Inhibition of cell cycle progression by rapamycin induces T cell clonal anergy even in the presence of costimulation

Costimulation (signal 2) has been proposed to inhibit the induction of T cell clonal anergy by either directly antagonizing negative signals arising from TCR engagement (signal 1) or by synergizing with signal 1 to produce IL-2, which in turn leads to proliferation and dilution of negative regulatory factors. To better define the cellular events that lead to the induction of anergy, we used the immunosuppressive agent rapamycin, which blocks T cell proliferation in late G1 phase but does not affect costimulation-dependent IL-2 production. Our data demonstrate that full T cell activation (signal 1 plus 2) in the presence of rapamycin results in profound T cell anergy, despite the fact that these cells produce copious amounts of IL-2. Similar to conventional anergy (induction by signal 1 alone), the rapamycin-induced anergic cells show a decrease in mitogen-activated protein kinase activation, and these cells can be rescued by culture in IL-2. Interestingly, the rapamycin-induced anergic cells display a more profound block in IL-3 and IFN-gamma production upon rechallenge. Finally, in contrast to rapamycin, full T cell activation in the presence of hydroxyurea (which inhibits the cell cycle in early S phase) did not result in anergy. These data suggest that it is neither the direct effect of costimulation nor the subsequent T cell proliferation that prevents anergy induction, but rather the biochemical events that occur upon progression through the cell cycle from G1 into S phase.     

Receptor-mediated activation of human B lymphocytes in a nonphosphotyrosine-dependent manner.

The B cell AgR regulates two signal transduction pathways: the tyrosine kinase and the phosphatidylinositol (PtdIns) pathways. Stimulation of B cells with Ag or anti-Ig antibody results in a rapid increase in tyrosine phosphorylation of multiple substrates. The AgR also mediates the activation of phospholipase C-gamma 1 (PLC-gamma 1) thus producing the second messengers, inositol trisphosphate and diacylglycerol. Although the detailed relationship between these two signaling pathways remains unclear, it has recently become apparent that PLC-gamma 1 might be a target for the AgR-associated protein tyrosine kinase. To address the question of whether tyrosine kinase activity is essential for B cell activation, we studied early biochemical changes and later cellular events induced by ligation of the purinoceptor (P2R). Ligation of ATP to its receptor on B cells has been previously shown to elicit increases in cytosolic free Ca2+ and inositol phosphate production as well as induction of c-fos mRNA expression and increased expression of IL-2 and transferrin receptors. We show here that ATP in a wide range of concentrations did not increase protein tyrosine kinase activity. In contrast with the AgR, P2R did not mediate tyrosine phosphorylation of PLC-gamma 1, thus suggesting that it may use another phosphoinositide-specific PLC that does not require phosphorylation on tyrosine residues for its activation. The results were supported by experiments with a specific tyrosine kinase inhibitor, tyrphostin AG-126. Preincubation with this inhibitor blocked AgR but not P2R-mediated inositol phosphate production, cytosolic free Ca2+ changes, and IL-2 and transferrin receptor expression. The results indicate that the PtdIns pathway may be sufficient to induce activation of B cells and that the tyrosine phosphorylation pathway is not necessary for nonantigenic B cell activation.     

Threshold signaling of human Th0 cells in activation and anergy: modulation of effector function by altered TCR ligand

Molecular interactions between TCR and its natural ligand, in the presence of costimulatory signals, elicit T cell effector functions, whereas subtle changes in the structure of antigenic peptides may induce only selected T cell effector function including anergy. In this study, we have investigated the immunological activity of an altered TCR ligand (p 2, 28-40A34,36) derived from the immunodominant T cell epitope of the group 2 allergen of house dust mite, in which residues at positions 34 and 36 were substituted by alanine. Elevated IFN-gamma synthesis was induced by equimolar concentrations of the analogue compared with native peptide (p 2, 28-40) and was paralleled by increased down-regulation of cell surface CD3. IL-5 and IL-10 production exhibit the same sensitivity to both peptides, implying that the induction of T cell effector functions are not all proportional to TCR occupancy. Both native peptide and the analogue bound to MHC class II (DRB1*1101) molecules with similar affinities. Furthermore, p 2, 28-40A34,36 induced T cell anergy at lower concentrations than native peptide. During the induction of anergy, TGF-beta production was comparable for both peptides, whereas IL-10 secretion was markedly increased but more so in response to p 2, 28-40A34,36. Membrane expression of costimulatory ligands CD80 and CD86 was similar for native peptide and p 2, 28-40A34,36 and increased in activation, whereas only CD86 was elevated during anergy. The modulation of T cell effector function with altered TCR ligands may have practical applications in reprogramming allergic inflammatory responses through the induction of T cell anergy and/or the promotion of Th1 cytokines.     

