Ethanol increases superoxide anion production stimulated with 4beta-phorbol 12-myristate 13-acetate in human polymorphonuclear leukocytes. Involvement of protein kinase C.

Stimulation of human polymorphonuclear leukocytes (PMNs) with PMA initiates a cascade of events leading to the production and release of superoxide anion (O-2), a major component in anti-bacterial defense. Generation of O-2 by PMA-stimulated PMNs occurs through the translocation and activation of protein kinase C (PKC). In this study, using freshly isolated PMNs, we examined the effect of ethanol on this response to PMA. Our results show that the basal production of O-2 was not affected by ethanol. In contrast, the response induced by PMA was potentiated by ethanol. This potentiation was observed even at high doses of PMA (200 nM) which alone had stimulated the O-2 response maximally. This enhanced response was not due to an increase of PMA uptake by PMNs. The maximal effect was obtained when the cells were preincubated with 80 mM of ethanol before PMA stimulation. Measurement of PKC activity in the cytosolic and membrane fractions showed that pretreatment of PMNs with ethanol increased twofold the PMA-stimulated PKC activity in the membrane fraction. Furthermore, Western blot analysis verified that this increase in PKC activity in the membrane fraction was linked to an increase in the translocation of PKC-alpha and -beta isoforms to the membrane. These results suggest that ethanol potentiates PMA-induced O-2 production through increasing PKC translocation and activity in PMNs.

The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Goralatide) protects from doxorubicin-induced toxicity: improvement in mice survival and protection of bone marrow stem cells and progenitors.

The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP or Goralatide), a physiological regulator of hematopoiesis, inhibits the entry into the S-phase of murine and human hematopoietic stem cells. It has been shown to reduce the damage to specific compartments in the bone marrow resulting from treatment with chemotherapeutic agents, ionizing radiations, hyperthermy, or phototherapy. The present study was performed to assess the therapeutic potential of AcSDKP in vivo in reducing both the toxicity and the hematopoietic damage induced by fractionated administration of doxorubicin (DOX), a widely used anticancer drug. Here we showed that AcSDKP could reduce DOX-induced mortality in mice and could protect particularly the long-term reconstituting cells (LTRCs) in addition to colony forming units-spleen, high proliferative potential colony-forming cells, and colony-forming units-granulocyte-macrophage (CFU-GM) from DOX toxicity. The protection against DOX-induced mortality in mice was improved when AcSDKP was administered for 3 days, at a dose of 2.4 micrograms/d, by continuous subcutaneous (SC) infusion or fractionated s.c. injections starting 48 hours before DOX treatment. Moreover, the recovery of the CFU-GM population in the AcSDKP-DOX-treated mice was optimized by the subsequent administration of granulocyte colony-stimulating factor (G-CSF). The coadministration of AcSDKP with DOX may improve its therapeutic index by reducing both acute hematotoxicity on late stem cells and progenitors and long-term toxicity on LTRCs. Optimization of these treatments combined with G-CSF may provide an additional approach to facilitate hematopoietic recovery after cancer chemotherapy.

Phosphatase activity regulates superoxide anion generation and intracellular signaling in human neutrophils.

Phosphorylation of components of the neutrophil NADPH oxidase plays a critical role in activation and maintenance of superoxide anion (O2-) generation. To investigate the role of dephosphorylation by phosphatases in regulating O2- production, human neutrophils were treated with calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, prior to stimulation. Calyculin A alone did not stimulate O2- production. However, neutrophils exposed to 50 nM calyculin A and the chemotactic peptide formyl-met-leu-phe (FMLP, 100 nM) displayed markedly enhanced O2- production in comparison to cells stimulated with FMLP alone (28.63 +/- 7.00 versus 8.69 +/- 3.69 nmol O2-/1.5 x 10(6) neutrophils/5 min, respectively, n = 18, p < 0.001), with an increased duration of O2- production. In contrast, phosphatase-inhibition decreased oxidative responsiveness to phorbol myristate acetate (PMA, > or = 16 nM). We next examined the effect of calyculin A on products of the phosphatidylcholine-specific phospholipase D (PLD) pathway by assaying the mass levels of phosphatidic acid (PA), choline and diacylglycerol (DAG). Calyculin A increased both PA and choline production to 224 +/- 28% and 315 +/- 61% of FMLP-stimulated controls, respectively (p < 0.01, n = 7) without significantly increasing DAG. Also, membrane protein kinase C activity increased more than 10-fold in FMLP-stimulated cells exposed to calyculin A but decreased in cells stimulated with PMA following calyculin A pre-treatment. These results suggest that phosphatases exert variable and stimulus-dependent effects on pathways leading to O2- production. Further, it appears that phospholipase D activity and PA generation represent important steps in the pathway for NADPH activation triggered by FMLP.

