Platelet-activating factor mediated effects on human neutrophil function are inhibited by pertussis toxin

Pretreatment of human neutrophils with pertussis toxin inhibits platelet-activating factor-mediated chemotaxis, superoxide generation, aggregation, and release of lysozyme. By contrast, superoxide generation observed in the presence of phorbol-12-myristate-13 acetate is unaffected. Our results suggest that a target protein for pertussis toxin, probably a GTP binding protein termed "Ni", is involved in the actions of platelet-activating factor on human neutrophils.     

Flow-induced prostacyclin production is mediated by a pertussis toxin-sensitive G protein.

Fluid flow and several other agonists induce prostacyclin (PGI2) production in endothelial cells. G proteins mediate the response of a large number of hormones such as histamine, but the transduction pathway of the flow signal is unclear. We found that GDP beta S and pertussis toxin inhibited flow-induced prostacyclin production in human umbilical vein endothelial cells. In addition, flow potentiated the histamine-induced production of PGI2. This suggests that flow stimulates prostacyclin production via a pertussis toxin-sensitive G protein and modulates the stimulus-response coupling of other agonists.     

Pleiotropic effects of Pasteurella multocida toxin are mediated by Gq-dependent and -independent mechanisms. involvement of Gq but not G11

Pasteurella multocida toxin (PMT) is a highly potent mitogen for a variety of cell types. PMT has been shown to induce various cellular signaling processes, and it has been suggested to function through the heterotrimeric G-proteins G(q)/G(11). To analyze the role of G(q)/G(11) in the action of PMT, we have studied the effect of the toxin in Galpha(q)/Galpha(11) double-deficient fibroblasts as well as in fibroblasts lacking only Galpha(q) or Galpha(11). Interestingly, formation of inositol phosphates in response to PMT was exclusively dependent on Galpha(q) but not on the closely related Galpha(11). Although Galpha(q)/Galpha(11) double-deficient and Galpha(q)-deficient cells did not respond with any production of inositol phosphates to PMT, PMT was still able to induce various other cellular effects in these cells, including the activation of Rho, the Rho-dependent formation of actin stress fibers and focal adhesions, as well as the stimulation of c-Jun N-terminal kinase and extracellular signal-regulated kinase. These data show that PMT leads to a variety of cellular effects that are mediated only in part by the heterotrimeric G-protein G(q).     

5-Fluorouracil enhances apoptosis sensitivity of T lymphocytes mediated by CD3 epsilon

Previous studies by our laboratory have reported that the T cell receptor (TCR) TCR/CD3 complex could mediate activation as well as apoptosis of T lymphocytes. Two tyrosine residues in the ITAM (immuno-receptor tyrosine-based activation motifs) of CD3 epsilon were required for apoptosis signalling of Jurkat T lymphocytes. Stable cell lines TJK and T3JK produced from CD8(-) Jurkat T lymphocytes by transfection with wild-type and mutant CD8 epsilon (fusion of the extracellular and transmembrane domains of human CD8 alpha to the intracellular domain of mouse CD3 epsilon), were used with CD8(-) Jurkat T lymphocytes for studying the role of single intact CD3 epsilon. 5-Fluorouracil (5-FU), a chemotherapeutic drug can induce cell death of many tumour cell lines. In the present experiments, we examined the expression of caspase-3, p53 and Bid in the three cell lines induced by 5-FU and/or anti-CD8 antibody. We found high expression of p53 during activation-induced cell death of TJK cells mediated by anti-CD8 antibody and apoptosis of TJK and T3JK induced by 5-FU, implicating p53 involvement in apoptosis of leukemia cells induced by anti-CD8 antibody and 5-FU. We also detected the active form of caspase-3 and Bid in apoptotic leukemia cells after treatment with 5-FU and/or anti-CD8 antibody, indicating that the drug and antibody induced cell death through caspase-3 and the signal pathway may involve the Bcl-2 protein family. Our results showed that combined treatment with 5-FU and anti-CD8 antibody could enhance the rate of apoptosis induced by 5-FU or anti-CD8 antibody through increased expression of p53 and by promoting activation of caspase-3 and Bid. This suggests that the combination of 5-FU and anti-CD8 antibody may play an important role in inducing apoptosis of leukemia cells.     

