC ϵ but not PKC α/β was involved in the production of IL‐12 and TNF‐α. Overall, these data provide evidence thatPKC inhibition impairs LPS signaling in DC and identify PKC ϵ as a potential target for the inhibition of Toll‐like receptor‐4‐mediated, IL‐12‐dependent Th1 type responses.

Helper T-cell responses elicited by Der p 1–pulsed dendritic cells and recombinant IL-12 in atopic and healthy subjects ☆ ☆☆

BACKGROUND Environmental allergens, such as Dermatophagoides pteronyssinus group 1 antigen (Der p 1), induce T(H2)-type responses in atopic patients, whereas healthy individuals have T(H1)-type responses to the same antigens. Because of their efficient synthesis of IL-12, dendritic cells (DCs) are potent inducers of T(H1)-type immune responses. OBJECTIVE We sought to determine whether DCs would skew allergen-specific T(H2)-type responses from atopic individuals. METHODS Purified CD4(+) T cells from healthy donors or atopic individuals were cultured in the absence or presence of recombinant (r)IL-12 with DCs derived from PBMCs and pulsed with Der p 1. Supernatants of DC-T cell cocultures were assayed by ELISA for IL-5 and IFN-gamma. RESULTS A T(H1)-type response developed in purified CD4(+) T cells from healthy donors in response to Der p 1-pulsed DCs, as indicated by high levels of IFN-gamma in culture supernatants. In contrast, CD4(+) T cells from atopic donors displayed a T(H2)-type profile characterized by high levels of IL-5 and low levels of IFN-gamma. The addition of rIL-12 (10 ng/mL) to DC-T cell cocultures resulted in the induction of IFN-gamma secretion by Der p 1-specific CD4(+) T cells from atopic patients, whereas their production of IL-5 was not inhibited. Using flow cytometry after intracytoplasmic staining, we found that IFN-gamma and IL-5 were secreted by distinct CD4(+) T-cell subpopulations. CONCLUSION The cytokine profile of Der p 1-specific T(H2)-like cells from atopic individuals is maintained when the allergen is presented by DCs, even in the presence of exogenous rIL-12.