Showing papers by "Richard A. Flavell published in 1997"

Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene

Abstract: Excitatory amino acids induce both acute membrane depolarization and latent cellular toxicity, which often leads to apoptosis in many neurological disorders. Recent studies indicate that glutamate toxicity may involve the c-Jun amino-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases. One member of the JNK family, Jnk3, may be required for stress-induced neuronal apoptosis, as it is selectively expressed in the nervous system. Here we report that disruption of the gene encoding Jnk3 in mice caused the mice to be resistant to the excitotoxic glutamate-receptor agonist kainic acid: they showed a reduction in seizure activity and hippocampal neuron apoptosis was prevented. Although application of kainic acid imposed the same level of noxious stress, the phosphorylation of c-Jun and the transcriptional activity of the AP-1 transcription factor complex were markedly reduced in the mutant mice. These data indicate that the observed neuroprotection is due to the extinction of a Jnk3-mediated signalling pathway, which is an important component in the pathogenesis of glutamate neurotoxicity.

Activation of Interferon-γ Inducing Factor Mediated by Interleukin-1β Converting Enzyme

Abstract: The interleukin-1beta (IL-1beta) converting enzyme (ICE) processes the inactive IL-1beta precursor to the proinflammatory cytokine. ICE was also shown to cleave the precursor of interferon-gamma inducing factor (IGIF) at the authentic processing site with high efficiency, thereby activating IGIF and facilitating its export. Lipopolysaccharide-activated ICE-deficient (ICE-/-) Kupffer cells synthesized the IGIF precursor but failed to process it into the active form. Interferon-gamma and IGIF were diminished in the sera of ICE-/- mice exposed to Propionibacterium acnes and lipopolysaccharide. The lack of multiple proinflammatory cytokines in ICE-/- mice may account for their protection from septic shock.

Interleukin (il)-6 directs the differentiation of il-4-producing cd4+ t cells

Abstract: Interleukin (IL)-4 is the most potent factor that causes naive CD4+ T cells to differentiate to the T helper cell (Th) 2 phenotype, while IL-12 and interferon γ trigger the differentiation of Th1 cells. However, the source of the initial polarizing IL-4 remains unclear. Here, we show that IL-6, probably secreted by antigen-presenting cells, is able to polarize naive CD4+ T cells to effector Th2 cells by inducing the initial production of IL-4 in CD4+ T cells. These results show that the nature of the cytokine (IL-12 or IL-6), which is produced by antigen-presenting cells in response to a particular pathogen, is a key factor in determining the nature of the immune response.

Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis.

Abstract: Protein tyrosine kinases activate the STAT (signal transducer and activator of transcription) signaling pathway, which can play essential roles in cell differentiation, cell cycle control, and development. However, the potential role of the STAT signaling pathway in the induction of apoptosis remains unexplored. Here we show that gamma interferon (IFN-gamma) activated STAT1 and induced apoptosis in both A431 and HeLa cells, whereas epidermal growth factor (EGF) activated STAT proteins and induced apoptosis in A431 but not in HeLa cells. EGF receptor autophosphorylation and mitogen-activated protein kinase activation in response to EGF were similar in both cell lines. The breast cancer cell line MDA-MB-468 exhibited a similar response to A431 cells, i.e., STAT activation and apoptosis correlatively resulted from EGF or IFN-gamma treatment. In addition, in a mutant A431 cell line in which STAT activation was abolished, no apoptosis was induced by either EGF or IFN-gamma. We further demonstrated that both EGF and IFN-gamma induced caspase 1 (interleukin-1beta converting enzyme [ICE]) gene expression in a STAT-dependent manner. IFN-gamma was unable to induce ICE gene expression and apoptosis in either JAK1-deficient HeLa cells (E2A4) or STAT1-deficient cells (U3A). However, ICE gene expression and apoptosis were induced by IFN-gamma in U3A cells into which STAT1 had been reintroduced. Moreover, both EGF-induced apoptosis and IFN-gamma-induced apoptosis were effectively blocked by Z-Val-Ala-Asp-fluoromethylketone (ZVAD) in all the cells tested, and studies from ICE-deficient cells indicated that ICE gene expression was necessary for IFN-gamma-induced apoptosis. We conclude that activation of the STAT signaling pathway can induce apoptosis through the induction of ICE gene expression.

