The Smoothened agonist SAG Modulates the Male and Female Peripheral Immune Systems Differently in an Immune Model of Central Nervous System Demyelination.

Both Hedgehog and androgen signaling pathways are known to promote myelin regeneration in the central nervous system. Remarkably, the combined administration of agonists of each pathway revealed their functional cooperation towards higher regeneration in demyelination models in males. Since multiple sclerosis, the most common demyelinating disease, predominates in women, and androgen effects were reported to diverge according to sex, it seemed essential to assess the existence of such cooperation in females. Here, we developed an intranasal formulation containing the Hedgehog signaling agonist SAG, either alone or in combination with testosterone. We show that SAG promotes myelin regeneration and presumably a pro-regenerative phenotype of microglia, thus mimicking the effects previously observed in males. However, unlike in males, the combined molecules failed to cooperate in the demyelinated females, as shown by the level of functional improvement observed. Consistent with this observation, SAG administered in the absence of testosterone amplified peripheral inflammation by presumably activating NK cells and thus counteracting a testosterone-induced reduction in Th17 cells when the molecules were combined. Altogether, the data uncover a sex-dependent effect of the Hedgehog signaling agonist SAG on the peripheral innate immune system that conditions its ability to cooperate or not with androgens in the context of demyelination.

Androgens show sex-dependent differences in myelination in immune and non-immune murine models of CNS demyelination.

Neuroprotective, anti-inflammatory, and remyelinating properties of androgens are well-characterized in demyelinated male mice and men suffering from multiple sclerosis. However, androgen effects mediated by the androgen receptor (AR), have been only poorly studied in females who make low androgen levels. Here, we show a predominant microglial AR expression in demyelinated lesions from female mice and women with multiple sclerosis, but virtually undetectable AR expression in lesions from male animals and men with multiple sclerosis. In female mice, androgens and estrogens act in a synergistic way while androgens drive microglia response towards regeneration. Transcriptomic comparisons of demyelinated mouse spinal cords indicate that, regardless of the sex, androgens up-regulate genes related to neuronal function integrity and myelin production. Depending on the sex, androgens down-regulate genes related to the immune system in females and lipid catabolism in males. Thus, androgens are required for proper myelin regeneration in females and therapeutic approaches of demyelinating diseases need to consider male-female differences.

P2X7 purinergic receptor plays a critical role in maintaining T-cell homeostasis and preventing lupus pathogenesis.

The severe lymphoproliferative and lupus diseases developed by MRL/lpr mice depend on interactions between the Fas lpr mutation and MRL genetic background. Thus, the Fas lpr mutation causes limited disease in C57BL/6 mice. We previously found that accumulating B220+ CD4-CD8- double negative (DN) T cells in MRL/lpr mice show defective P2X7 receptor ( P2X7)-induced cellular functions, suggesting that P2X7 contributes to T-cell homeostasis, along with Fas. Therefore, we generated a B6/lpr mouse strain (called B6/lpr-p2x7KO) carrying homozygous P2X7 knockout alleles. B6/lpr-p2x7KO mice accumulated high numbers of FasL-expressing B220+ DN T cells of CD45RBhighCD44high effector/memory CD8+ T-cell origin and developed severe lupus, characterized by leukocyte infiltration into the tissues, high levels of IgG anti-dsDNA and rheumatoid factor autoantibodies, and marked cytokine network dysregulation. B6/lpr-p2x7KO mice also exhibited a considerably reduced lifespan. P2X7 is therefore a novel regulator of T-cell homeostasis, of which cooperation with Fas is critical to prevent lymphoaccumulation and autoimmunity.

Hypercholesterolemia Negatively Regulates P2X7-Induced Cellular Function in CD4+ and CD8+ T-Cell Subsets from B6 Mice Fed a High-Fat Diet.

We have previously showed that plasma membrane cholesterol and GM1 ganglioside content are responsible for the opposite sensitivity of mouse leukemic T cells to ATP. We also reported that the sensitivity of CD4+ and CD8+ T cells to ATP depends on their stage of differentiation. Here, we show that CD4+ and CD8+ T cells from B6 mice express different levels of membrane GM1 and P2X7 but similar levels of cholesterol. Thus, in CD4+ T cells, membrane cholesterol content negatively correlated with ATP/P2X7-induced CD62L shedding but positively correlated with pore formation, phosphatidylserine externalization, and cell death. By contrast, in CD8+ T cells, cholesterol, GM1, and P2X7 levels negatively correlated with all these ATP/P2X7-induced cellular responses. The relationship between cholesterol and P2X7-induced cellular responses was confirmed by modulating cholesterol levels either ex vivo or through a high-fat diet. Membrane cholesterol enrichment ex vivo led to a significant reduction in all P2X7-induced cellular responses in T cells. Importantly, diet-induced hypercholesterolemia in B6 mice was also associated with decreased sensitivity to ATP in CD4+ and CD8+ T cells, highlighting the relationship between cholesterol intake and the amplitudes of P2X7-induced cellular responses in T cells.

