Nutrients and gene expression.
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Animals↗
Biomedical subjects
A D Procopio
Publications and source records attributed to A D Procopio.
Endothelial cell transfections: a way for studying promoter activity of genes involved in atherogenesis.
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Arteriosclerosis↗
GTP-binding proteins transduce signals generated via human FC gamma receptor IIIA (CD16).
This study demonstrates that GTP-binding proteins regulate Fc gamma RIII-mediated signal transduction and inositol phosphate (IPn) generation in human NK cells. In addition the cross-linking of CD16 by mAb, guanosine 5'-o-3-thiophosphate induced 1,4,5 inositol trisphosphate (IP3) release in permeabilized NK cells and their membranes. By contrast, guanosine 5'-o-2-thiophosphate, almost completely inhibited IP3 generation induced by cross-linking with anti-CD16 mAb. Pretreatment of NK cells with 10 to 100 ng/ml Vibrio cholerae toxin (Ctx) almost completely inhibited the generation of IP3 and of other Ipn as well as Fc gamma RIII-operated cell functions such as antibody-dependent cell-mediated cytotoxicity against antibody-coated P815 mastocytoma cells. Isolated B subunit of Ctx was inactive. Bordetella pertussis toxin (0.1 to 1 microgram/ml) only marginally affected IP3 release and antibody-dependent cell-mediated cytotoxicity. Ctx increased cAMP levels in NK cells. However, inhibition of IP3 release preceded the rise of cAMP. Moreover, cAMP analogues (8-chlor-cAMP, 8-bromo-cAMP, dibutiryl-cAMP), as well as intracellular cAMP-enhancing agents (PGE1, PGE2, and forskolin) did not mimicked the effects of Ctx on IP3 generation, suggesting that the adenylate cyclase pathway is not responsible for the early effects of Ctx on Fc gamma RIII-mediated signalling. Overall these results demonstrate that signal transduction via Fc gamma RIII is mediated by Ctx-sensitive cellular membrane GTP-binding protein.
Adenylate Cyclase Toxin↗
Effects of protein kinase C (PK-C) activators and inhibitors on human large granular lymphocytes (LGL): role of PK-C on natural killer (NK) activity.
The role of protein kinase C (PK-C) in the early metabolic events involved in human natural killer (NK) cell activation has been studied through the action of PK-C-specific activators and inhibitors. Highly purified human large granular lymphocytes (LGL) were treated for 1 hr with the diacylglycerol analog 1-oleoyl-2-acetyl glycerol (OAG) (10(-4)-10(-5) g/ml) or with 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-8)-10(-10) g/ml), both specific activators of PK-C. Both these agents consistently increased NK activity against K562 target cells. Suboptimal doses of either OAG or TPA also synergized with Ca2+ ionophores to augment spontaneous cytotoxic activity. Pretreatment of LGL with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrocloride (H7) (5-40 microM), a potent PK-C inhibitor, greatly reduced NK activity in a time- and dose-dependent fashion. By contrast, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA 1004), a potent cAMP- and cGMP-dependent PK inhibitor with almost no effect on PK-C, marginally reduced NK activity. Moreover, almost complete NK activity inhibition was observed when H7 (10 microM), but not HA 1004 (50 microM), was present in the NK assay. Finally, 48 hr stimulation of LGL with TPA (10(-6) g/ml), a treatment able to inactivate most of the PK-C cellular pool, almost completely abrogated NK activity. This functional evidence was supported by phosphorylation of several endogenous substrates which occurs within 5 min in TPA-treated LGL. Two proteins of 70 and 56 kDa have been identified as major PK-C substrates, together with other phosphorylated proteins with MW ranging from 177 to 43 kDa. H7, but not HA 1004, almost completely inhibited the TPA-induced phosphorylation of all of these proteins in the NK cells. These data strongly suggest that selective activation of PK-C plays an essential role in the mechanisms of NK cell activation.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Comparative evaluation of multiple lymphoid and recombinant human interleukin-2 preparations.