Targeted disruption of the MKK4 gene causes embryonic death, inhibition of c-Jun NH2-terminal kinase activation, and defects in AP-1 transcriptional activity

Abstract: MKK4 is a member of the mitogen-activated protein kinase kinase group of dual specificity protein kinases that functions as an activator of the c-Jun NH2-terminal kinase (JNK) in vitro. To examine the function of MKK4 in vivo, we investigated the effect of targeted disruption of the MKK4 gene. Crosses of heterozygous MKK4 (+/−) mice demonstrated that homozygous knockout (−/−) animals die before embryonic day 14, indicating that the MKK4 gene is required for viability. The role of MKK4 in JNK activation was examined by investigation of cultured MKK4 (+/+) and MKK4 (−/−) cells. Disruption of the MKK4 gene blocked JNK activation caused by: (i) the mitogen-activated protein kinase kinase kinase MEKK1, and (ii) treatment with anisomycin or heat shock. In contrast, JNK activation caused by other forms of environmental stress (UV-C radiation and osmotic shock) was partially inhibited in MKK4 (−/−) cells. Regulated AP-1 transcriptional activity, a target of the JNK signal transduction pathway, was also selectively blocked in MKK4 (−/−) cells. Complementation studies demonstrated that the defective AP-1 transcriptional activity was restored by transfection of MKK4 (−/−) cells with an MKK4 expression vector. These data establish that MKK4 is a JNK activator in vivo and demonstrate that MKK4 is an essential component of the JNK signal transduction pathway.

Evidence that the multidrug resistance protein (MRP) functions as a co-transporter of glutathione and natural product toxins.

Abstract: The MRP (multidrug resistance protein) gene, a member of the ubiquitous superfamily of ATP-binding cassette transporters, is associated with the multidrug resistance of mammalian cells to natural product anticancer agents. We have previously shown that abrogation of MRP expression by gene targeting leads to hypersensitivity to several drugs. In two independently produced MRP double knockout clones, the baseline export of glutathione (GSH) was one-half that of wild-type embryonic stem (ES) cells. The export of GSH from wild-type ES cells, but not from the MRP double knockout clones, increased in the presence of etoposide (VP-16) and sodium arsenite, accompanied by equivalent decreases in intracellular levels of GSH. In the two MRP double knockout clones, the intracellular steady-state concentration of etoposide was twofold greater than that in wild-type cells. Depletion of intracellular GSH by D,L-buthionine sulfoximine increased the intracellular accumulation of radiolabeled etoposide in parental ES cells up to the level present in the two MRP knockout clones but did not change etoposide levels in the MRP knockout clones. These observations provide evidence that: (a) MRP exports GSH physiologically, presumably in association with an endogenous compound(s); (b) baseline MRP expression protects cells from the toxic effects of xenobiotics by effluxing the xenobiotics and GSH from the intracellular compartment into the extracellular medium by a co-transport mechanism; and (c) disruption of the gene encoding MRP abrogates the cotransport of xenobiotics and GSH.

A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion.

Abstract: Mucus hypersecretion and plugging of lower respiratory tract airways contributes to the morbidity and mortality associated with asthma. Interleukin (IL)-4 plays a putative role in some forms of asthma. Thus, transgenic mice that overexpress murine IL-4 selectively within the lung were used to study the effect of IL-4 on mucus glycoprotein gene expression and mucin release. Histologic examination of lung sections from IL-4 mice revealed that nonciliated epithelial cells from conducting airways were hypertrophic, due at least in part to the accumulation of mucus glycoprotein. The cytoplasm of these cells stained positively for glycoproteins using mucicarmine, alcian blue (AB), and periodic acid-Schiff (PAS). Ciliated cells were also enlarged but did not show any mucin-specific staining. Inclusion granules typically found in nonciliated (Clara) cells of control mice were absent in the IL-4 transgenic mice. Northern blot analysis of total RNA from lung tissue revealed that the expression of the MUC5AC, but not MUC2, mucin gene was distinctly upgraded in IL-4 transgenic mice compared to transgene-negative controls. In addition, a 5- to 10-fold increase in AB- and PAS-positive material was found in lavage fluid from IL-4 overexpressing mice compared to transgene-negative controls. Thus, the overexpression of IL-4 locally within the lung enhances mucus glycoprotein synthesis by altering gene expression, results in the accumulation of mucus glycoprotein in nonciliated epithelial cells, and induces the release of mucus into the airway lumen. We therefore hypothesize that the overproduction of mucus seen in some patients with asthma may be a direct result of the action of IL-4 within the inflamed lung.