Clonal anergy is induced in vitro by T cell receptor occupancy in the absence of proliferation.

Murine Th1 clones that receive signals through their TCR in the absence of APC-derived co-stimulatory signals do not produce IL-2 and instead become anergic, i.e., they are subsequently unable to produce IL-2 in response to Ag and normal APC. The critical cellular event required to prevent the induction of this anergic state appears to be T cell proliferation. Anergy was induced when T cell clones were stimulated under conditions where both TCR occupancy and costimulatory signals were provided but where proliferation in response to the IL-2 produced was prevented. Once induced, anergy could be reversed if the T cells were allowed to undergo multiple rounds of cell division. These results show that anergy is induced as a consequence of TCR occupancy in the absence of cell division; this can be achieved either by limiting IL-2 production because of deficient provision of co-stimulatory signals or by preventing response to IL-2.     

Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody

The effect of stimulating normal type 1 murine T cell clones with anti-CD3 antibody was examined in vitro. In the absence of accessory cells, anti-CD3 antibody immobilized on plastic plates stimulated inositol phosphate production, suboptimal proliferation, IL-2 and IL-3 production, and maximal IFN-gamma production. Addition of accessory cells augmented lymphokine production and proliferation when the effects of "high-dose suppression" were relieved by removing the T cells from the antibody-coated plates. Exposure of type 1 T cell clones to immobilized anti-CD3 antibody alone rapidly induced long-lasting proliferative unresponsiveness (anergy) to Ag stimulation that could be prevented by accessory cells. This anergic state was characterized by a lymphokine production defect, not a failure of the T cells to respond to exogenous IL-2 or to express surface Ti/CD3 complexes. In addition, anergy could not be induced in the presence of cyclosporine A. These results suggest that under certain conditions anti-CD3 antibodies may have potent immunosuppressive effects independent of Ti/CD3 modulation. Furthermore, our results support a two-signal model of type 1 T cell activation in which Ti/CD3 occupancy alone (signal 1) induces anergy, whereas Ti/CD3 occupancy in conjunction with a costimulatory signal (signal 2) induces a proliferative response.     

Interleukin-1beta, Src- and non-Src tyrosine kinases, and nitric oxide synthase induction in rat aorta in vitro

We studied the potential roles for endogenous interleukin-1beta (IL-1beta) and for several signaling pathways in the spontaneous induction in vitro of inducible nitric oxide synthase (iNOS) in endothelium-denuded rat aorta rings. Added IL-1beta augmented, whereas the IL-1beta receptor antagonist IL-1ra blocked, spontaneous iNOS induction. Furthermore, increases in IL-1beta mRNA preceded those of iNOS mRNA. Mitogen-activated protein kinase kinase and phosphatidyl inositol 3' kinase inhibition did not block iNOS induction, whereas nuclear factor kappaB inhibition did. The sarcoma virus tyrosine kinase (Src) family-selective inhibitor 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) blocked the upregulation of IL-1beta mRNA and the subsequent induction of iNOS but not the induction of iNOS stimulated by exogenously added IL-1beta. In contrast, the non-Src inhibitors TP 47/AG 213 and genistein and the tyrosine phosphatase inhibitor vanadate did not affect the spontaneous upregulation of IL-1beta mRNA but blocked both the IL-1beta-mediated and spontaneous induction of iNOS. We conclude that 1) the upregulation of tissue IL-1beta, via a signaling pathway involving a Src family kinase, plays a key role in rat vascular iNOS induction and 2) non-Src tyrosine kinases play roles downstream from IL-1beta for iNOS induction.     