Six lymphoid human interleukin-2s (nIL-2s) [four from peripheral blood mononuclear cells (PBMC) and two from JURKAT cells] and six recombinant IL-2s (rIL-2s) were obtained for comparative evaluation. The main issues addressed were possible differences among the preparations in potency in T cell growth assays and other functional assays, and the possible presence of other cytokine activities or contaminants. Each preparation was assigned a standardized IL-2 activity in reference units (RU) by comparing its T cell growth promoting activity against the Biological Response Modifiers Program IL-2 (JURKAT) reference reagent. Relative to the IL-2 unitage indicated by the suppliers, the RU varied from 110-fold less to 38.5-fold more for the various preparations. Two nIL-2s and two rIL-2s contained significant levels of endotoxin. One nIL-2 contained low levels of both alpha and gamma interferon (IFN), and one nIL-2 had a high level of gamma IFN. All other IL-2s were negative for IFN activity. All IL-2 preparations significantly augmented human natural killer (NK) activity, although the amount of RU required varied from 0.1 to 50 RU. Four nIL-2s and three rIL-2s induced human PBMC to produce gamma IFN, whereas two nIL-2s and one rIL2 did not. All nIL-2s had substantial amounts of B cell growth factor activity, whereas none of the rIL-2s consistently displayed this activity. All IL-2s stimulated the tritiated thymidine [3H]TdR incorporation of human PBMC in the absence of other stimuli, in addition to augmenting the response to mitogen or alloantigens. Some nILs and IL-2s had effects on human monocytes such as inhibiting migration, inducing cytotoxic or growth inhibitory activity against tumor cells, and causing changes in cell surface markers. The IL-2s were also tested for activity in vitro and in vivo in mice. Although there was a 12-fold variation in activity among the preparations, all but one of the IL-2s showed augmentation of the mixed lymphocyte reaction activity and all IL-2s tested stimulated macrophage cytotoxicity in vitro. All IL-2s tested enhanced the mixed lymphocyte-allogeneic tumor cell reaction resulting in greater production of cytotoxic T cells. However, significant quantitative differences in potency were evident among the various IL-2 preparations, especially the nIL-2s. Only very high doses of IL-2 (intraperitoneal injection of 100,000 RU/animal) induced in vivo augmentation of splenic or peritoneal NK cells, although all IL-2s tested increased NK activity against tumor target cells in vitro with substantially lower doses (10-100 RU/ml).(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Production of multiple cytokines by clones of human large granular lymphocytes.
LGL in addition to mediating natural killer (NK) activity, can secrete a variety of lymphokines, depending on the stimulus used: interleukin-1 (IL-1), interleukin-2 (IL-2), interferon alpha and gamma (IFN), and B-cell growth factor (BCGF). To define more directly whether cells with NK activity can also secrete one or more cytokines, we obtained clones by limiting dilution assays from highly purified preparations of human LGL and cultured them in IL-2-containing medium for several weeks. All the clones tested spontaneously produced detectable levels of IFN-gamma and 35 of 40 clones (87%) produced higher levels when stimulated with PHA. A smaller proportion (16%) of clones (9 of 54) secreted IL-1 after stimulation with LPS, while 34% of the clones (17 of 49) produced IL-2 in response to PHA stimulation. Cytokine production was associated with both cytotoxic and noncytotoxic clones and did not correlate with their surface phenotype, as has been observed for fresh LGL. The ability to produce IL-1 or IL-2 was not usually found within the same clones following PHA and LPS stimulation, respectively; however two clones produced both IL-1 and IL-2 when stimulated in different experiments, but not at the same time. In addition, two of nine cloned LGL simultaneously produced IFN gamma and IL-1. These results indicate that LGL-derived clones have the ability to produce multiple cytokines, suggesting that the LGL population may play an important immunoregulatory role and may also be capable of self-regulation of cytolytic activity.
Antigens, Surface↗
Noncytotoxic functions of natural killer (NK) cells: large granular lymphocytes (LGL) produce a B cell growth factor (BCGF).
Highly purified human large granular (LGL), depleted of any detectable contaminant T and B cells or monocytes, were found to be potent producers in vitro of a soluble B cell growth factor (BCGF) able to sustain proliferation of B cells activated by anti-mu. Activation by lectins (phytohemagglutinin, PHA, concanavalin A, Con A; and pokeweed mitogen, PWM) was required to induce the production of high levels of this BCGF from cultured LGL. Production of BCGF was also detected after the binding of LGL with natural killer (NK)-sensitive (K562) but not with NK-resistant (RL male 1) target cells. In contrast to T cells, LGL did not need the additional presence of accessory cells to reach optimal production of BCGF by 72 hr of culture. The subpopulation of LGL responsible for the production of BCGF had phenotypic characteristics associated with NK cells (3G8+, HNK1+/OKT11+, DR-, OKT3-, Leu-M1-), and separated cells with these markers exerted high levels of NK activity. Selective production of BCGF also was obtained from cytotoxic clones derived from LGL. A partial characterization of the LGL-derived BCGF was performed by gel filtration. BCGF activity was detected in fractions with estimated m.w. of 20,000 and 45,000. The LGL-derived BCGF activity was resistant to reduction with 2-mercaptoethanol and was stable at -20 degrees C for months. Conversely, heating (56 degrees C for 1 hr) or digestion with trypsin greatly reduced the LGL-derived BCGF activity. These findings strongly suggest that LGL including those with NK activity can play an important positive role in the early events of the B cell-mediated immune response.
Adult↗