Response to local inflammation of IL-1 beta-converting enzyme- deficient mice.

Abstract: IL-1 beta-converting enzyme (ICE) cleaves pro-IL-1 beta to the mature, released form. Although other proteases can process pro-IL-1 beta, ICE-deficient (ICE -/-) mice do not release mature IL-1 beta in response to endotoxin. The purpose of our study was to investigate the response of ICE -/- mice in two models of local inflammation, turpentine-induced tissue damage and zymosan-induced peritonitis. No differences were observed in the development of the systemic acute phase response after turpentine administration between wild-type and ICE -/- mice, but this response was completely impaired in IL-1 beta -/- mice. Accordingly, the levels of mature IL-1 beta produced in response to turpentine did not differ between wild-type and ICE -/- mice. In contrast, following zymosan-induced peritonitis, the levels of mature IL-1 beta were significantly lower in ICE -/- mice. This was associated with a 50% decrease in cellular infiltrate in ICE -/- mice compared with that in wild-type controls. The reduced production of zymosan-induced mature IL-1 beta in ICE -/- mice was also observed from cultured peritoneal or spleen cells. Our results demonstrate that in turpentine-induced tissue necrosis, precursor IL-1 beta is processed by non-ICE proteases, but in complement-mediated inflammation, ICE participates in the processing of the IL-1 beta precursor.

Disruption of the murine MRP (multidrug resistance protein) gene leads to increased sensitivity to etoposide (VP-16) and increased levels of glutathione.

Abstract: The mrp (multidrug resistance protein) gene has been associated with the multidrug resistance of cancer cells in vitro and in vivo. To gain information on its physiological role, embryonic stem cells were used to generate mice homozygous for a disruption of the mrp gene, resulting in complete abrogation of mrp expression. No physiological abnormalities were observed, at least up to 4 months of age. Viability, fertility, and a range of histological, hematological, and serum-chemical parameters were similar in mrp(+/+) and mrp(-/-) mice. mrp(-/-) mice displayed an increased sensitivity to etoposide phosphate (2-fold) accompanied by greater bone marrow toxicity, whereas the acute toxicity of sodium arsenite was equivalent in mrp(+/+) and mrp(-/-) mice. Tissue levels of glutathione (GSH) were elevated in breast, lung, heart, kidney, muscle, colon, testes, bone marrow cells, blood mononuclear leukocytes, and blood erythrocytes of mrp(-/-) mice and were unchanged in organs known to express little if any mrp, such as the liver and small intestine. The increase in GSH was not due to an increase in the activity of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis. The findings demonstrate that mrp is dispensable for development and growth but exerts a role in drug detoxification and GSH metabolism.

Blocking activator protein-1 activity, but not activating retinoic acid response element, is required for the antitumor promotion effect of retinoic acid

Abstract: Retinoic acid is one of the most promising drugs for chemotherapy and chemoprevention of cancer. Either blocking activator protein-1 (AP-1) activity or activating retinoic acid response element (RARE) have been proposed to be responsible for its antitumor activity. However, evidence for this hypothesis is lacking in vivo studies. To address this issue, we used an AP-1-luciferase transgenic mouse as a carcinogenesis model and new synthetic retinoids that are either selective inhibitors of AP-1 activation or selective activators of the RARE. The results showed that the SR11302, an AP-1 inhibition-specific retinoid, and other AP-1 inhibitors such as trans-retinoic acid and fluocinolone acetonide, markedly inhibit both 12-O-tetradecanoylphorbol-13-acetate-induced papilloma formation and AP-1 activation in 7,12-dimethyl benz(a)anthracene-initiated mouse skin (P 0.05). These results provide the first in vivo evidence that the antitumor effect of retinoids is mediated by blocking AP-1 activity, but not by activation of RARE.