[TGF-ß : A key player in age-related loss of skin innate immunity]. / TGF-ß : un acteur essentiel de la perte d'immunité innée du tissu cutané au cours du vieillissement - L'actualité scientifique vue par les étudiants du Master Biologie Santé, module physiopathologie de la signalisation, Université Paris-Saclay.

TITLE: TGF-ß : un acteur essentiel de la perte d'immunité innée du tissu cutané au cours du vieillissement - L'actualité scientifique vue par les étudiants du Master Biologie Santé, module physiopathologie de la signalisation, Université Paris-Saclay. ABSTRACT: Pour la sixième année, dans le cadre du module d'enseignement « Physiopathologie de la signalisation ¼ proposé par l'université Paris-sud, les étudiants du Master « Biologie Santé ¼ de l'université Paris-Saclay se sont confrontés à l'écriture scientifique. Ils ont sélectionné une quinzaine d'articles scientifiques récents dans le domaine de la signalisation cellulaire présentant des résultats originaux, via des approches expérimentales variées, sur des thèmes allant des relations hôte-pathogène aux innovations thérapeutiques, en passant par la signalisation hépatique et le métabolisme. Après un travail préparatoire réalisé avec l'équipe pédagogique, les étudiants, organisés en binômes, ont ensuite rédigé, guidés par des chercheurs, une Nouvelle soulignant les résultats majeurs et l'originalité de l'article étudié. Ils ont beaucoup apprécié cette initiation à l'écriture d'articles scientifiques et, comme vous pourrez le lire, se sont investis dans ce travail avec enthousiasme ! Trois de ces Nouvelles sont publiées dans ce numéro, les autres le seront dans des prochains numéros.

Functional cooperation of the hedgehog and androgen signaling pathways during developmental and repairing myelination.

Hedgehog morphogens control fundamental cellular processes during tissue development and regeneration. In the central nervous system (CNS), Hedgehog signaling has been implicated in oligodendrocyte and myelin production, where it functions in a concerted manner with other pathways. Since androgen receptor (AR) plays a key role in establishing the sexual phenotype of myelin during development and is required for spontaneous myelin regeneration in the adult CNS, we hypothesized the existence of a possible coordination between Hedgehog and androgen signals in oligodendrocyte and myelin production. Here, we report complementary activities of both pathways during early postnatal oligodendrogenesis further revealing that persistent Hedgehog signaling activation impedes myelin production. The data also uncover prominent pro-myelinating activity of testosterone and involvement of AR in the control of neural stem cell commitment toward the oligodendroglial lineage. In the context of CNS demyelination, we provide evidence for the functional cooperation of the pathways leading to acceleration of myelin regeneration that might be related to their respective role on microglial and astroglial responses, higher preservation of axonal integrity, lower neuroinflammation, and functional improvement of animals in an immune model of CNS demyelination. Strong decreases of deleterious cytokines in the CNS (GM-CSF, TNF-α, IL-17A) and spleen (IL-2, IFN-γ) stand as unique features of the combined drugs while the potent therapeutic activity of testosterone on peripheral immune cells contributes to increase tolerogenic CD11c+ dendritic cells, reduce the clonal expansion of conventional CD4+ T cells and increase CD4+ Foxp3+ regulatory T cells. Altogether, these data might open promising perspectives for demyelinating diseases.

CD8+, but not CD4+ effector/memory T cells, express the CD44highCD45RBhigh phenotype with aging, which displays reduced expression levels of P2X7 receptor and ATP-induced cellular responses.