Role of calcium in the activation of mouse peritoneal macrophages: induction of NO synthase by calcium ionophores and thapsigargin.

Bacterial lipopolysaccharide (LPS) has been recognized as one of the most potent activating signals for mouse peritoneal macrophages. In macrophages primed by interferon-gamma (IFN-gamma) or trehalose dimycolate (TDM), LPS induces NO synthase and the events associated with a high nitric oxide output: antitumor and antiparasitic activities. In the present report, it is shown that drugs (calcium ionophores or thapsigargin) which elevate the concentration of cytosolic calcium, [Ca2+]i, induce NO synthase and antitumor activities in primed macrophages, mimicking LPS action. Calcium ionophores and thapsigargin trigger NO synthase activity in macrophages primed in vivo by TDM, in thioglycollate-elicited macrophages primed in vitro by IFN-gamma, and in IFN-gamma-treated EMT6 adenocarcinoma cells. However, activation of TDM-primed macrophages by LPS does not seem to involve calcium fluxes: (i) no change in [Ca2+]i was detectable in TDM-primed macrophages loaded with Fura-2 and exposed to LPS, and (ii) activation of TDM-primed macrophages by LPS can be obtained in the presence of 4 mM EGTA. NO synthase expression is thus controlled in primed macrophages by two different pathways; calcium ionophores can replace LPS but do not act through the same intracellular cascade.

LPS-induced activation of primed murine peritoneal macrophages is modulated by prostaglandins and cyclic nucleotides.

Murine peritoneal macrophages primed in vivo by trehalose dimycolate (TDM) express cytostatic activity against tumor cells after treatment in vitro with 10 ng/ml lipopolysaccharide (LPS) during a 4-hr period (activation step). There is a strict correlation (P < 0.0001) between acquisition of antitumoral activity and induction of NO synthase quantified by its end products citrulline and NO2-. LPS also stimulates the release of cyclooxygenase products which exert a retroinhibitory action on NO synthase and cytostatic activities, as judged by an increase of both parameters by indomethacin (1 microM) and a decrease by externally added PGE2 (1 microM). LPS increases cellular and extracellular cAMP levels through an indomethacin-sensitive pathway, pointing to cAMP as a second messenger in the retroinhibitory action of LPS-induced prostaglandins. In fact, the addition of 8-bromo-cAMP or of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine during the activation step decreases NO synthase activity; however, at the same time these drugs increase the apparent efficiency of NO as an antitumor agent.

Arachidonate metabolism triggered in primed macrophages by signals inducing antitumor activity.

The acquisition of antitumoral functions by mouse peritoneal macrophages is controlled by the addition of activating agents (lipopolysaccharide (LPS), muramyldipeptide (MDP) or A23187), on appropriately primed macrophages. The release of eicosanoids during this activation step was examined by radio-HPLC. We demonstrated that the induction of antitumor activity in primed macrophages by LPS or MDP was associated with the release of 20:4 derivatives; arachidonic acid was metabolized predominantly via the cyclooxygenase pathway to PGE2 and thromboxane. The production of PGE2, quantified by an enzyme immunoassay, was sustained and important (up to 20 ng/ml/h/10(6) macrophages). However, PGE2 and thromboxane did not seem essential to the activation process: induction of antitumor activity took place and was even enhanced in the presence of indomethacin, whereas it was decreased by exogenous PGE2. During culture in vitro, primed macrophages released spontaneously significant amounts of 20:4 metabolites and became unresponsive to activation stimuli. Again indomethacin had a positive effect: it protected primed macrophages against this loss of activability. Cyclooxygenase metabolites released in response to activating stimuli or spontaneously seem to trigger deactivation pathways.

MDP and LPS act synergistically to induce arginine-dependent cytostatic activity in rat alveolar macrophages.

Rat alveolar macrophages can be activated in vitro for cytostatic activity against tumor cells by MDP and LPS acting in synergy. MDP can be substituted for by analogs active as adjuvants. Macrophage activation correlates with an increased production of nitrite and citrulline. NG-monomethyl-L-arginine, a specific inhibitor of the L-arginine metabolism having nitrite and citrulline as end products, abolishes the cytostatic activity. We therefore conclude that, in this model, the main effector mechanism of the cytostatic activity is mediated by molecules derived from L-arginine through the newly described NOo-generating pathway.