Blockade of CD40 ligand-CD40 interaction impairs CD4+ T cell-mediated alloreactivity by inhibiting mature donor T cell expansion and function after bone marrow transplantation.

Abstract: Alloreactive T cells require costimulatory signals via CD40 ligand (CD40L). The tissue-destructive properties of allogeneic CD4+ but not CD8+ T cells were inhibited by anti-CD40L mAb. Fewer CD4+ thoracic duct lymphocytes (TDL) were obtained in mAb-treated recipients. Kinetic studies revealed that CD4+ T cell expansion was reduced or delayed which may account, in part, for the partial graft-vs-host disease protective effect of anti-CD40L mAb. TDL were found to have diminished anti-host-specific proliferative responses. The frequency of donor TDL and splenocytes that expressed the Th1 cytokines IL-2, IL-12 p40, and IFN-gamma mRNA was markedly diminished in mAb-treated recipients, demonstrating that Th1-driven alloresponses were susceptible to CD40L targeting. Perforin mRNA-expressing T cells were undetectable in mAb-treated recipients, consistent with reduced in vivo lethality after the adoptive transfer of allogeneic CD4+ T cells. Similar findings were observed in both B cell-replete or -deficient recipients, indicating that allogeneic T cell expansion and priming can be sustained by a non-B cell, CD40+ host cell population. Mice receiving CD40L-deficient allogeneic CD4+ T cells had survival rates comparable to the rates of those given anti-CD40L mAb treatment. Because anti-CD40L mAb also was found to prevent host anti-donor-mediated marrow allograft rejection, in vivo blockade of CD40L-CD40 interactions may provide a highly beneficial approach to improving the outcome of allogeneic bone marrow transplantation.

Polarization of IL-4- and IFN-gamma-producing CD4+ T cells following activation of naive CD4+ T cells.

Abstract: Naive CD4+ T cells initially transcribe both IL-4 and IFN-gamma when stimulated with either the mitogen Con A or Ag in the presence of IL-4 or IL-12 and, therefore, appear uncommitted as to the pathway of differentiation they will follow. However, when stimulated either with Con A or with Ag in the presence of IL-4, CD4+ T cells become primed to follow the Th2 differentiation pathway, and we show now that by 48 h of culture in this environment these cells extinguish IFN-gamma gene transcription. Likewise, priming in the presence of IL-12 leads to the development of Th1 cells, which switch off the expression of the IL-4 gene. To clarify the Th1 differentiation pathway, we performed ablation studies using IL-4 thymidine kinase transgenic mice. When the antiviral drug ganciclovir was added 1 day after primary stimulation in the presence of IL-12, IFN-gamma- and IL-4-producing cells were ablated. In contrast, when ganciclovir was added 2 days after primary stimulation, IL-4-producing cells, but not IFN-gamma-producing cells, were ablated. Thus, our studies show that by 48 h after activation, Th1 or Th2 cells have already become polarized to the differentiation pathway that they will follow. As the differentiation toward Th1 and Th2 effector cells proceeds, substantial amounts of IFN-gamma and IL-4 mRNA accumulate, while the mRNAs of the corresponding lineage (i.e., IFN-gamma in the case of Th2 cells, and IL-4 in the case of Th1 cells) diminish to undetectable levels. IL-4R is up-regulated during T cell differentiation by a mechanism mediated mainly by IL-4. The fact that IL-12 priming does not suppress IL-4-dependent IL-4R up-regulation shows that both IL-4 mRNA and cytokine are produced by IL-12-primed naive CD4+ T cells during differentiation into Th1 cells. Naive CD4+ T cells, therefore, begin as uncommitted cells which express both Th1 and Th2 cytokines that rapidly extinguish the expression of the inappropriate cytokine as the commitment toward the effector lineages is made.

Role of CD4+ T cells in pathogenesis associated with Leishmania amazonensis infection.