Induction of polyclonal CD8+ T cell activation and effector function by Pertussis toxin

Pertussis toxin (PTX) has pronounced adjuvant activity and strongly enhances innate and adaptive immune responses, including increased antibody production and Th1/Th2 cytokine production. Adjuvant effects of PTX on Th1 and Th2 cells are primarily mediated via CD80/86 costimulation via enhanced expression of these molecules by APCs. However, it has remained unresolved whether PTX modulates the expression of costimulatory and inhibitory molecules on CD4+ and CD8+ T cells. To address this question, we determined the expression kinetics of CD28, CTLA-4, and CD40L on spleen CD4+ and CD8+ T cells after incubation with PTX. The results show that PTX upregulated the expression of CD28 by CD8+ T cells, but not by CD4+ T cells. In contrast, the expression of CTLA-4 and CD40L was not substantially altered on CD4+ or CD8+ T cells. CD28 upregulation by CD8+ T cells was paralleled by upregulation of CD69 and the induction of IFN-γ, Granzyme B (GrB), and IL-17. CD8+ T cell activation and cytokine production could be substantially blocked with anti-CD80 and CD86 antibodies, consistent with CD28 mediated signaling. Treatment of highly purified CD8+ T cells with PTX resulted in upregulation of CD28 and CD69, and production of IFN-γ. Incubation with CD28 mAb further enhanced this effect, suggesting that PTX has direct effects on CD8+ T cells which are enhanced by CD80/86-mediated costimulation provided by APCs.     

Neutrophil activation by surface bound IgG is via a pertussis toxin insensitive G protein

Pre-treatment of neutrophils with either pertussis or cholera toxins does not inhibit neutrophil activation by surface bound IgG. In contrast, pretreatment with the phorbol ester, phorbol myristate acetate, results in a dose dependent inhibition of degranulation by surface bound IgG. This inhibition is similar to that seen with soluble ligands where it is thought to be due to interference with the interaction of an activated guanine nucleotide binding protein with phospholipase C (J. Biol. Chem.,262,6121,1987). More directly, GTP binding and GTPase activity are enhanced when human neutrophil membranes are incubated in wells containing surface bound IgG. Neither of these G protein functions were inhibited when membranes were prepared in the presence of pertussis toxin, suggesting that neutrophil activation by surface bound IgG proceeds by a mechanism that involves a pertussis toxin insensitive G protein.     

The effects of pertussis toxin and cholera toxin on mitogen-induced interleukin-2 production: evidence for G protein involvement in signal transduction

GTP-binding proteins, known as G proteins, play important roles in transducing signals generated by the binding of specific ligands to cell surface receptors. We examined the possibility that a G protein is involved in transducing the concanavalin A (Con A) signal for IL-2 production using a T-cell hybridoma, FS6-14.13, and the bacterial toxins, pertussis toxin (PTX) and cholera toxin (CTX). These toxins are known to interact with and modify the functions of G proteins. High concentrations of PTX (25-50 micrograms/ml) stimulated IL-2 production in the FS-6 cells in the absence of Con A, presumably due to the ability of its B subunit to crosslink membrane proteins. However, in the presence of Con A, PTX inhibited IL-2 production at concentrations ranging from 0.05 to 50 micrograms/ml. It is unlikely that this inhibition was due to a competitive interaction between Con A and PTX for binding sites at the cell surface, since high concentrations of PTX only minimally reduced Con A-FITC binding, evaluated by FACS analysis. In addition, concentrations of PTX which were not able to stimulate IL-2 production in the absence of Con A, retained their ability to inhibit IL-2 production in the presence of Con A. These data suggest the involvement of the PTX A subunit in this activity. In support of this possibility, PTX catalyzed ADP-ribosylation of a Mr = 41,000-Da protein in FS-6 membranes. This strongly suggests that a PTX substrate is involved in transducing the Con A signal for IL-2 production in FS-6 cells. CTX also inhibited Con A-induced IL-2 production, an effect mimicked by the addition of dibutyryl-cAMP. This suggests that a CTX substrate linked to the adenylyl cyclase-cAMP pathway is probably not involved in transducing the stimulatory Con A signal, but may play a role in downregulating T-cell activation.     

Effects of insulin,pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.     

Effects of antibodies to the filamentous haemagglutinin and to the pertussis toxin of Bordetella pertussis on adherence and toxic effects to 3T3 cells

Abstract Adherence of B. pertussis to NIH3T3 mouse fibroblasts was efficiently inhibited by a mouse immune serum reacting specifically with the filamentous haemagglutinin (FHA), whereas a mouse immune serum reacting specifically with the pertussis toxin (Ptx) produced partial inhibition only significant after 3 h infection. Protection against cytopathic effects on infected 3T3 cells with anti-FHA antibodies was at least as effective (83.3%± 7.5) as with anti-Ptx antibodies (75%± 4). This suggests that adherence of B. pertussis to eukaryotic receptors is a primary mechanism determining both bacterial proliferation and toxic effects in susceptible cells, and that prevention of B. pertussis attachment to cell receptors might be sufficient to protect against both infectious and toxic processes in whooping cough.     

Functional modification by cholera-toxin-catalyzed ADP-ribosylation of a guanine-nucleotide-binding regulatory protein serving as the substrate of pertussis toxin.