Surfactant protein A regulates pulmonary surfactant secretion via activation of phosphatidylinositol 3-kinase in type II alveolar cells

Pulmonary surfactant is secreted by the type II alveolar cells of the lung, and this secretion is induced by secretagogues of several types (e.g., ionomycin, phorbol esters, and terbutaline). Secretagogue-induced secretion is inhibited by surfactant-associated protein A (SP-A), which binds to a specific receptor (SPAR) on the surface of type II cells. The mechanism of SP-A-activated SPAR signaling is completely unknown. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 rescued surfactant secretion from inhibition by SP-A. In order to directly demonstrate a role for PI3K in SPAR signaling, PI3K activity was immunoprecipitated from type II cell extracts. PI3K activity increased rapidly after SP-A addition to type II cells. Since many receptors that activate PI3K do so through tyrosine-specific protein phosphorylation, antisera to phosphotyrosine, insulin-receptor substrate-1 (IRS-1), or SPAR were also examined. These antisera coimmunoprecipitated PI3K activity that was stimulated by SP-A. In addition, the tyrosine-specific protein kinase inhibitors genistein and herbimycin A blocked the action of SP-A on surfactant secretion. We conclude that SP-A signals to regulate surfactant secretion through SPAR, via pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of PI3K. This activation leads to inhibition of secretagogue-induced secretion of pulmonary surfactant.     

Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.     

Hepatitis C virus core protein induces an anergic state characterized by decreased interleukin-2 production and perturbation of mitogen-activated protein kinase responses

Alterations of cytokine responses are thought to favor the establishment of persistent hepatitis C virus (HCV) infections, enhancing the risk of liver cirrhosis and hepatocellular carcinoma. Here we demonstrate that the expression of the HCV core (C) protein in stably transfected T cells correlates with a selective reduction of interleukin-2 (IL-2) promoter activity and IL-2 production in response to T-cell receptor triggering, whereas the activation of IL-4, IL-10, gamma interferon, and tumor necrosis factor alpha was moderately increased. This altered cytokine expression profile was associated with a perturbation of mitogen-activated protein (MAP) kinase responses. Extracellular regulated kinase and p38 were constitutively phosphorylated in C-expressing cells, while triggering of the costimulatory c-Jun N-terminal kinase (JNK) signaling cascade and activation of the CD28 response element within the IL-2 promoter appeared to be impaired. The perturbations of MAP kinase phosphorylation could be eliminated by cyclosporine A-mediated inhibition of nuclear factor of activated T cells, suggesting that the inactivation of JNK signaling and hyporesponsiveness to IL-2 induction were downstream consequences of C-induced Ca(2+) flux in a manner that mimics the induction of clonal anergy.     

Gangliosides GD1a and GM3 induce interleukin-10 production by human T cells

Gangliosides are sialic acid-containing glycosphingolipids and exhibit various physiologic functions. Gangliosides GD1a and GM3 strongly induced interleukin-10 (IL-10) protein secretion and mRNA expression in T cells from normal human subjects while the other gangliosides were ineffective. IL-10 induction by both gangliosides was completely blocked by protein tyrosine kinase (PTK) inhibitors, herbimycin A, genistein, and tyrphostin AG 1288, but not by other signal transduction inhibitors. These results suggest that GD1a and GM3 may induce IL-10 production in T cells by regulating the PTK-dependent signaling pathway. These gangliosides may thus act as important immunoregulators via IL-10.     

The role of tyrosine phosphorylation in signal transduction through surface Ig in human B cells. Inhibition of tyrosine phosphorylation prevents intracellular calcium release.