Abstract: Most inbred strains of mice are susceptible to Leishmania amazonensis infection. We have examined the mechanism(s) underlying this generalized susceptibility using mice deficient in T cell development or in the expression of either MHC class I or class II. In contrast to wild-type C57BL/6 (B6) mice that uniformly developed large ulcerating lesions, mice lacking functional CD4+ T cells (due to targeted disruption of genes for either MHC class II trans-activator or I-A beta) showed no signs of lesion development for up to 12 to 14 wk postinfection and contained significantly lower numbers of parasites in lesions. Similarly, both B6 nude and RAG2 -/- mice failed to develop lesions. However, RAG2 -/- mice reconstituted with naive wild-type CD4+ T cells and beta2m -/- mice did develop lesions. Lesions of MHC class II -/- mice contained minimal numbers of CD8+ T cells, a marked reduction of monocytes/macrophages, and evident extracellular parasites. The inability to mount an inflammatory response in MHC class II -/- mice correlated with the failure to produce lymphokines that lead to the recruitment of monocytes/granulocytes. These results demonstrate that CD4+ T cells are the primary lymphocyte subset that mediates cellular infiltration, lesion pathology, and therefore, susceptibility to L. amazonensis infection. The disease-promoting CD4+ T cells in L. amazonensis-infected mice have the characteristics of Th1 cells. The striking differences in the course of infection between MHC class II -/- mice infected with L. amazonensis and Leishmania major suggest that these parasites may have adapted different strategies regarding the CD4-dependent immune response.

Cleavage of FLICE (caspase-8) by granzyme B during cytotoxic T lymphocyte-induced apoptosis

Abstract: Cytotoxic T lymphocytes induce apoptosis in target cells through the CD95(APO-1/Fas) and the perforin/granzyme B (GrB) pathway. The exact substrate of GrB in vivo is still unknown, but to induce apoptosis GrB requires the activity of caspases in target cells. We show here that in HeLa target cells induction of apoptosis through the perforin/GrB pathway resulted in minor direct cleavage of CPP32 (caspase-3) by GrB. Most caspase-3 cleavage resulted from activation of an upstream caspase. Moreover, target cells derived from caspase-3(-/-) mice displayed GrB-induced poly(ADP-ribose) polymerase (PARP) cleavage with only partially reduced efficiency compared to wild-type target cells. This indicates that other PARP-cleaving caspases can be activated during perforin/GrB-induced cell death. In contrast to caspase-3, FLICE (caspase-8) was directly cleaved by GrB in HeLa cells. We therefore conclude that FLICE not only plays a central role in CD95(APO-1/Fas)-induced apoptosis but can also be directly activated during perforin/GrB-induced apoptosis.

In vivo properties of monocyte chemoattractant protein-1.

Abstract: Monocyte chemoattractant protein-i (MCP- i) attracts monocytes, memory T lymphocytes, and natural killer (NK) cells in vitro. Its expression has been documented in disorders characterized by mono- nuclear cell infiltrates, suggesting that it may contrib- ute to the inflammatory component of such diseases as atherosclerosis, multiple sclerosis, or rheumatoid ar- thritis. To prove a causal association, the in vivo prop- erties of MCP-1 must be understood. Several lines of transgenic mice have been constructed to address this question. A transgenic line in which MCP-1 expression is controlled by the MMTV-LTR expressed high levels of MCP-i in multiple organs but showed no evidence for monocyte infiltration. Instead, these mice were more susceptible to infection by the intracellular pathogens, Listeria monocytogenes and Mycobacte- rium tuberculosis. These mice had high serum levels of MCP-i, suggesting that their circulating monocytes may have been desensitized or that MCP-1 stimulated a Th2-dominant response. In contrast, another model in which MCP-1 expression was controlled by the in- sulin promoter demonstrated a monocytic infiltrate in pancreatic islets. These results indicate that MCP-i ex- pression at low levels in an anatomically confined area results in monocyte infiltration, suggesting that when properly expressed, MCP-i's in vitro properties are reproduced in vivo. This justifies the examination of MCP-i-deficient mice in disease models in order to ex- plore MCP-i's role in pathogenesis. J. Leukoc. Biol. 62: 577-580; 1997.