Previously we reported that the sensitivity of CD4+ T cells to ATP does not depend on P2X7 receptor (P2X7R) expression levels but on their activation and differentiation stages. Therefore, here we have investigated a potential relationship between the sensitivity of CD8+ T cells to ATP and their stages of differentiation. Thus, the CD8+ subpopulation exhibits a drastically reduced sensitivity to ATP with aging, which parallels the strong increase of an effector/memory CD8+ subset expressing high levels of CD44 cell adhesion molecule and CD45RB transmembrane phosphatase (CD44hiCD45RBhi). Using l-selectin/CD62L, CC-chemokine receptor 7, and CD127/IL-7 receptor-α markers, we showed that effector/memory CD8+ T cells belong to a central or effector memory subset. In contrast, the CD44hiCD45RBhi effector/memory subset is absent or poorly expressed in the CD4+ T subpopulation regardless of age. While ATP treatment can trigger channel and pore formation, CD62L shedding, phosphatidylserine exposure, and cell death in the CD44loCD45RBhi-naive CD8+ subset, it is unable to induce these cellular activities in the CD44hiCD45RBhi effector/memory CD8+ subset. Importantly, both CD44loCD45RBhi-naive and CD44hiCD45RBhi effector/memory subsets express similar low levels of P2X7R, demonstrating that the sensitivity of CD8+ T cells to ATP depends on the stage of differentiation instead of P2X7R expression levels.-Mellouk, A., Bobé, P. CD8+, but not CD4+ effector/memory T cells, express the CD44highCD45RBhigh phenotype with aging, which displays reduced expression levels of P2X7 receptor and ATP-induced cellular responses.

Variations in Cellular Responses of Mouse T Cells to Adenosine-5'-Triphosphate Stimulation Do Not Depend on P2X7 Receptor Expression Levels but on Their Activation and Differentiation Stage.

A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5'-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RBlow Tconvs, but not CD45RBhigh Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RBlow Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RBhighCD44low to effector/memory CD45RBlowCD44high stage. Maximum levels of upregulation are reached on recently activated CD69+ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69+ CD45RBhighCD44low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RBhighCD44low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RBlow Tconvs, CD45RBlowFoxp3+ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different abilities of ATP-treated Tconvs to form pore or cleave CD62L depending on their activation and differentiation state suggests that P2X7R signaling varies according to the physiological role of T convs during antigen activation in secondary lymphoid organs or trafficking to inflammatory sites.

P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells.

P2X7 receptor activation contributes to inflammation development in different pathologies. We previously reported that the P2X7 receptor is over-expressed in the gut mucosa of patients with inflammatory bowel disease, and that P2X7 inhibition protects against chemically induced colitis. Here, we investigated in detail the role of the P2X7 receptor in inflammatory bowel disease development, by treating P2X7 knockout (KO) and WT mice with two different (and established) colitis inductors. P2X7 KO mice were protected against gut inflammation induced by 2,4,6-trinitrobenzenesulfonic acid or oxazolone, with no weight loss or gut histological alterations after treatment. P2X7 receptor knockout induced regulatory T cell accumulation in the colon, as evaluated by qRT-PCR for FoxP3 expression and immunostaining for CD90/CD45RBlow. Flow cytometry analysis of mesenteric lymph node cells showed that P2X7 activation (by ATP) triggered regulatory T cell death. In addition, such cells from P2X7 KO mice expressed more CD103, suggesting increased migration of regulatory T cells to the colon (relative to the WT). Our results show that the P2X7 has a key role during inflammation development in inflammatory bowel disease, by triggering the death and retention in the mesenteric lymph nodes of regulatory T cells that would otherwise promote immune system tolerance in the gut.

Sensitivity of leukemic T-cell lines to arsenic trioxide cytotoxicity is dependent on the induction of phosphatase B220/CD45R expression at the cell surface.

