Differential effects of propranolol on responses to receptor-dependent and receptor-independent stimuli in human neutrophils.

Recent evidence suggests that the hydrolysis of phosphatidylcholine (PC) by phospholipase D (PLD) may mediate superoxide anion (O2-) production in human neutrophils. To define the role of the PC-specific PLD products phosphatidic acid (PA) and diacylglycerol (DAG) in O2- production in response to agonists which activate the PLD pathway, we blocked the metabolism of PA to DAG with propranolol, an inhibitor of PA phosphohydrolase. Propranolol (150 microM) enhanced the production of O2- in response to the receptor agonists n-formyl-methionyl-leucyl-phenylalanine (FMLP, 292 +/- 94% of controls), platelet-activating factor (PAF, 932 +/- 215%) and leukotriene B4 (LTB4, 1305 +/- 475%). In the presence of propranolol, total O2- production in response to PAF and LTB4, which are potent priming stimuli but very weak direct agonists, was similar to that obtained with FMLP. IN contrast, responses to receptor-independent agonists phorbol myristate acetate (PMA) and ionomycin were inhibited (81 +/- 8% and 87 +/- 5% inhibition, respectively). The effects of propranolol were demonstrable in the absence of cellular calcium and were shared by both stereoisomers of the drug. These data are consistent with the hypothesis that PA produced through the hydrolysis of PC by PLD is an important mediator of O2- production in response to receptor-dependent agonists. However, the inhibitory effects of propranolol on receptor-independent stimuli suggest that PA generated through the PLD pathway plays a different role in the signal transduction mechanisms of these agonists or that propranolol may have additional effects beyond inhibition of PA phosphohydrolase.

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.

High-dose cytosine arabinoside and etoposide: an effective regimen without anthracyclines for refractory childhood acute non-lymphocytic leukemia.

The purpose of this report is to describe the tolerability and activity of the combination of high-dose cytosine arabinoside (Ara-C) given at the maximum tolerated dose of 36 g/m2, together with high doses of etoposide in relapsed and refractory childhood acute leukemias. Eighteen children with relapsed or refractory acute leukemia were treated with Ara-C 3 g/m2 every 12 h on days 1-6, followed by etoposide 400 mg/m2 on days 7-9 (HDAC/VP-16). Eight children with refractory disease received HDAC/VP-16 as salvage induction therapy after failing conventional induction regimens; four of five refractory ANLL patients (80%) had a complete response (CR) after HDAC/VP-16 therapy. Ten patients received HDAC/VP-16 as post-remission intensification therapy; five patients (four ANLL, one relapsed ALL) remain in second CR at 56, 26, 9, 5 and 2 months. Toxicities were primarily hematologic and dermatologic. Seven patients (39%) developed bacterial or fungal infections; four patients developed grade 3 or 4 acral erythema. No patient died of therapy-related toxicity. The combination of 36 g/m2 cytosine arabinoside and 1200 mg/m2 etoposide is an effective regimen for children with relapsed or refractory acute nonlymphocytic leukemia, with tolerable toxicities; the absence of anthracyclines makes this regimen suitable for patients who have previously received maximal doses of anthracyclines or who have evidence of cardiac dysfunction. Further evaluation of this regimen in acute nonlymphocytic leukemia is presently being investigated.

Chemotactic peptide enhancement of phorbol ester-induced protein kinase C activity in human neutrophils.