The alpha subunits of Gi (Gi alpha) and Gs (guanine-nucleotide-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in differentiated HL-60 cell membranes upon stimulation of chemotactic receptors by fMLF (fM, N-formylmethionine). The ADP-ribosylation site of Gi alpha modified by cholera toxin appeared to be different from that modified by pertussis toxin [Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M. & Katada, T. (1989) J. Biol. Chem. 264, 21,394-21,400]. This allowed us to investigate how the two types of ADP-ribosylation influence the function of the signal-coupling protein. The major findings observed in HL-60 cell membranes, where the same Gi alpha molecule was ADP-ribosylated by treatment of the membranes with either toxin, are summarized as follows. (a) More fMLF bound with a high affinity to cholera-toxin-treated membranes than to the control membranes. The high-affinity binding was, however, not observed in pertussis-toxin-treated membranes. (b) Although fMLF stimulated guanine nucleotide binding and GTPase activity in control membranes, stimulation was almost completely abolished in pertussis-toxin-treated membranes. In contrast, fMLF-dependent stimulation of GTPase activity, but not that of guanine nucleotide binding was attenuated in cholera-toxin-treated membranes. (c) Gi alpha, once modified by cholera toxin, still served as a substrate of pertussis-toxin-catalyzed ADP-ribosylation; however, the ADP-ribosylation rate of modified Gi was much lower than that of intact Gi. These results suggested that Gi ADP-ribosylated by cholera toxin was effectively capable of coupling with fMLF receptors, resulting in formation of high-affinity fMLF receptors, and that hydrolysis of GTP bound to the alpha subunit was selectively impaired by its ADP-ribosylation by cholera toxin. Thus, unlike the ADP-ribosylation of Gi by pertussis toxin, cholera-toxin-induced modification would be of great advantage to the interaction of Gi with receptors and effectors that are regulated by the signal-coupling protein. This type of modification might also be a candidate for unidentified G proteins which were less sensitive to pertussis toxin and appeared to be involved in some signal-transduction systems.     

Regression of a murine gammaherpesvirus 68-positive b-cell lymphoma mediated by CD4 T lymphocytes

Murine gammaherpesvirus 68-infected S11 cells were injected subcutaneously into nude mice. Adoptively transferred restimulated lymphocytes consistently elicited the regression of S11 tumors. CD4 T lymphocytes were most effective in preventing tumor formation, and immunohistochemistry highlighted populations of CD4 T cells in regressing tumors.     

Pertussis toxin activates protein kinase C and a tyrosine protein kinase in the human T cell line Jurkat

Pertussis toxin activates T lymphocytes by a mechanism that is independent of its ADP-ribosylation activity. The toxin stimulates increases in diacylglycerol and intracellular calcium apparently by interacting with a cell surface receptor. Consistent with the production of these second messengers we have found that pertussis toxin activates protein kinase C in the Jurkat cell line. The toxin was also found to activate a tyrosine protein kinase in these cells in a manner similar to that observed with phytohemagglutinin. These results provide evidence that the mechanism of activation of T cells by pertussis toxin involves stimulating the activity of protein kinase C and a tyrosine protein kinase.     

Vagotonic effects of enkephalin are not mediated by sympatholytic mechanisms

This study examined the hypothesis that vagotonic and sympatholytic effects of cardiac enkephalins are independently mediated by different receptors. A dose-response was constructed by administering the delta-receptor opioid methionine-enkephalin-arginine-phenylalanine (MEAP) by microdialysis into the interstitium of the canine sinoatrial node during vagal and sympathetic stimulation. The right cardiac sympathetic nerves were stimulated as they exited the stellate ganglion at frequencies selected to increase heart rate approximately 35 bpm. The right cervical vagus was stimulated at frequencies selected to produce a two-step decline in heart rate of 25 and 50 bpm. A six-step dose-response was constructed by recording heart rates during nerve stimulation as the dose of MEAP was increased between 0.05 pmol/min and 1.5 nmol/min. Vagal transmission improved during MEAP at 0.5 pmol/min. However, sympathetically mediated tachycardia was unaltered with any dose of MEAP. In Study 2, a similar dose-response was constructed with the kappa-opioid receptor agonist trans(+/-)-3-4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide-HCl (U-50488H) to illustrate an independent sympatholytic effect and to verify its kappa-receptor character. U-50488H gradually suppressed the sympathetic tachycardia, with a significant effect obtained only at the highest dose (1.5 nmol/min). U-50488H had no effect on vagally mediated bradycardia. Surprisingly, the sympatholytic effect was not reversed by withdrawing U-50488H or by the subsequent addition of the kappa-antagonist 17,17'-(dichloropropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-binorphinan-3,4',14,14'-tetroldi-hydrochloride (norBNI). Study 3 was conducted to determine whether the sympatholytic effect of U-50488H could be prevented by norBNI. NorBNI blocked the sympatholytic effect of the U50488H for 90 mins. When norBNI was discontinued afterward and U-50488H was continued alone, a sympatholytic effect emerged within 30 mins. Collectively these observations support the hypothesis that the vagotonic influence of MEAP is not dependent on a sympatholytic influence. Furthermore, the sympatholytic effect is mediated independently by kappa-receptors. The sympatholytic effect of sustained kappa-receptor stimulation appears to evolve gradually into a functional state not easily reversed.     

