Cyclin D3 regulates proliferation and apoptosis of leukemic T cell lines.

Activation of the T cell receptor in leukemic T cell lines or T cell hybridomas causes growth inhibition. A similar growth inhibition is seen when protein kinase C is activated through addition of phorbol myristate acetate. This inhibition is due to an arrest of cell cycle progression in G(1) combined with an induction of apoptosis. Here we have investigated the mechanism by which these stimuli induce inhibition of proliferation in Jurkat and H9 leukemic T cell lines. We show that expression of cyclin D3 is reduced by each of these stimuli, resulting in a concomitant reduction in cyclin D-associated kinase activity. This reduction in cyclin D3-expression is crucial to the observed G(1) arrest, since ectopic expression of cyclin D3 can abrogate the G(1) arrest seen with each of these stimuli. Moreover, ectopic expression of cyclin D3 also prevents the induction of programmed cell death by phorbol myristate acetate and T-cell receptor activation, leading us to conclude that cyclin D3 not only plays a crucial role in progression through the G(1) phase, but is also involved in regulating apoptosis of T cells.

CD28 induces cell cycle progression by IL-2-independent down-regulation of p27kip1 expression in human peripheral T lymphocytes.

CD28 is the primary T cell costimulatory receptor, and upon ligation with its ligands, it enhances T cell proliferation and IL-2 synthesis. In this study we examined the role of CD28 in the initial proliferative response and cell cycle entry of T lymphocytes. Stimulation through CD3 alone resulted in a poor proliferative response, while in the presence of CD28 costimulation a strong increase in the number of cells in S-phase could be detected after 48 h of stimulation. CD28 costimulation enhanced expression of cyclin D3 and induced down-regulation of p27kip1 expression. Cross-linking CD28 was much more effective in inducing cyclin D3 expression and in down-regulating p27kip1 expression than addition of IL-2. Blocking experiments, using antibodies that neutralize IL-2 or the IL-2 receptor, showed that the effects induced by CD28 are independent of endogenous IL-2. Moreover, using a variety of immunosuppressants that interfere with IL-2 signaling pathways, we were able to show that IL-2 is not required for cell cycle entry induced by CD28 costimulation. From these experiments it can be concluded that CD28 and IL-2 use different signaling pathways for down-regulation of p27kip1 expression. We hypothesize that costimulation through CD28 is responsible for initial cell cycle entry of T lymphocytes, while IL-2, which is produced after costimulation, might be involved in sustaining proliferation.

Characterization of exocytosis in electropermeabilized neutrophils by flow cytometric analysis: difference in sensitivity to calcium and guanosine-5'-[gamma-thio]triphosphate.

When rabbit neutrophils were subjected to two electrical discharges of 4.75 kV/cm, the cells became permeable to propidium iodide. Measurement of propidium iodide fluorescence using flow cytometry showed that all cells in the suspension were permeabilized. The cells remained permeable for > 20 min when the cells were stored at 0 degree C. When exocytosis was induced by Ca2+ alone, the orthogonal light scatter (a sensitive parameter for cell granularity) of the complete population changed depending on the concentration. All the cells were equally sensitive to Ca2+ and showed a similar degree of exocytosis at the same time. In the presence of a fixed concentration of Ca2+ and a variable concentration of guanosine-5'-[v-thio]triphosphate (GTP gamma S), a division of the cell population was observed in the orthogonal light scatter histogram. At low GTP gamma S concentrations, a part of the population showed complete exocytosis and a part of the population showed almost no exocytosis. With increasing GTP gamma S concentrations, the light scatter pattern of the population changed indicating that the cells were gradually sensitive to GTP gamma S. Electropermeabilized neutrophils showed an equal sensitivity to Ca2+ and a graded sensitivity to GTP gamma S. Flow cytometry is considered as an ideal tool to study such an effect on a cell-to-cell basis.

Neutrophil chemotaxis induced by the diacylglycerol kinase inhibitor R59022.

The diacylglycerol kinase inhibitor R59022 induced chemotaxis in neutrophils. The response to R59022 was primarily chemotactic and only very little chemokinetic. Pretreatment with the protein kinase C inhibitors staurosporine and AMG-C16 inhibited chemotaxis induced by R59022 indicating the involvement of protein kinase C. In contrast, chemotaxis induced by fMet-Leu-Phe was only slightly inhibited by staurosporine and AMG16. The effects of R59022 were comparable to the effects of the protein kinase C activators DiC8 and PMA and suggest an involvement of protein kinase C. Pretreatment with pertussis toxin inhibited R59022-induced migration, fMet-Leu-Phe-induced migration, and random migration. GTP gamma S, which stimulates migration of electropermeabilized neutrophils by itself, causes an additive increase of migration in electropermeabilized neutrophils stimulated with a suboptimal concentration R59022, but causes a synergistic increase of migration in cells stimulated with a suboptimal concentration fMet-Leu-Phe. The effects of GTP gamma S on migration are completely inhibited by AMG-C16. This suggests that the GTP-binding protein involved in R59022-activated migration is the G protein that is associated with random migration.