Chemotactic factors and phorbol esters may act synergistically to evoke neutrophil responses, but the mechanism of such interaction is not entirely clear. We investigated the combined effects of the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA) on protein kinase C (PKC) activity in human neutrophils. FMLP had little effect on the sharp decrease in cytosolic PKC activity induced by PMA. However, cells exposed to FMLP and PMA exhibited a synergistic increase in particulate PKC activity (1 +/- 1 pmol 32P/10(7) PMNs/min in unstimulated cells, 53 +/- 12 pmol 32P with 20 ng/ml PMA, 6 +/- 3 pmol 32P with 10(-7) M FMLP, and 191 +/- 17 pmol 32P with FMLP and PMA). FMLP also markedly increased calcium/phospholipid-independent protein kinase activity in particulate fractions of control and PMA-treated cells. Enhancement of PKC activity required the presence of cytochalasin B during cell stimulation. Cellular calcium was crucial to the FMLP effect since enhancement was decreased in cells incubated with EGTA or Quin2. These results suggest that chemotactic factors and phorbol esters may mediate synergistic effects on neutrophil responses through enhancement of particulate PKC activity. The enhancing effect is probably mediated through chemoattractant-mediated increases in intracellular calcium.

The behavioural and neurochemical profile of the putative dopamine D3 receptor agonist, (+)-PD 128907, in the rat.

The functional relevance of the dopamine D3 receptor is still unresolved, largely because of the absence of selective D3 receptor ligands. In the present study we have examined the in vivo profile of (+)-PD 128907, a potent and functionally selective D3 receptor agonist. Low doses of (+)-PD 128907 reduced spontaneous locomotor activity in the rat (ED50 = 13 +/- 3 micrograms/kg, s.c.) a response which was comparable with the non-selective D2,3 receptor agonist apomorphine (ED50 = 13 +/- 1.6 micrograms/kg, s.c.). In addition (+)-PD 128907 impaired prepulse inhibition of the acoustic startle response, with significant effects observed at doses of 30 micrograms/kg when appropriate prepulse intensities were used. Higher doses reversed gamma-butyrolactone-induced catecholamine synthesis (ED50 = 95 +/- 22 and 207 +/- 37 micrograms/kg in accumbens and striatum respectively) and induced yawning (100-300 micrograms/kg), penile grooming (30-1000 micrograms/kg) and sniffing (> or = 300 micrograms/kg) although doses 3- to 10-fold greater than apomorphine were required to produce maximal effects. In contrast to apomorphine, however, (+)-PD 128907 failed to induce intense stereotyped licking and biting in the rat. In view of the potency and selectivity of (+)-PD 128907 for the D3 receptor, a role in the control of locomotor activity is suggested. In addition, the observation that (+)-PD 128907 disrupts prepulse inhibition, a phenomenon which is also impaired in schizophrenic subjects, may indicate the pathological importance of this receptor subtype.

Neutrophil Priming Mechanisms of Sulfolipid-I and N-Formyl-Methionyl-Leucyl-Phenylalanine.

The principal sulfatide of virulent Mycobacterium tuberculosis, sulfolipid-I (SL-I), both directly stimulates neutrophil superoxide (O(-)(2)) release and, at substimulatory concentrations, primes these cells for markedly enhanced oxidative responsiveness to other stimuli. The present study was undertaken to clarify the priming mechanisms by comparing cellular events following priming doses of SL-I with those following priming with N-formyl-methionyl-leucylphenylalanine (FMLP). We compared the involvement of the calcium cation (Ca(2+)), as well as membrane protein kinase C (PKC) activity and the translocation of NADPH oxidase-cytosolic cofactor effected by priming levels of the two agonists. The investigation led to two important conclusions. First, we clearly demonstrate that priming by both SL-I and FMLP results from activation of cellular processes that are not involved in direct oxidative activation. For example, whereas direct induction of O(-)(2) generation by FMLP and SL-I required increases in intracellular Ca(2+), an increase in intracellular calcium concentration ([Ca(2+)](i)) above basal levels was not required for priming. Second, we identified key differences in the cellular responses to priming doses of SL-I and FMLP. Whereas increased membrane PKC activity caused by priming doses of FMLP was only partially blocked by chelation of intracellular Ca(2+), Ca(2+) chelation completely inhibited the increase in membrane PKC activity caused by SL-I. NADPH oxidase-cytosolic factor translocation to plasma membranes was completely blocked by pertussis toxin when priming doses of SL-I were used. This guanine-nucleotide-binding protein inhibitor had no effect on FMLP-dependent translocation of the oxidase cofactors. The comparative approach introduced in this report provides a valuable and novel method to discern the complex interactions of various cellular processes that regulate the state of activation of stimulated cells. Copyright 1994 S. Karger AG, Basel

Differential inhibition of neutrophil superoxide generation by nonsteroidal antiinflammatory drugs.

