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R A Flavell

Publications and source records attributed to R A Flavell.

At least 91 records · Page 5Linked to original sources
PubMed2000

The lack of NF-kappa B transactivation and PKC epsilon expression in CD4(+)CD8(+) thymocytes correlates with negative selection.

Deletion of autoreactive thymocytes at the DP stage is the basis for tolerance to thymus-expressed self antigens. In this study we investigated whether distinct signalling pathways are induced in DP thymocytes as compared to mature T cells upon stimulation with antigen. Using triple transgenic mice expressing a TCR transgene, dominant negative ras/Mek proteins and a reporter gene construct with AP-1 or NF-kappa B binding sites, we showed a complete lack of transcriptional activity of NF-kappa B but not AP-1 in DP thymocytes, whereas both were transcriptionally active in mature T cells after antigenic stimulation. Lack of NF-kappa B induction correlated with increased death in response to antigen. AP-1 induction was dependent on the integrity of the ras/Mek pathway indicating that this pathway was activated in the DP thymocytes. In contrast, we found a complete lack of constitutive expression of the epsilon isoform of Protein Kinase C (PKC) in DP thymocytes, although it was present in mature thymocytes and peripheral T cells. Taken together the results suggest that the lack of PKC epsilon in DP thymocytes could lead to the absence of NF-kappa B activity after antigenic stimulation contributing to negative selection. Cell Death and Differentiation (2000) 7, 1253 - 1262.

Animals↗
PubMed2000

Conference highlight: do T cells care about the mitogen-activated protein kinase signalling pathways?

Mitogen-activated protein (MAP) kinases, which include the extracellular response kinases, p38 and c-Jun amino terminal kinases (JNK), play a significant role in mediating signals triggered by cytokines, growth factors and environmental stress. The JNK and p38 MAP kinases have been involved in growth, differentiation and cell death in different cell types. In the present paper, we describe how the JNK and p38 MAP kinase signalling pathways are regulated and their role during thymocyte development and the activation and differentiation of T cells in the peripheral immune system. The results from these studies demonstrate that the JNK and p38 MAP kinase signalling pathways regulate different aspects of T-cell mediated immune responses.

Animals↗
PubMed2000

Chronic murine colitis is dependent on the CD154/CD40 pathway and can be attenuated by anti-CD154 administration.

BACKGROUND & AIMS: Experimental colitis in most animal models is caused by dysregulation of T lymphocytes that display a T helper 1 (Th1) phenotype. CD154 (CD40L/gp39), a member of the tumor necrosis factor (TNF) family, is up-regulated on T cells on activation and has been shown to play a key role in the induction of a Th1 response. We investigated whether chronic experimental colitis is dependent on the CD154/CD40 pathway and whether disease can be prevented by anti-CD154 antibody treatment. METHODS: Two models of chronic colitis were used: CD45Rb(hi) cell transfer into recombination activation gene-deficient (Rag(-/-)) mice and bone marrow transplant of tgepsilon26 animals. In both models, mice were reconstituted with cells from CD154-deficient animals. In another series of experiments, wild-type CD45Rb(hi) T cell-reconstituted recipients were treated with anti-CD154, either from the start of the experiment or after onset of disease. RESULTS: T cells deficient in CD154 induced a milder clinical disease, less weight loss, and fewer histologic signs of colitis than wild-type cells. The level of interleukin 12 in the serum of CD154-deficient T-cell recipients was 5-fold less than that of wild-type cell recipients. Nevertheless, no signs of deviation from a Th1 phenotype were observed. Treatment with anti-CD154 antibodies substantially impaired disease development, even when started after the onset of colitis. CONCLUSIONS: The CD154/CD40 pathway plays a critical role in Th1-induced chronic experimental colitis. Blocking CD154, even after the onset of disease, ameliorates colitis but does not induce a T helper 2 (Th2) phenotype.

Animals↗
PubMed2000

Amyloid beta-induced neuronal death is bax-dependent but caspase-independent.