Regulated overexpression of interleukin 11 in the lung. Use to dissociate development-dependent and -independent phenotypes.

Abstract: Standard overexpression transgenic approaches are limited in their ability to model waxing and waning diseases and frequently superimpose development-dependent and -independent phenotypic manifestations. We used the clara cell 10-kD protein (CC10) promoter and the reverse tetracycline transactivator (rtTA) to create a lung-specific, externally regulatable, overexpression transgenic system and used this system to express human interleukin 11 (IL-11) in respiratory structures. Gene induction could be achieved in utero, in neonates and in adult animals. Moreover, gene expression could be turned off by removal of the inducing stimulus. When gene activation was initiated in utero and continued into adulthood, subepithelial airway fibrosis, peribronchiolar mononuclear nodules, and alveolar enlargement (emphysema) were noted. Induction in the mature lung caused airway remodeling and peribronchiolar nodules, but alveolar enlargement was not appreciated. In contrast, induction in utero and during the first 14 d of life caused alveolar enlargement without airway remodeling or peribronchiolar nodules. Thus, IL-11 overexpression causes abnormalities that are dependent (large alveoli) and independent (airway remodeling, peribronchiolar nodules) of lung growth and development, and the CC10-rtTA system can be used to differentiate among these effector functions. The CC10-rtTA transgenic system can be used to model waxing and waning, childhood and growth and development-related biologic processes with enhanced fidelity.

Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1.

Abstract: Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that attracts monocytes and T lymphocytes in vitro; however, its in vivo functions are poorly understood. To address this question, we constructed transgenic mice expressing MCP-1 controlled by an insulin promoter. These mice developed a chronic insulitic infiltrate composed of F4/80+ monocytes with minor populations of CD4+, CD8+, and B220+ cells. Despite persistent transgene expression, the insulitis never progressed, and blood glucose levels remained normal. Thus, MCP-1 alone is sufficient to elicit a monocytic infiltrate, but not to activate elicited cells. These results differ from those obtained with another transgenic model using the mouse mammary tumor virus long terminal repeat, in which mice expressed substantial MCP-1 in several organs but had no infiltrates. However, mice expressing both transgenes had minimal insulitis, indicating that high systemic levels of MCP-1 prevented monocytes from responding to local MCP-1. Thus, the ability of MCP-1 to elicit monocytic infiltration depends on its being expressed at low levels in an anatomically restricted area.

The CD40 ligand. At the center of the immune universe

Abstract: For several years, the primary function of CD40 ligand (CD40L) has been believed to be in regulation of contact-dependent, CD40-CD40L-mediated signals between B- and T-cells, which are essential for the regulation of thymus-dependent (TD) humoral immune responses. Recently, a flurry of reports indicate that CD40 is expressed by variety of cell types other than B-cells that include dendritic cells, follicular dendritic cells, monocytes, macrophages, fibroblasts, and endothelial cells. These studies show that CD40-CD40L interactions are important in inflammatory process. For the past few years, through the availability of CD40L-knockout mice, new data have emerged to support the belief that CD40L has many more functions than its role in TD humoral immunity. CD40L-deficient mice have provided significant information towards our understanding of the in vivo role of CD40L. The current picture that emerges indicates that CD40-CD40L interactions mediate many cell-mediated immune responses and T-cell-mediated effector functions that are required for proper functioning of the host defense system. This article focuses on the in vivo role of the CD40L in regulation of cell-mediated effector functions.

Reciprocal regulation of CD30 expression on CD4+ T cells by IL-4 and IFN-gamma.