GTP[S] stimulates migration of electropermeabilized neutrophils via a pertussis toxin-sensitive G-protein.

Electropermeabilized neutrophils were used to study the role of G-proteins in neutrophil migration. Rabbit neutrophils, under specific conditions, retained their ability to migrate after electropermeabilization. Introduction of guanosine-5'-[3-thio] triphosphate (GTP[S]) into the cell interior stimulated random migration and enhanced migration activated by a suboptimal concentration of formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) (10(-11) M). GTP[S] had no effect on random migration by intact cells, or on migration of intact cells activated with a suboptimal concentration of fMet-Leu-Phe, indicating that the effect of GTP[S] was intracellular. The effects of GTP[S] were inhibited by pertussis toxin and by guanosine-5'-[2-thio] diphosphate (GDP beta S) indicating that a pertussis toxin-sensitive G-protein was involved. GTP stimulated random migration to the same extent as GTP[S], but had only a small effect on migration activated by a suboptimal concentration of fMet-Leu-Phe (10(-11) M). Several other nucleotides tested had no effect on random migration or migration activated with 10(-11) M fMet-Leu-Phe. The results show that neutrophil migration can be potentiated by direct activation of a pertussis toxin-sensitive G-protein, and the results obtained with GTP suggest that possibly more than one G-protein is involved in this process.

Activation of neutrophil migration by dioctanoyl-sn-glycerol and fMet-Leu-Phe is controlled by different pathways.

Migration activated by fMet-Leu-Phe is inhibited by GTP[S] and is little affected by protein kinase C inhibitors. We investigated the effects of GTP[S] and the protein kinase C inhibitor AMG-C16 on dioctanoyl-sn-glycerol (DiC8)-activated migration of rabbit neutrophils and compared them with the effects on fMet-Leu-Phe-activated migration and random migration. GTP[S] did not inhibit DiC8-activated migration or random migration but inhibited fMet-Leu-Phe-activated migration. AMG-C16 gave a strong inhibition of DiC8-activated migration but had only a small effect on fMet-Leu-Phe-activated migration and random migration. When fMet-Leu-Phe and DiC8 were added together in suboptimal concentrations an additive effect was found. Pretreatment with the diacylglycerol kinase inhibitor R59022 enhanced random migration. The enhancement was completely inhibited by AMG-C16 and was unaffected by GTP[S]. These findings suggest that DiC8-activated migration and fMet-Leu-Phe-activated migration are controlled by different pathways.

Strontium and barium induce exocytosis in electropermeabilized neutrophils.

Calcium, strontium and barium induced an exocytotic response in electropermeabilized rabbit neutrophils while magnesium was without any effect. The extent of enzyme release was found to depend upon the concentration of these cations. For all cations, an optimum concentration was found with the same maximum enzyme release. At concentrations higher than optimum a decrease in lysozyme release was observed. Efficiency to induce enzyme release was in the order: Ca2+ > Sr2+ > Ba2+. Enzyme release was significantly enhanced by guanosine-5'-[gamma-thio]triphosphate (GTP gamma S) resulting in a shift to the left of the dose/response curve. The enhancement by GTP gamma S was strongest with Ca2+, was less with Sr2+, and was very little with Ba2+. The time course of lysozyme release was the same for Ca2+, Sr2+, and Ba2+ in the presence and absence of GTP gamma S when suboptimal cation concentrations were used. A decrease in responsiveness to the effectors after electropermeabilization was observed with Ca2+, Sr2+ and Ba2+ in the presence and absence of GTP gamma S. The lysozyme release induced by the different cations was not inhibited by the protein kinase C inhibitor staurosporine and was slightly affected by pertussis toxin. Ca2+ and Sr2+, but not Ba2+, potentiated formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) induced enzyme release in intact neutrophils. The divalent cation ionophore A23187 induced enzyme release in the presence of Ca2+ and Sr2+ but not in the presence of Ba2+. The results obtained with electropermeabilized neutrophils indicate that Sr2+ and Ba2+ can act as substitutes for Ca2+ in activating exocytosis, and that permeabilized neutrophils provide the best tool to investigate the effects of alkaline earth ions in exocytosis.