BACKGROUND: Arsenic trioxide (As2O3) is highly effective in treating acute promyelocytic leukemia (APL), but shows more variable therapeutic efficacy for other types of hematological malignancies. Previously, we reported that As2O3 selectively eliminates pathogenic B220-expressing T cells in autoimmune MRL/lpr mice. We investigated herein the relationship between As2O3 sensitivity of leukemic T-cell lines and the expression levels of the B220 isoform of transmembrane tyrosine phosphatase CD45. METHODS: GSH content, O2(-) production, and B220, HSP70, Fas and FasL membrane expression was measured by flow cytometry. Subcellular localization of B220 was determined by imaging flow cytometry. Cell death was analyzed by morphological changes, annexin V and propidium iodide staining, and caspase 8 and 9 activation. B220 mRNA expression was analyzed by RT-PCR. Activated NF-κB p50 was quantified by a DNA binding ELISA. RESULTS: We selected human (Jurkat, Jurkat variant J45.01, HPB-ALL) and mouse (EL-4, BW5147, L1210) T-cell lines for their marked differences in As2O3 sensitivity over a large range of doses (1 to 20 µM). Differences in redox status cannot explain the dramatic differences in As2O3 sensitivity observed among the T-cell lines. Unexpectedly, we found that B220 is differentially induced on As2O3-treated T-cell lines. As2O3 treatment for 24 h induced low (HPB-ALL), intermediate (Jurkat) and high (EL-4, BW5147) levels of B220 membrane expression, membrane-bound HSP70 and cell death, but inhibited NF-κB p50 nuclear translocation. When high levels of B220 expression were achieved with low doses of As2O3, the T-cell lines died by apoptosis only. When high doses of As2O3 were required to induce B220 expression, leukemic T cells died by both apoptosis and necrosis. CONCLUSIONS: Cellular redox status is not essential for As2O3 sensitivity of leukemic T cells, suggesting the existence of additional factors determining their sensitivity to As2O3 cytotoxicity. Phosphatase B220 could be such a factor of sensitivity. As2O3 treatment inhibits NF-κB p50 nuclear translocation, and induces B220 expression and cell death in a dose and time dependent manner. The levels of B220 induction on leukemic T cells strictly correlate with both the extent and form of cell death, B220 might therefore play a checkpoint role in death pathways.

Leukocyte population dynamics and detection of IL-9 as a major cytokine at the mouse fetal-maternal interface.

Despite much interest in the mechanisms regulating fetal-maternal interactions, information on leukocyte populations and major cytokines present in uterus and placenta remains fragmentary. This report presents a detailed and quantitative study of leukocyte populations at the mouse fetal-maternal interface, including a comparison between pregnancies from syngeneic and allogeneic crosses. Our results provide evidence for drastic differences not only in the composition of leukocyte populations in the uterus during pregnancy, but also between uterine and placental tissues. Interestingly, we have observed a significant decrease in the number of myeloid Gr1+ cells including monocytes, and myeloid CD11c+ cells including DCs in placenta from an allogeneic pregnancy. In addition, we have compared the expression levels of a panel of cytokines in non-pregnant (NP) or pregnant mouse uterus, in placenta, or in their isolated resident leukocytes. Qualitative and quantitative differences have emerged between NP, pregnant uterus and placenta. Unexpectedly, IL-9 was the major cytokine in NP uterus, and was maintained at high levels during pregnancy both in uterus and placenta. Moreover, we have found that pregnancy is associated with an increase in uterine IL-1a and a significant decrease in uterine G-CSF and GM-CSF. Comparing allogeneic versus syngeneic pregnancy, less allogeneic placental pro-inflammatory cytokines CCL2 (MCP-1), CXCL10 (IP-10) and more IL1-α in whole uterus was reproducibly observed. To our knowledge, this is the first report showing a detailed overview of the leukocyte and cytokine repertoire in the uterus of virgin females and at the fetal-maternal interface, including a comparison between syngeneic and allogeneic pregnancy. This is also the first evidence for the presence of IL-9 in NP uterus and at the maternal-fetal interface, suggesting a major role in the regulation of local inflammatory or immune responses potentially detrimental to the conceptus.

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.

ADAMs 10 and 17 represent differentially regulated components of a general shedding machinery for membrane proteins such as transforming growth factor alpha, L-selectin, and tumor necrosis factor alpha.

Protein ectodomain shedding is a critical regulator of many membrane proteins, including epidermal growth factor receptor-ligands and tumor necrosis factor (TNF)-alpha, providing a strong incentive to define the responsible sheddases. Previous studies identified ADAM17 as principal sheddase for transforming growth factor (TGF)-alpha and heparin-binding epidermal growth factor, but Ca++ influx activated an additional sheddase for these epidermal growth factor receptor ligands in Adam17-/- cells. Here, we show that Ca++ influx and stimulation of the P2X7R signaling pathway activate ADAM10 as sheddase of many ADAM17 substrates in Adam17-/- fibroblasts and primary B cells. Importantly, although ADAM10 can shed all substrates of ADAM17 tested here in Adam17-/- cells, acute treatment of wild-type cells with a highly selective ADAM17 inhibitor (SP26) showed that ADAM17 is nevertheless the principal sheddase when both ADAMs 10 and 17 are present. However, chronic treatment of wild-type cells with SP26 promoted processing of ADAM17 substrates by ADAM10, thus generating conditions such as in Adam17-/- cells. These results have general implications for understanding the substrate selectivity of two major cellular sheddases, ADAMs 10 and 17.

