Pierre Bobé

Arsenic enhances the activation of Stat1 by interferon gamma leading to synergistic expression of IRF-1.

Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia (APL), including those who have relapsed after treatment with all-trans-retinoic acid (RA). In vitro studies with the APL-derived NB4 cell line showed that As2O3 exerts a dose-dependent dual effect, which induces apoptosis at 1 μM, whereas at a lower concentration of 0.1 μM, a partial differentiation of APL is observed. In non-APL cells, interferon (IFN) α and 1 μM As2O3 act synergistically to induce apoptosis. In this report, we show that in NB4 cells and in two RA-resistant NB4-derived cell lines, NB4-R1 and NB4-R2, IFNα or IFNγ combined with 0.1 μM As2O3 lead to an increased maturation effect. Moreover, IFNγ alone is able to differentiate RA-sensitive and -resistant cells with a higher maturation effect on NB4-R2 cells. In contrast, all these cells underwent apoptosis in the presence of the cytokine and a higher concentration of As2O3. IFNγ boosted As2O3-induced apoptosis in APL cells as tested by TUNEL, Annexin V staining and activation of caspase 3. As2O3 differently altered IFN-induced gene products; it downregulated PML/RARα and PML, did not alter PKR and Stat1, and upregulated interferon regulatory family (IRF)-1. Synergism by IFNγ and arsenic on IRF-1 expression is mediated by a composite element in the IRF-1 promoter that includes an IFNγ-activation site (GAS) overlapped by a nonconsensus site for nuclear factor kappa B (NFκB). Arsenic has no effect on NFκB, whereas it enhances the activation of Stat1 by IFNγ in NB4 cells leading to an increase in IRF-1 expression.

B lymphocytes mediate Fas‐dependent cytotoxicity in MRL/lpr mice

The Fas/Fas ligand (FasL) pathway is one of the two major effector mechanisms of T cell‐mediated cytotoxicity. To prevent nonspecific killing by lymphoid cells, FasL expression on the cell surface of immune effector cells is strictly regulated. However, MRL/lpr autoimmune‐prone mice massively overexpress FasL on their T lymphocytes, which render them able to kill Fas+ targets in vitro and in vivo. It is surprising that we show in the present work that B lymphocytes purified from MRL/lpr spleen cells express FasL to the same extent as T cells at the mRNA and protein level. These B cells are potent cytotoxic effectors against Fas+ but not Fas− targets. The B lymphocyte effectors were used ex vivo without any in vitro activation by B cell stimuli. Furthermore, we found that MRL/lpr B lymphocytes have the same cytotoxic potential as natural killer cells, which have been characterized as potent, Fas‐mediated, cytotoxic effectors. The level of membrane‐anchored FasL increases with the size of the B cell and cell‐surface activation marker CD69 expression, indicating that the expression of FasL is up‐regulated in parallel with the activation state of the B cell. The activated B cell population contained the major cytotoxic activity, and a minor part was associated with CD138/Syndecan‐1+ plasma cells.

Respective roles of TNF-|[alpha]| and IL-6 in the immune response-elicited by adenovirus-mediated gene transfer in mice

The immunogenicity of recombinant adenoviruses (Ad) constitutes a major concern for their use in gene therapy. Antibody- and cell-mediated immune responses triggered by adenoviral vectors hamper long-term transgene expression and efficient viral readministration. We previously reported that interleukin (IL)-6 and tumor necrosis factor (TNF)-α play an essential role in both the acute phase and antibody response against Ad, respectively. As TNF-α controls the immune response and the development of the immune system, we examined here the consequence of blockade of TNF-α activity through Ad-mediated gene delivery of a dimeric mouse TNFR1-IgG fusion protein on transgene expression from a second Ad. Ad encoding TNFR1-IgG (AdTNFR1-Ig) was injected intravenously along with Ad encoding β-galactosidase or α1-antitrypsin transgene in wild-type (IL-6+/+) but also in IL-6-deficient mice (IL-6−/−) to analyze how TNF-α and IL-6 diminish liver gene transfer efficacy. Blockade of TNF-α leads to increased transgene expression in both wild-type and IL-6−/− mice due to a reduced inflammatory response and to diminished recruitment of macrophages and NK cells towards the liver. Antibody responses against adenoviral particles and expressed transgenes were only delayed in AdTNFR1-Ig-treated wild-type mice, but were markedly reduced in AdTNFR1-Ig-treated IL-6−/− mice. Finally, treatment of mice with etanercept, a clinically approved anti-TNF-α drug, confirmed the importance of controlling proinflammatory cytokines during gene therapy by adenoviral vectors.

Unusual expression of LINE-1 transposable element in the MRL autoimmune lymphoproliferative syndrome-prone strain.

LINE-1 are endogenous mobile genetic elements that have dispersed and accumulated in the genomes of eukaryotes via germline transposition, with up to 100 000 copies in mammalian genomes. LINE-1 elements transpose by reverse transcription of their own transcript. Transposition requires synthesis of a full-length, sense-strand transcripts and proteins encoded by open reading frame (ORF) 1 and ORF2. Although severely repressed in most normal tissues, LINE-1 occasionally leads to disease by insertional mutagenesis. In the present study, Northern blot and in situ hybridization analyses revealed a template-strand transcription of LINE-1 ORF2 (encoding reverse transcriptase, RT) in lymphoid organs and the liver from MRL-+/+ and Fas-deficient MRL/lpr strains and their normal ancestors. While these sense transcripts are restricted to the nucleus in hepatocytes, they are also found in the cytoplasm in splenocytes. In contrast to transcription, ORF2 translation was detected only in MRL strains, as shown by the cytoplasmic labelling of splenic cells obtained with a monoclonal antibody recognizing the LINE-1 RT. This antibody coprecipitated two proteins of 45 and 12 kDa from MRL/lpr lymphoid organ lysates that were removed by pretreatment with anti-β2-microglobulin antiserum, suggesting a structural association between a LINE-1 product and a major histocompatibility complex class I or class I-like molecule.

Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220 + Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice

Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220+ CD4–CD8– (DN) T lymphocytes. The precise ontogeny of B220+ DN T cells is unknown. B220+ DN T lymphocytes could be derived from effector CD4+ and CD8+ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220+ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220+ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220+ T cells observed in normal MRL+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220+ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.