Fibrillar amyloid beta (Abeta) peptides are major constituents of senile plaques in Alzheimer disease (AD) brain and cause neuronal apoptosis in vitro. Bax and caspase-3 have been implicated in the pathogenesis of AD and are components of a well-defined molecular pathway of neuronal apoptosis. To determine whether Abeta-induced neuronal apoptosis involves bax and/or caspase-3 activation, we examined the effect of Abeta on wild-type, bax-deficient, and caspase-3-deficient telencephalic neurons in vitro. In wild-type cultures, Abeta produced time- and concentration-dependent caspase-3 activation, apoptotic nuclear changes, and neuronal death. These neurotoxic effects of Abeta were not observed in bax-deficient cultures. Caspase-3 deficiency, or pharmacological inhibition of caspase activity, prevented caspase-3 activation and blocked the appearance of apoptotic nuclear features but not Abeta-induced neuronal death. Neither calpain inhibition nor microtubule stabilization with Taxol protected telencephalic neurons from Abeta-induced caspase activation or apoptosis. These results have potential implications regarding the underlying pathophysiology of AD and towards AD treatment strategies.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Interleukin 18 restores defective Th1 immunity to Candida albicans in caspase 1-deficient mice.

Caspase 1, formerly designated interleukin 1beta (IL-1beta)-converting enzyme, processes pro-IL-1beta and pro-IL-18 to yield active cytokines that play a pivotal role in inflammation and cell activation. We show here the effect of caspase 1 deficiency on the inflammatory and adaptive immune responses to the fungus Candida albicans. Caspase 1 deficiency did not affect susceptibility to primary systemic infection with the fungus, as revealed by survival and fungal growth. However, Th1-mediated resistance to reinfection was greatly impaired in caspase 1-deficient mice, and this correlated with low-level production of IL-12 and gamma interferon. Early in infection, production of these cytokines and that of tumor necrosis factor alpha, IL-6, and, interestingly, IL-1beta occurred normally in caspase 1-deficient mice, while that of IL-18 was severely impaired. Exogenous administration of IL-18, more than IL-12, restored the Th1-mediated resistance to the infection. We conclude that, while caspase 1 is not indispensable for release of mature IL-1beta in candidiasis, the caspase 1-dependent production of IL-18 may represent an important and novel pathway for the expression of sustained Th1 reactivity to the fungus.

Animals↗
PubMed2000

Serologic diagnosis of Lyme borreliosis by using enzyme-linked immunosorbent assays with recombinant antigens.

Class-specific enzyme-linked immunosorbent assays (ELISAs) with purified recombinant antigens of Borrelia burgdorferi sensu stricto and Western blot analyses with whole cells of this spirochete were used to test human sera to determine which antigens were diagnostically important. In analyses for immunoglobulin M (IgM) antibodies, 14 (82%) of 17 serum samples from persons who had erythema migrans reacted positively by an ELISA with one or more recombinant antigens. There was frequent antibody reactivity to protein 41-G (p41-G), outer surface protein C (OspC), and OspF antigens. In an ELISA for IgG antibodies, 13 (87%) of 15 serum samples had antibodies to recombinant antigens; reactivity to p22, p39, p41-G, OspC, and OspF antigens was frequent. By both ELISAs, serum specimens positive for OspB, OspE, and p37 were uncommon. Analyses of sera obtained from persons who were suspected of having human granulocytic ehrlichiosis (HGE) but who lacked antibodies to ehrlichiae revealed IgM antibodies to all recombinant antigens of B. burgdorferi except OspB and IgG antibodies to all antigens except OspE. Immunoblotting of sera from the study group of individuals suspected of having HGE reaffirmed antibody reactivity to multiple antigens of B. burgdorferi. There was minor cross-reactivity when sera from healthy subjects or persons who had syphilis, oral infections, or rheumatoid arthritis were tested by ELISAs with p37, p41-G, OspB, OspC, OspE, and OspF antigens. Although the results of class-specific ELISAs with recombinant antigens were comparable to those recorded for assays with whole-cell antigen and for individuals with confirmed clinical diagnoses of Lyme borreliosis, immunoblotting is still advised as an adjunct procedure, particularly when there are low antibody titers by an ELISA.

Antigens, Bacterial↗
PubMed2000

Follicular dendritic cells and dissemination of Creutzfeldt-Jakob disease.