Abstract: Prior studies have implicated CD30 as a marker for Th2 cells, but the mechanism that underlies this correlation was unknown We show here that CD30 was expressed on activated CD4+ T cells in the presence of IL-4 In the absence of endogenously produced IL-4, however, even Th2 lineage cells lost CD30 expression Thus, CD30 is not an intrinsic marker of Th2 cells, but is inducible by IL-4 CD30 was also found to be down-regulated by IFN-gamma Committed Th1 effector cells do not express CD30, although differentiating Th1 lineage cells temporarily express CD30 The transient expression of CD30 on differentiating Th1 lineage cells was mainly the result of endogenously produced IL-4 induced by IL-12 Culture of IL-12-primed cells under conditions that reverse the phenotype (Ag plus IL-4) resulted in two cell populations based upon their ability to express CD30 One population responded to IL-4 upon restimulation and became a CD30-positive, Th0-like cell population, while the other remained CD30 negative and synthesized only IFN-gamma Thus, CD30 expressed on CD4+ T cells reflected the ability of CD4+ T cells to respond to IL-4

Roles of IL-4 and IFN-gamma in stabilizing the T helper cell type 1 and 2 phenotype.

Abstract: It has previously been reported that Th1 CD4 T cell populations can be converted to IL-4 producers, whereas Th2 populations are refractory to IL-12-mediated IFN-gamma production. We have recently shown that CD30 is a marker for the IL-4 response and have therefore used CD30 here to study Th1 and Th2 commitment. We show that Th2 cell populations normally show a stable phenotype and fail to respond to IL-12 because of endogenous IL-4 production. IFN-gamma abrogates this antagonistic effect of IL-4 and permits the conversion of Th2 populations to IFN-gamma producers by IL-12. In the complete absence of IL-4, however, IFN-gamma is not required for this transformation, and Th1 cells generated by IL-12 become committed to the Th1 pathway and lose the ability to respond to IL-4. Thus, the balance between local IL-4 and IFN-gamma in an immune response is a key factor in determining the outcome of the CD4 effector T cell response.

Reprogramming the signalling requirement for AP‐1 (activator protein‐1) activation during differentiation of precursor CD4+ T‐cells into effector Th1 and Th2 cells

Abstract: Upon antigenic stimulation, precursor CD4+ helper T-cells differentiate into two subsets of effector cells, Th1 and Th2. These two subpopulations are defined by the pattern of cytokine expression that distinguishes these differentiated cells from their precursors. We have used reporter transgenic mice here to show that, during differentiation of precursor T-cells into effector Th1 or Th2 cells, high levels of preformed activator protein (AP)-1 complexes are accumulated. However, upon stimulation, the preformed AP-1 complexes in effector Th2 cells, but not in Th1 cells, are able to induce high levels of AP-1 transcriptional activity. Furthermore, in contrast to precursor T-cells, the induction of AP-1 transcriptional activity is independent of calcium and co-stimulatory signals in effector Th2 cells. This AP-1 transcriptional activity appears to correlate with the presence of JunB complexes, which accumulate differentially in effector Th2 cells, but not in precursor CD4+ T-cells or effector Th1 cells. Unlike precursor cells, the activation of AP-1 does not appear to be mediated by c-Jun N-terminal kinase (JNK) in effector Th2 cells. These results indicate that during differentiation of T-cells, and probably other cell types, the signal requirements for the AP-1 transcription machinery are reprogrammed to enable the differentiated cells to perform their specialized functions.

Transcription mediated by NFAT is highly inducible in effector CD4+ T helper 2 (Th2) cells but not in Th1 cells.

Abstract: Transcriptional factors of the NFAT family play an important role in regulating the expression of several cytokine genes during the immune response, such as the genes for interleukin 2 (IL-2) and IL-4, among others. Upon antigen stimulation, precursor CD4+ T helper (pTh) cells proliferate and differentiate into two populations of effector cells (eTh1 and eTh2), each one expressing a specific pattern of cytokines that distinguishes them from their precursors. eTh2 cells are the major source of IL-4, while gamma interferon is produced by eTh1 cells. Here we have used reporter transgenic mice to show that DNA binding and transcriptional activities of NFAT are transiently induced during the differentiation of pTh cells into either eTh1 or eTh2 cells to mediate the expression of IL-2 as a common growth factor in both pathways. However, although NFAT DNA binding is similarly induced in both eTh1 and eTh2 cells upon antigen stimulation, only the NFAT complexes present in eTh2 cells are able to mediate high-level transcription, and relatively little NFAT transcriptional activity was induced in eTh1 cells. In contrast to activated pTh cells, neither eTh1 nor eTh2 cells produced significant IL-2 upon stimulation, but the high levels of NFAT transcriptional activities directly correlate with the IL-4 production induced in response to antigen stimulation in eTh2 cells. These data suggest that activated NFAT is involved in the effector function of eTh2 cells and that the failure of eTh1 cells to produce IL-4 in response to an antigen is due, at least partially, to a failure to induce high-level transcription of the IL-4 gene by NFAT. Regulation of NFAT could be therefore a critical element in the polarization to eTh1 or eTh2.