[New therapeutic perspectives for arsenic: from acute promyelocytic leukemia to autoimmune diseases]. / De nouvelles perspectives thérapeutiques pour l'arsenic: De la leucémie aiguë promyélocytaire aux maladies auto-immunes.

Since 1996, arsenic trioxide (As2O3) is used to treat patients with acute promyelocytic leukemia. We have recently shown that As2O3 is a novel promising therapeutic agent for the autoimmune diseases (human lupus-like syndrome) and the massive lymphoproliferation (human autoimmune lymphoproliferative-like syndrome) developed by MRL/lpr mice. As2O3 is able to achieve an almost complete regression of antibody- and cell-mediated manifestations in MRL/lpr mice. As2O3 eliminated the activated T lymphocytes responsible for lymphoproliferation and skin, lung, and kidney lesions. This treatment also markedly reduced anti-DNA autoantibodies, rheumatoid factor, IL-18, IFN-gamma, nitric oxide metabolites, TNF-alpha, Fas ligand and IL-10 levels, and immune-complex deposits in glomeruli, leading to significantly prolonged survival rates.

Arsenic trioxide: A promising novel therapeutic agent for lymphoproliferative and autoimmune syndromes in MRL/lpr mice.

MRL/lpr mice develop a human lupuslike syndrome and, as in autoimmune lymphoproliferative syndrome (ALPS), massive lymphoproliferation due to inactivation of Fas-mediated apoptosis. Presently, no effective therapy exists for ALPS, and long term, therapies for lupus are hazardous. We show herein that arsenic trioxide (As2O3) is able to achieve quasi-total regression of antibody- and cell-mediated manifestations in MRL/lpr mice. As2O3 activated caspases and eliminated the activated T lymphocytes responsible for lymphoproliferation and skin, lung, and kidney lesions, leading to significantly prolonged survival rates. This treatment also markedly reduced anti-DNA autoantibody, rheumatoid factor, IL-18, IFN-gamma, nitric oxide metabolite, TNF-alpha, Fas ligand, and IL-10 levels and immune-complex deposits in glomeruli. As2O3 restored cellular reduced glutathione levels, thereby limiting the toxic effect of nitric oxide, which is overproduced in MRL/lpr mice. Furthermore, As2O3 protected young animals against developing the syndrome and induced almost total disease disappearance in older affected mice, thereby demonstrating that it is a novel promising therapeutic agent for autoimmune diseases.

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.

An essential role for IFN-alpha in the overexpression of Fas ligand on MRL/lpr lymphocytes and on their spontaneous Fas-mediated cytotoxic potential.

Lymphocytes from aged autoimmune MRL/lpr mice overexpress Fas ligand (FasL), and are cytotoxic against Fas+ target cells. This cytotoxic potential is only partly due to FasL, as wild-type MRL+/+ lymphocytes are not able to kill Fas+ targets after induction of FasL. In addition, serum levels of interferon-alpha (IFN-alpha) increase in parallel with the Fas-dependent cytotoxic potential of lymphocytes from MRL/lpr mice as they age. To understand the mechanisms underlying these observations, combined suppression subtractive hybridization (SSH) and RT-PCR were used to study differential gene expression in splenocytes from MRL/lpr mice compared with splenocytes from MRL+/+ mice. Twenty-two genes were upregulated transcriptionally in MRL/lpr splenocytes compared with their MRL+/+ counterparts. Furthermore, 9 of these genes were also upregulated after treatment of MRL/lpr splenocytes with IFN-alpha, and 4 were strongly downregulated. MRL/lpr lymphocytes were also found to be hyperresponsive to IFN-alpha. Thus, MRL/lpr lymphocytes overexpressed mRNA for the IFN-alpha receptor (IFNAR-1 and IFNAR-2) chains of the IFN-alpha/beta receptor and exhibited high endogenous levels of both Stat1 and phosphorylated Stat1 proteins. Lymphocytes from young MRL/lpr mice, with low Fas-dependent cytotoxic activity, were found to become highly cytotoxic against Fas+ targets after treatment with IFN-alpha. These data suggest that IFN-alpha plays an important role in the physiopathology of the systemic lupus erythematosus (SLE)-like syndrome that occurs in MRL/lpr mice.