The contribution of immune system cells to the propagation of transmissible encephalopathies is not well understood. To determine how follicular dendritic cells (FDC) may act, we challenged lymphotoxin beta null and wild-type (wt) controls with a Creutzfeldt-Jakob disease (CJD) agent. There was only a small difference in incubation time to clinical disease even after peripheral challenge with low infectious doses (31 in a total of 410 days). Brain pathology with extensive microglial infiltration, identified by keratan sulfate, as well as astrocytic hypertrophy, was also equivalent in all groups despite the fact that null mice had neither FDC nor splenic metallophilic macrophages that filter particulate antigen. Because FDC accumulate pathologic prion protein (PrP) in infected but not wt mice, we studied the cellular distribution of PrP by confocal microscopy. The majority of pathologic PrP collected on the plasma membrane of FDC, as identified by the Ca(+2)-binding protein S100A. This surface distribution suggested that agent aggregated with pathologic PrP might spread by cell-to-cell contacts. While several types of leukocytes may be involved in agent dissemination, cells of myeloid lineage were found to be infectious. Moreover, perivascular tracks of microglia and abnormal PrP after intraperitoneal inoculation were consistent with hematogenous spread. In summary, FDC are not required for CJD agent spread from the periphery, although FDC may enhance spread through surface accumulation of pathologic PrP. While it is still not clear where the infectious agent replicates, macrophages can sequester appreciable levels of infectivity and hence act as reservoirs for prolonged latent infection.

Animals↗
PubMed2000

c-Jun NH(2)-terminal kinase inhibits targeting of the protein phosphatase calcineurin to NFATc1.

The protein phosphatase calcineurin is a critical mediator of calcium signals during T-cell activation. One substrate of calcineurin is the transcription factor NFATc1, which is retained in the cytoplasm of quiescent cells. NFATc1 activation requires the translocation of the transcription factor into the nucleus, a process that is mediated by calcineurin. This interaction with calcineurin requires a targeting domain (PxIxIT motif) located in the NH(2)-terminal region of NFATc1. Here we demonstrate that the calcineurin targeting domain of NFATc1 is phosphorylated and inactivated by the c-Jun NH(2)-terminal kinase (JNK). This disruption of calcineurin targeting inhibits the nuclear accumulation and transcription activity of NFATc1 and accounts for the observation that Jnk1(-/-) T cells exhibit greatly increased NFATc1-dependent nuclear responses.

Amino Acid Motifs↗
PubMed2000

Airway hyperresponsiveness and airway obstruction in transgenic mice. Morphologic correlates in mice overexpressing interleukin (IL)-11 and IL-6 in the lung.

Understanding the sources of variation in airway reactivity and airflow is important for unraveling the pathophysiology of asthma, obstructive lung disease, and other pulmonary disorders. Transgenic expression of two closely related cytokines in the mouse lung produced opposite effects on these parameters. Interleukin (IL)-6 did not alter basal airways resistance and decreased methacholine responsiveness, whereas IL-11 caused airways obstruction and increased airway responses to methacholine. To clarify these differences we examined histologic sections and used morphometry to compare bronchiolar and parenchymal dimensions in 1- to 2-mo-old transgenic mice expressing IL-6 or IL-11 and littermate control mice. Both transgenic strains showed similar emphysema-like airspace enlargement, nodular peribronchiolar collections of mononuclear cells, thickening of airway walls, and subepithelial airway fibrosis. When compared with littermate control mice, the IL-6 mice showed an approximately 50% increase in the caliber of their bronchioles and an increase in airway wall thickness that was in proportion to the increase in the size of their airways. In contrast, the remodeling response was more robust in the IL-11 transgenic mice. It was also seen in airways with normal external and luminal diameters and thus was out of proportion to the caliber of their airways. These results support the hypothesis that structural alterations and resulting caliber changes of respiratory airways can have important effects on airway physiology and reactivity.

Airway Obstruction↗
PubMed1999

Expression of activated CDC42 induces T cell apoptosis in thymus and peripheral lymph organs via different pathways.