Exocytosis in electropermeabilized neutrophils. Responsiveness to calcium and guanosine 5'-[gamma-thio]triphosphate.

Electropermeabilized neutrophils were used to study the exocytotic response in rabbit neutrophils. Enzyme release from electropermeabilized neutrophils could be induced by elevating the Ca2+ concentration. Ca(2+)-induced secretion was significantly enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in a concentration-dependent manner. The effect of GTP[S] could be blocked by guanosine 5'-[beta-thio]diphosphate (GDP[S]) and was not affected by pertussis toxin. GTP[S] did not induce enzyme release in the absence of Ca2+. Induction of an exocytotic response did not require addition of ATP. However, neutrophils permeabilized in the absence of ATP became refractory to stimulation due to a reduction in their affinity for Ca2+. Responsiveness to the effectors Ca2+ or Ca2+ + GTP[S] could be prolonged or restored by ATP. ATP was not the only agent that prolonged responsiveness; other nucleotides and inorganic phosphates were also effective. The protein kinase C inhibitors staurosporine and 1-O-hexadecyl-2-methyl-sn-glycerol did not inhibit exocytosis and had only a small effect on the prolongation and restoration of responsiveness by ATP. A hypothesis is presented suggesting that the loss of responsiveness is caused by dephosphorylation and that the restoration or prolongation of responsiveness is not mediated by protein kinase C. It is possible that an as yet unidentified Ca(2+)-binding protein is dephosphorylated, resulting in a decrease in Ca2+ affinity.

Inhibition of neutrophil chemotaxis by purinoceptor agonists.

Adenine nucleotides inhibited fMet-Leu-Phe-activated chemotaxis by rabbit neutrophils in the micromolar concentration range. The sequence of inhibitory effectiveness was ATP[S] greater than ATP greater than ADP greater than AMP greater than adenosine. In the presence of EDTA, inhibition of chemotaxis by ATP occurred to the same degree as in the presence of Ca2+ (and Mg2+), indicating that neither ectonucleotidases nor Ca2+ fluxes across the plasma membrane are of importance for the inhibitory effect. The inhibitory effect of ATP persisted when the nucleotide was removed after preincubation, before the cells were submitted to chemotaxis. Exposure of neutrophils to ApCpp results in desensitization towards inhibition by ATP. Other nucleoside triphosphates, such as XTP, GTP, ITP, CTP and UTP, also inhibited neutrophil migration. The relative potency of the nucleotides, which are used to discriminate between the subtypes of the P2 purinoceptor, was (at a concentration of 50 microM) ATP greater than ApCpp greater than AppNp greater than MeSATP greater than AppCp. The results suggest that inhibition of neutrophil chemotaxis by purinoceptor agonists is mediated by P2 purinoceptors and that the subtype is different from the P2x or P2y purinoceptor.

The role of calcium in neutrophil migration: the effect of calcium and calcium-antagonists in electroporated neutrophils.

Chemotaxis by electroporated rabbit peritoneal neutrophils in the absence of Ca2+ is only slightly different from that in the presence of Ca2+. Pretreatment of neutrophils with quin2-AM causes inhibition of chemotaxis. Calcium antagonists as nitrendipine and verapamil are inhibitory in nanomolar concentrations, while 10(5) times higher concentrations are required for inhibition of chemotaxis by neutrophils which were not electroporated. The results support the hypothesis that Ca2+ from Ca(2+)-storing organelles is of importance for chemotaxis, but that chemotaxis is not dependent on changes in cytoplasmic Ca2+ concentrations.

Cytochalasin B-induced superoxide production in polycation-treated neutrophils.

Cytochalasin B alone induces little superoxide production in intact rabbit peritoneal neutrophils. The cytochalasin causes a strong production of superoxide in cells treated with membrane-permeabilizing polycations. Several polycations were able to express the activating effect of cytochalasin B. Especially the poly-L-arginine with a molecular weight of 24,000 proved to be effective. The effectiveness of some polycations is limited because they inactivate the superoxide-generating oxidase system of the neutrophil. Cytochalasin B-induced superoxide production starts at poly-L-arginine concentrations that cause a change of membrane permeability. At the concentrations of cytochalasin B used in our experiments, the binding of [3H]cytochalasin B is not enhanced in poly-L-arginine-treated cells as compared with control cells. Activation of superoxide production by cytochalasin B in polycation-treated neutrophils occurs both in the presence or absence of extracellular Ca2+. When the cells are pretreated with agents that known to interfere with intracellular Ca2+, the subsequent activation is strongly inhibited, suggesting a role for intracellular Ca2+ in cytochalasin B-induced activation. It is suggested that cytochalasin B alone is not able to activate all the steps that eventually result in complete activation of the superoxide-generating oxidase and that membrane perturbation by polycation provides activation of the remaining steps.