Cross-linking surface Ig on human B cells, or the TCR complex on T cells leads to the rapid appearance of newly tyrosine phosphorylated proteins. This is associated with inositol phospholipid turnover and a rise in intracellular calcium. Incubation of human B or T lymphocytes with the tyrosine kinase inhibitors, herbimycin and genistein, inhibits new tyrosine phosphorylation after receptor-linked activation. This is associated with complete abrogation of the increase in intracellular calcium in these lymphocytes and inhibition of inositol phospholipid turnover. Herbimycin- and genistein-treated lymphocytes are nevertheless still capable of responding to aluminum fluoride with a rise in intracellular calcium. These data support the contention that a B cell-associated protein tyrosine kinase regulates signal transduction via phospholipase C. CD45, the membrane associated protein tyrosine phosphatase, and PMA that activates protein kinase C, both inhibit the calcium response in B lymphocytes induced by receptor cross-linking. PMA and cross-linking CD45 both induced the appearance of tyrosine phosphorylated proteins in human B cells, although the pattern is quite distinct from that seen when surface lg is cross-linked. However, the induction of new tyrosine phosphorylation by anti-mu does not appear to be affected by these reagents. Although this may reflect an insensitivity of the tyrosine phosphorylation assay, it could indicate that regulation of the calcium response and regulation of the tyrosine kinase can be independent processes.     

Agents that mimic antigen receptor signaling inhibit proliferation of cloned murine T lymphocytes induced by IL-2

We have shown previously that stimulation of cloned murine T lymphocytes via the TCR inhibits their responsiveness to rIL-2. Signaling via the TCR is believed to result in a variety of biochemical events that include a rise in intracellular free calcium and activation (translocation) of protein kinase C. These two signals also can be generated by calcium ionophores, such as ionomycin, and by activators of protein kinase C, such as PMA. We report here that treatment of cloned murine T lymphocytes with PMA, ionomycin, or the combination led to a dose-dependent inhibition of IL-2-dependent proliferation but did not inhibit lymphokine secretion. Concentrations of PMA and ionomycin that maximally inhibited proliferation stimulated maximal lymphokine secretion and increased mitochondrial activity as assessed by measurement of cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium-bromide. Furthermore, PMA, ionomycin, the combination, or immobilized anti-CD3 mAb added after 12 to 16 h of culture with IL-2 could inhibit proliferation. These results demonstrate that PMA and ionomycin mimic stimulation of the TCR by high concentrations of immobilized anti-TCR mAb in that proliferation is inhibited and lymphokine secretion is induced. In addition, PMA or ionomycin could independently inhibit proliferation of some cells. These findings suggest that alternative mechanisms exist to regulate proliferation. Either increased levels of intracellular calcium or the physiologic events corresponding to those induced by PMA can inhibit IL-2-dependent replication of T lymphocytes.     

Induction of T cell anergy by low numbers of agonist ligands.

Engagement of TCR by its ligand, the MHC/peptide complex, causes T cell activation. T cells respond positively to stimulation with agonists, and are inhibited by antagonist MHC/peptide ligands. Failure to induce proper conformational changes in the TCR or fast TCR/MHC dissociation are the leading models proposed to explain anergy induction by antagonist ligands. In this study, we demonstrate that presentation of between 1 and 10 complexes of agonist/MHC II by unfixed APC induces T cell anergy that persists up to 7 days and has characteristics similar to anergy induced by antagonist ligand or TCR occupancy without costimulation. Furthermore, anergy-inducing doses of hemagglutinin 306-318 peptide led to the engagement of less than 1000 TCR/CD3 complexes. Thus, engagement of a subthreshold number of TCR by either a low density of agonist/MHC or a 2-3 orders of magnitude higher density of antagonist/MHC causes anergy. Moreover, we show that anergy induced by low agonist concentrations is inhibited in the presence of IL-2 or cyclosporin A, suggesting involvement of the calcineurin signaling pathway.     