CDC42, a Ras-related small GTP binding protein, is involved in diverse cellular functions in lymphocytes. We generated transgenic mice expressing constitutively active murine CDC42 (Q61L) under the control of the human CD2 promoter. Transgenic mice showed smaller thymi with a dramatic reduction of CD4+CD8+, CD4+ and CD8+ thymocytes and with increase of CD4-CD8- thymocytes at CD25-CD44+ and CD25+ stage. A high percentage of the transgenic thymocytes were apoptotic, explaining the reduction of cellularity and size of the thymus. Mature T cells (TCR alphabeta+) in peripheral lymph organs, spleen and lymph node, were also dramatically reduced, and exhibited massive apoptosis. Expression of Fas and Fas ligand on both thymocytes and peripheral T cells was upregulated in transgenic mice, but the increased apoptosis in the thymus was independent of Fas (CD95), whereas peripheral spleen and lymph node T cell apoptosis was Fas dependent. Thus, activated CDC42 triggers distinct apoptotic pathways in thymocytes and peripheral T cells.

Amino Acid Substitution↗
PubMed1999

Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation.

Alzheimer's disease (AD) has a substantial inflammatory component, and activated microglia may play a central role in neuronal degeneration. CD40 expression was increased on cultured microglia treated with freshly solublized amyloid-beta (Abeta, 500 nanomolar) and on microglia from a transgenic murine model of AD (Tg APPsw). Increased tumor necrosis factor alpha production and induction of neuronal injury occurred when Abeta-stimulated microglia were treated with CD40 ligand (CD40L). Microglia from Tg APPsw mice deficient for CD40L demonstrated reduction in activation, suggesting that the CD40-CD40L interaction is necessary for Abeta-induced microglial activation. Finally, abnormal tau phosphorylation was reduced in Tg APPsw animals deficient for CD40L, suggesting that the CD40-CD40L interaction is an early event in AD pathogenesis.

Alzheimer Disease↗
PubMed1999

Differential requirements for NF-kappaB and AP-1 trans-activation in response to minimal TCR engagement by a partial agonist in naive CD8 T cells.

We investigated the basis for partial reactivity of naive CD8 T cells expressing an alloreactive transgenic TCR in response to a mutant alloantigen. When unstimulated APCs were used, IFN-gamma as well as IL-2 and cell proliferation were observed in response to wild-type Ag, whereas mutant Ag induced only IFN-gamma. DNA binding and reporter gene assays showed that the response to mutant Ag involved NF-kappaB, but not AP-1 activation, whereas wild-type Ag activated both transcription factors. Increasing the contribution of costimulatory signals by using LPS-activated APCs partially corrected the activation by mutant Ag, because proliferation and weak IL-2 production could be measured. This also led to AP-1 activation, albeit with delayed kinetics, in response to mutant Ag. To explain how engagement of the same TCR by distinct ligands results in different T cell responses, it may be proposed, in line with models stressing the importance of the kinetics of Ag/TCR interaction, that two types of signals be distinguished: a "fast" short-lived signal is sufficient to activate NF-kappaB; whereas a "slow" signal obtained after prolonged TCR engagement is required for AP-1 activation. Failure to activate AP-1 in limiting conditions (unstimulated mutant APC) was partially corrected by increasing costimulation.

Animals↗
PubMed1999

Essential role of phosphoinositide metabolism in synaptic vesicle recycling.

Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.

Animals↗
PubMed1999

CD40-CD40 ligand costimulation is required for generating antiviral CD4 T cell responses but is dispensable for CD8 T cell responses.

This study documents a striking dichotomy between CD4 and CD8 T cells in terms of their requirements for CD40-CD40 ligand (CD40L) costimulation. CD40L-deficient (-/-) mice made potent virus-specific CD8 T cell responses to dominant as well as subdominant epitopes following infection with lymphocytic choriomeningitis virus. In contrast, in the very same mice, virus-specific CD4 T cell responses were severely compromised. There were 10-fold fewer virus-specific CD4 T cells in CD40L-/- mice compared with those in CD40L+/+ mice, and this inhibition was seen for both Th1 (IFN-gamma, IL-2) and Th2 (IL-4) responses. An in vivo functional consequence of this Th cell defect was the inability of CD40L-/- mice to control a chronic lymphocytic choriomeningitis virus infection. This study highlights the importance of CD40-CD40L interactions in generating virus-specific CD4 T cell responses and in resolving chronic viral infection.