Inhibition of neutrophil migration by guanine nucleotides.

Guanine nucleotides have, besides an activating effect on exocytosis and respiratory burst in permeabilized neutrophils, a modulating effect on some functions in intact neutrophils. We investigated the effect of guanine nucleotides on fMet-Leu-Phe-induced migration of rabbit neutrophils using the Boyden chamber technique. GTP gave a moderate inhibition of fMet-Leu-Phe-induced neutrophil migration. The GTP analogue GTP[S] had a stronger inhibitory effect than GTP. Other nucleotides, such as GDP, GMP, and guanosine were less effective inhibitors than GTP and GTP[S]. Maximal inhibition was achieved at nucleotide concentration of about 40 microM; higher concentration gave only little additional inhibition. The inhibitory effect persisted when the nucleotide was removed after pretreatment of the neutrophil with that nucleotide. Guanine nucleotide induced inhibition was not due to an interference with the fMet-Leu-Phe receptor as casein-induced migration was equally inhibited. We recently found that ATP inhibited neutrophil chemotaxis. The results obtained with guanine nucleotides resembles the inhibitory effects of ATP and its analogues. It is conceivable that guanine nucleotide-induced inhibition of neutrophil migration is mediated by an interaction of these nucleotides with purinergic receptors.

Cytochalasin-induced nitroblue tetrazolium dye (NBT) reduction in polymorphonuclear leukocytes.

Cytochalasins induce little cytochrome c reduction in intact polymorphonuclear leukocytes (PMNs). A strong activation of the respiratory burst is induced by cytochalasin B or by the other cytochalasins, when the dye nitro blue tetrazolium (NBT) is used to measure the burst. Cytochalasin B-induced NBT reduction is dependent on calcium, which is mainly derived from intracellular stores. In the presence of NBT an enhanced binding of [3H]cytochalasin B can be observed. The enhanced reduction of NBT by cytochalasin B-treated cells is completely inhibited by pretreatment of the PMNs with pertussis toxin, suggesting the involvement of a pertussis toxin-sensitive G-protein in cytochalasin-induced activation of the respiratory burst.

Induction of superoxide production in rabbit neutrophils by a stable analogue of GTP.

Treatment of rabbit neutrophils with the stable GTP analogue GTP gamma S results in generation of superoxide. Whereas poly-L-arginine, an agent known to damage the plasma membrane, has only a small effect by itself, a strong and synergistic enhancement of superoxide generation is obtained in the presence of GTP gamma S and poly-L-arginine. The effect is potentiated in the presence of NADPH. Other nucleotides, such as GTP, are not effective in inducing superoxide generation. The results indicate that the stable guanine nucleotide GTP gamma S may activate superoxide production in rabbit neutrophils, and that this effect is evident if the plasma membrane has been permeabilized by poly-L-arginine, allowing an easy entry of GTP gamma S into the cell.

Activity of two types of Fc receptors, Fc gamma RI and Fc gamma RII, in human monocyte cytotoxicity to sensitized erythrocytes.

We investigated the cytotoxicity of human monocytes mediated by two types of receptors for the Fc portion of IgG, Fc gamma RI and Fc gamma RII. Erythrocytes sensitized with human IgG (EA-human IgG) were used to assay Fc gamma RI function, and erythrocytes sensitized with mouse IgG1 (EA-mouse IgG1) were used to assay Fc gamma RII. Both types of Fc gamma R were observed to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), which was further characterized by using different monoclonal anti-Fc gamma R antibodies (MoAb) and monomeric IgG. Lysis of EA-human IgG was inhibited by both monomeric human IgG and mouse IgG2a in a dose-dependent way, and also by anti-Fc gamma RI MoAb 10.1. Cytolysis of EA-mouse IgG1 was inhibited by monomeric mouse IgG1 and by two anti-Fc gamma RII MoAb, IV.3 and CIKM5. Antibodies of the mouse IgG2b isotype affected neither type of ADCC. The effectiveness of cytotoxicity mediated by either of the Fc gamma R was studied by means of targets sensitized with a calibrated number of IgG molecules. At least 20 times more IgG molecules per target cell were necessary to obtain half-maximal cytotoxicity mediated by Fc gamma RII than for Fc gamma RI-mediated cytolysis. Furthermore, the previously described polymorphism of Fc gamma RII was also reflected in Fc gamma RII-dependent cytotoxicity. These studies demonstrate that Fc gamma RII can mediate ADCC, although a higher degree of target cell sensitization is required than for Fc gamma RI-mediated ADCC.