Regulation of the maintenance of peripheral T-cell anergy by TAB1-mediated p38 alpha activation

In anergic T cells, T-cell receptor (TCR)-mediated responses are functionally inactivated by negative regulatory signals whose mechanisms are poorly understood. Here, we show that CD4(+) T cells anergized in vivo by superantigen Mls-1(a) express a scaffolding protein, transforming growth factor beta-activated protein kinase 1-binding protein 1 (TAB1), that negatively regulates TCR signaling through the activation of mitogen-activated protein kinase p38 alpha. TAB1 was not expressed in naive and activated CD4(+) T cells. Inhibition of p38 activity in anergic T cells by a chemical inhibitor resulted in the recovery of interleukin 2 (IL-2) and the inhibition of IL-10 secretion. T-cell hybridoma 2B4 cells transduced with TAB1-containing retrovirus (TAB1-2B4 cells) showed activated p38 alpha, inhibited extracellular signal-regulated kinase (ERK) activity, culminating in reduced IL-2 levels and increased IL-10 production. The use of a p38 inhibitor or cotransfection of a dominant-negative form of p38 in TAB1-2B4 cells resulted in the recovery of ERK activity and IL-2 production. These results imply that TAB1-mediated activation of p38 alpha in anergic T cells regulates the maintenance of T-cell unresponsiveness both by inhibiting IL-2 production and by promoting IL-10 production.     

Induction of competence to respond to IL-4 by CD4+ T helper type 1 cells requires costimulation.

Rested murine CD4+ Th1 clones do not produce IL-4, but have previously been shown to be capable of responding to IL-4 if they are first activated with Ag and APC. In this study, we have examined the activation requirements for induction of competence to respond to IL-4 in these clones. TCR occupancy alone (given either as chemically fixed APC and Ag, anti-CD3, Con A, or ionomycin and PMA) was inadequate, but the addition of a source of costimulation to any of these stimuli resulted in complete induction of competence to respond to IL-4. Pretreatment of the Th1 clones with TCR occupancy alone induced an anergic state from which subsequent full stimulation with Ag and APC failed to give IL-4 responsiveness. Pretreatment of the cells with IL-2 alone was an inadequate signal to induce IL-4 responsiveness and only a partial response was obtained when TCR occupancy was combined with IL-2. Addition of anti-IL-2 and anti-IL-2R antibodies during full activation with APC and Ag gave a 50% inhibition of competence induction. These results demonstrate that costimulation, in addition to its role in IL-2 production, is an important second signal for inducing T cells to become competent to respond to IL-4.     

An islet activating protein-sensitive G protein is involved in dopamine inhibition of angiotensin and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells

In primary culture of anterior pituitary cells, dopamine inhibited the angiotensin (AII)-stimulated inositol phosphate production by 28 +/- 2.5% (n = 14), with an EC50 of 660 +/- 228 nM (n = 8). This effect was blocked by (+)-butaclamol, a specific dopamine receptor antagonist. RU 24926, a D2 specific agonist, but not SKF 38393, a specific D1 agonist, inhibited AII-stimulated inositol phosphate production, suggesting that this dopamine effect is mediated through a dopamine receptor of the D2 subtype. Dopamine also partially inhibited (25%) inositol phosphate production stimulated by thyrotropin-releasing hormone (TRH). Our results suggest that the dopamine-mediated inhibition of hormonally stimulated inositol phosphate production is probably not mediated through the known inhibitory effects of dopamine on cAMP and Ca2+ intracellular concentrations. Although unknown, the mechanism by which dopamine inhibited the AII and TRH-stimulated inositol phosphate production implicates a GTP binding protein sensitive to the islet activating protein (IAP) since dopamine effects were blocked by this toxin. The alpha subunit of the GTP binding protein involved could be one of the three ADP-ribosylated proteins found in anterior pituitary cells in primary cultures, the alpha o (39 kDa), the alpha i (41 kDa), and an alpha subunit of 40 kDa. Indeed, we show here that this 40-kDa IAP substrate, already described in a few tissues, is present in anterior pituitary cells. The negative coupling between dopamine receptors and the AII or TRH inositol phosphate production systems, could be implicated in the dopamine inhibition of the AII- and TRH-stimulated prolactin release since such an inhibition is blocked by IAP. Our results suggest that the negative regulation of inositol phosphate production is one of the mechanisms by which dopamine controls hormonally stimulated prolactin release.