In order to further characterize the effects of nonsteroidal antiinflammatory drugs on neutrophil superoxide (O2-) generation, human neutrophils were incubated in the presence of sulfinpyrazone, phenylbutazone, and indomethacin prior to exposure to a variety of oxidative stimuli. Stimuli used included the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 5.0 X 10(-7) M), NaF (20 mM), phorbol myristate acetate (PMA, 3.2 X 10(-7) M), and opsonized zymosan (250 micrograms/ml). Superoxide release induced by FMLP was inhibited by all three drugs with half-maximal inhibition (KI50) at 2.5, 30, and 120 microM for sulfinpyrazone, phenylbutazone, and indomethacin, respectively. This inhibition was not due to drug interference with the assay system since comparable inhibition was not observed in a cell-free O2- -generating system. The neutrophil's response to NaF was blunted by sulfinpyrazone (KI50 = 400 microM) and phenylbutazone (KI50 = 65 microM), but was unaffected by indomethacin. A similar inhibitory pattern was observed when zymosan was used as the oxidative stimulus. Sulfinpyrazone and phenylbutazone inhibited the response to zymosan (KI50s of 425 and 32 microM, respectively), whereas indomethacin augmented it. PMA stimulation evoked O2- production which was inhibited by phenylbutazone (KI50 = 350 microM) but not by sulfinpyrazone or indomethacin in concentrations up to 1 mM. The results support the hypothesis that the enzyme system responsible for neutrophil O2- generation can be activated by more than one mechanism. The results also emphasize the need to evaluate pharmacologic modulation of neutrophil responses in light of the stimulus used to evoke the response.

Stimulation by lipoxygenase products of superoxide anion production in FMLP-treated neutrophils.

Our recent observation that leukotriene B4 (10(-9)M) is a potent enhancer of FMLP-initiated neutrophil superoxide anion formation prompted an evaluation of the ability of other lipoxygenase products and related compounds to modulate this response. The results indicate that FMLP-evoked O-2 may be enhanced by 10(-8)-10(-7)M levels of a number of lipids, in addition to LTB4, including 5-HPETE, 5-HETE, 5,15-DiHPETE and by higher levels of other 15-series lipoxygenase products and arachidonic acid. It is of interest that the relative potency of these agents in potentiating the superoxide response to FMLP approximately parallels their reported ability to induce chemotactic activity in leukocytes.

Differential effects of lipoxygenase products on FMLP and LTB4 evoked neutrophil aggregation.

There is evidence that the endogenous biosynthesis of LTB4 is involved in the aggregation of human neutrophils induced by the chemotactic peptide f-met-leu-phe (FMLP). If LTB4 mediates this aggregatory response, then agents which desensitize neutrophils to LTB4 should inhibit the cellular response to FMLP. Since many lipoxygenase products modulate other neutrophil responses to LTB4 and FMLP, we have investigated the effects of lipoxygenase products on LTB4- and FMLP-initiated aggregation. Prior exposure to low concentrations of LTB4 (0.5-10 nM) inhibited subsequent aggregation to the same agent (50 nM), but it did not influence the response to FMLP (10(-7) M). Relatively high concentrations of 5-HETE (5-50 microM) inhibited aggregation initiated by either stimulus. Although the hydroperoxy derivative 5-HPETE also inhibited the response to LTB4, in the relatively narrow concentration range of 1-4 microM it stimulated FMLP-induced aggregation. This latter effect was confirmed using 12 cell preparations from six separate donors; it (the activity of 5-HPETE) was not mimicked by other 5-lipoxygenase products, including LTB4, nor the dihydroperoxide 8,15-DiHPETE. Our results indicate that neutrophil aggregation in response to LTB4 or FMLP can be selectively potentiated or inhibited. On the basis of these data we conclude that the endogenous synthesis of LTB4 is not directly involved in the neutrophil aggregatory response to FMLP, although the hydroperoxy intermediate 5-HPETE may act to enhance the cellular response.