Animals↗
PubMed1999

Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is required for tumor promotion.

Activator protein-1 (AP-1) is a transcription factor that consists of either a Jun-Jun homodimer or a Jun-Fos heterodimer. Transactivation of AP-1 is required for tumor promoter-induced transformation in mouse epidermal JB6 cells and for progression in mouse and human keratinocytes. Until now, the question of whether AP-1 transactivation is required for carcinogenesis in vivo has remained unanswered, as has the issue of functionally significant target genes. To address these issues we have generated a transgenic mouse in which transactivation mutant c-jun (TAM67), under the control of the human keratin-14 promoter, is expressed specifically in the basal cells of the epidermis where tumor induction is initiated. The keratin-14-TAM67 transgene was expressed in the epidermis, tongue, and cervix, with no apparent abnormalities in any tissue or organ. TAM67 expression blocked 12-O-tetradecanoylphorbol 13-acetate (TPA, phorbol 12-tetradecanoate 13-acetate) induction of the AP-1-regulated luciferase in AP-1 luciferase/TAM67 mice, but did not inhibit induction of candidate AP-1 target genes, collagenase-1 or stromelysin-3. More interestingly, TAM67 expression did not inhibit TPA-induced hyperproliferation. In two-stage skin carcinogenesis experiments, the transgenic animals showed a dramatic inhibition of papilloma induction. We conclude that transactivation of a subset of AP-1-dependent genes is required for tumor promotion and may be targeted for cancer prevention.

Animals↗
PubMed1999

Lymphotoxin-beta-deficient mice show defective antiviral immunity.

Lymphotoxin beta (LTbeta), a member of the tumor necrosis factor family, plays an important role in lymphoid organogenesis. In order to determine whether LTbeta is involved in cellular immunity, we investigated the antiviral immune response of LTbeta-deficient (LTbeta -/-) mice to lymphocytic choriomeningitis virus (LCMV). Cytotoxic T lymphocyte (CTL) responses to LCMV were severely diminished, leading to viral persistence in brain and kidney. However, major functions of LTbeta-deficient T lymphocytes and dendritic cells were intact. Reconstitution of irradiated LTbeta +/+ mice with LTbeta -/- bone marrow induced a disorganized splenic structure, accompanied by impairment of the LCMV-specific CTL response. These data indicate that the absence of LTbeta does not affect the intrinsic function of T lymphocytes or of dendritic cells but that the structural integrity of the spleen is strongly associated with generation of antiviral immunity.

Adoptive Transfer↗
PubMed1999

The TCR zeta-chain immunoreceptor tyrosine-based activation motifs are sufficient for the activation and differentiation of primary T lymphocytes.

The TCR complex signals through a set of 10 intracytoplasmic motifs, termed immunoreceptor tyrosine-based activation motifs (ITAMs), contained within the gamma-, delta-, epsilon-, and zeta-chains. The need for this number of ITAMs is uncertain. Limited and contradictory studies have examined the ability of subsets of the TCR's ITAMs to signal into postthymic primary T lymphocytes. To study signaling by a restricted set of ITAMs, we expressed in transgenic mice a chimeric construct containing the IAs class II MHC extracellular and transmembrane domains linked to the cytoplasmic domain of the TCR zeta-chain. Tyrosine phosphorylation and receptor cocapping studies indicate that this chimeric receptor signals T cells independently of the remainder of the TCR. We show that CD4+ and CD8+ primary T cells, as well as naive and memory T cells, are fully responsive to stimulation through the IAs-zeta receptor. Further, IAs-zeta stimulation can induce primary T cell differentiation into CTL, Th1, and Th2 type cells. These results show that the zeta-chain ITAMs, in the absence of the gamma, delta, and epsilon ITAMs, are sufficient for the activation and functional maturation of primary T lymphocytes. It also supports the isolated use of the zeta-chain ITAMs in the development of surrogate TCRs for therapeutic purposes.

Amino Acid Sequence↗