Effect of ras-gene transformation on the inhibition of NIH3T3 cell growth by pertussis toxin

To investigate the relationship between the effects of a pertussis toxin-inhibitable class of G-proteins and the ras family of protooncogenes on cell growth, we isolated multiple cell lines transformed by oncogenic Hras or Nras genes and measured the ability of pertussis toxin to inhibit their growth rate. Although all of the cell lines were morphologically transformed and could grow in agar suspension, there was considerable variability in their resistance to pertussis toxin, ranging from cell lines completely resistant to pertussis toxin to cell lines as sensitive to pertussis toxin as the parental cells from which they derived. For those lines resistant to pertussis toxin, this resistance is not due to an inability of pertussis toxin to reach or react with its intracellular target; pertussis toxin could be shown to ADP-ribosylate the endogenous G-proteins of all lines tested regardless of whether it affected their growth rate. There was a strong correlation between the level of active ras protein expressed in the different lines and the degree of resistance to pertussis toxin (r = 0.80). Although the Hras-transformed cell lines were more resistant to pertussis toxin as a group than the Nras-transformed cell lines, we believe that this is not a primary difference between Nras and Hras, but, rather, is due to a higher average level of expression of ras in the cell lines receiving Hras. We suggest that the consequences of ras transformation vary with the concentration of oncogenic ras present in the cell, and that different assays or different properties of transformation show different sensitivities to the level of ras expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatitis C virus infection of human T lymphocytes is mediated by CD5

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease. Although infection of hepatocytes is mainly responsible for manifestations of hepatitis C, the virus also invades the immune system by a yet-to-be-identified mechanism. Using human T cell lines and primary T lymphocytes as targets and patient-derived HCV as inocula, we aimed to identify how HCV gains entry into these cells. HCV replication was determined by detection of the HCV RNA replicative (negative) strand and viral proteins, while specific antibodies, knocking down gene expression and making otherwise-resistant cells prone to HCV, were employed to identify a receptor molecule determining T lymphocyte permissiveness to HCV infection. The results revealed that T cell susceptibility to HCV requires CD5, a lymphocyte-specific glycoprotein belonging to the scavenger receptor cysteine-rich family. Blocking of T cell CD5 with antibody or silencing with specific short hairpin RNA (shRNA) decreased cell susceptibility to HCV, while increasing CD5 expression by mitogen stimulation had the opposite effect. Moreover, transfection of naturally CD5-deficient HEK-293 fibroblasts with CD5 facilitated infection of these otherwise HCV-resistant cells. In contrast to T cells, hepatocytes do not express CD5. The data revealed that CD5 is a molecule important for HCV entry into human T lymphocytes. This finding provides direct insight into the mechanism of HCV lymphotropism and defines a target for potential interventions against HCV propagating in this extrahepatic compartment.     

Opposing effects of ICOS on graft-versus-host disease mediated by CD4 and CD8 T cells

ICOS, a CD28 family member expressed on activated CD4(+) and CD8(+) T cells, plays important roles in T cell activation and effector function. Here we studied the role of ICOS in graft-vs-host disease (GVHD) mediated by CD4(+) or CD8(+) T cells in allogeneic bone marrow transplantation. In comparison of wild-type and ICOS-deficient T cells, we found that recipients of ICOS(-/-) CD4(+) T cells exhibited significantly less GVHD morbidity and delayed mortality. ICOS(-/-) CD4(+) T cells had no defect in expansion, but expressed significantly less Fas ligand and produced significantly lower levels of IFN-gamma and TNF-alpha. Thus, ICOS(-/-) CD4(+) T cells were impaired in effector functions that lead to GVHD. In contrast, recipients of ICOS(-/-) CD8(+) T cells exhibited significantly enhanced GVHD morbidity and accelerated mortality. In the absence of ICOS signaling, either using ICOS-deficient donors or ICOS ligand-deficient recipients, the levels of expansion and Tc1 cytokine production of CD8(+) T cells were significantly increased. The level of expansion was inversely correlated with the level of apoptosis, suggesting that increased ability of ICOS(-/-) CD8(+) T cells to induce GVHD resulted from the enhanced survival and expansion of those cells. Our findings indicate that ICOS has paradoxical effects on the regulation of alloreactive CD4(+) and CD8(+) T cells in GVHD.