Differential Transcription Directed by Discrete Gamma Interferon Promoter Elements in Naive and Memory (Effector) CD4 T Cells and CD8 T Cells

Abstract: Acquisition of the ability to produce gamma interferon (IFN-gamma) is a fundamental property of memory T cells and enables one subset (T helper 1 [TH1]) to deliver its effector functions. To examine regulation of IFN-gamma gene expression in a model system which recapitulates TH1 differentiation, we prepared reporter transgenic mice which express the luciferase gene under the control of proximal and distal regulatory elements (prox.IFN gamma and dist.IFN gamma) from the IFN-gamma promoter. Memory T cells, but not naive T cells, secreted IFN-gamma and expressed both prox.IFN gamma and dist.IFN gamma transcriptional activities. Naive T cells required priming to become producers of IFN-gamma and to direct transcription by these elements. While both CD4+ and CD8+ T cells produced IFN-gamma, only CD4+ T cells expressed prox.IFN gamma transcriptional activity. Induction of transcriptional activity was inhibited by known antagonists of effector T-cell populations. Cyclosporin A inhibited transcriptional activity directed by both elements in effector T cells. Elevated cyclic AMP inhibited transcriptional activity directed by prox.IFN gamma in primed CD4+ T cells but enhanced transcriptional activity directed by dist.IFN gamma in primed CD8+ T cells. Taken together, these data show that prox.IFN gamma and dist.IFN gamma transcriptional activities mirror IFN-gamma gene expression in naive and memory CD4+ T cells but suggest that differences exist in regulation of IFN-gamma gene expression in CD4+ and CD8+ T-cell subsets.

alpha beta T cell regulation and CD40 ligand dependence in murine systemic autoimmunity.

Abstract: To explore the mechanisms by which alpha beta T cells and gamma delta T cells regulate systemic autoimmunity, lupus-prone mice were rendered deficient in CD40 ligand and/or alpha beta T cells by intercrossing CD40L -/- and TCR-alpha -/- knockouts, generating CD40L-intact or -deficient (CD40L+ or CD40L-), alpha beta T cell-intact or -deficient (alpha beta+ or alpha beta-) MRL-lpr/lpr animals As expected, CD40L+ alpha beta+ mice developed high titer autoantibodies along with severe renal and cutaneous disease CD40L+ alpha beta- animals developed lower levels of autoantibodies, accompanied by less severe or delayed renal and cutaneous disease CD40L- alpha beta+ mice developed even lower titers of autoantibodies and less severe renal disease yet developed cutaneous lesions indistinguishable from those of CD40L+ alpha beta+ disease Most surprisingly, CD40L- alpha beta- animals developed higher levels of some autoantibodies than did CD40L- alpha beta+ mice and developed renal disease similar in severity to CD40L+ alpha beta- counterparts; however, they failed to develop skin disease Thus, disruption of CD40L and alpha beta T cells provides a novel dissection of the physiology and pathology of murine lupus; while these data confirm previous findings demonstrating a role for CD40L-dependent, alpha beta T cell-dependent mechanisms in autoantibody production and renal disease in murine lupus, they also: 1) establish that alpha beta T cells may drive autoimmune skin disease by a CD40L-independent mechanism; 2) identify a role for CD40L in non-alpha beta T cell-dependent autoantibody production and autoimmune skin disease; and 3) suggest a role for alpha beta T cells in the down-regulation of autoimmunity driven by other T cells Thus, both alpha beta and non-alpha beta T cells, such as gamma delta T cells, regulate systemic autoimmunity by CD40L-dependent and -independent mechanisms