Profiling the health service needs of populations using diagnosis-based classification systems.

Managed care presents the challenge to deliver health care services to populations from a fixed pool of dollars and be accountable for doing so. From a health plan and a community perspective, it is critical to have classification systems that adequately describe the health service needs of the population. This article describes the design and structure of the National Association of Children's Hospitals and Related Institutions Classification of Congenital and Chronic Health Conditions and illustrates its management applications from its first testing on a full-service, paid claims database. Capabilities and limitations of diagnosis-based classification systems and the International Classification of Diseases, Ninth Edition, Clinical Modification diagnosis codes that underpin such systems are discussed.

Mechanism and regulation of neutrophil priming by platelet-activating factor.

Although a weak direct stimulus of superoxide anion (O2-) production, platelet-activating factor (PAF) markedly enhances responses to chemotactic peptides (such as n-formyl-met-leu-phe, FMLP) and phorbol esters (such as phorbol myristate acetate, PMA) in human neutrophils. The mechanism of priming was explored first through inhibition of steps in the signal transduction pathway at and following PAF receptor occupation. Priming was not altered by pertussis toxin or intracellular calcium chelation, but the PAF receptor antagonist WEB 2086 and the protein kinase C (PKC) inhibitors sphinganine and staurosporine significantly inhibited the primed response. In order to study the regulation of PAF priming, the effect of PAF alone was desensitized by exposure to escalating doses of PAF prior to exposure to the secondary stimuli. The priming effect of PAF was not desensitized under these conditions. The role of PKC in desensitization was also studied. Prior exposure to PAF also desensitized the increase in membrane PKC activity evoked by a single concentration of PAF. However, when the PAF response was desensitized, PKC priming of the response to FMLP or PMA still occurred, suggesting that PKC activity may play a role in the maintenance of the primed state despite PAF desensitization. These data suggest that: (1) PAF priming is receptor- and PKC-mediated but is independent of pertussis toxin-inhibitable G-proteins or intracellular calcium, (2) during migration in vivo, neutrophils may be desensitized to the direct effects of PAF but maintain the capacity for enhanced responses to other stimuli, (3) desensitization of PAF-induced particulate PKC activity also occurs, but PAF primes PKC activity despite PAF desensitization, and (4) distinct mechanisms govern the direct and priming effects of PAF on oxidative metabolism.

Priming of neutrophil oxidative responses by platelet-activating factor.

Platelet-activating factor is a potent proinflammatory lipid mediator which directly stimulates neutrophil chemotaxis, degranulation, aggregation, and superoxide radical (O2-) production. PAF also modifies or 'primes' neutrophil responses to other agents. Although a relatively weak direct oxidative agonist, PAF markedly enhances O2- release evoked by phorbol myristate acetate (PMA) and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), increasing the maximal rate of O2- production by a calcium-dependent mechanism. PAF also increases protein kinase activity in the membrane fraction of neutrophils. In search of a mechanism for oxidative priming by PAF, we investigated the role of PAF in modifying PMA-induced activation/translocation of protein kinase C (PKC) in human neutrophils. In the presence of PAF and PMA both PKC and calcium-, phospholipid-independent protein kinase activities in particulate fractions increase markedly over activities detected in the presence of PMA alone. The increase in particulate protein kinase activities requires the presence of cytochalasin B and is calcium-dependent. The PKC-enhancing effect of PAF may be involved in the mechanism whereby the phospholipid 'primes' neutrophils for augmented oxidative responses to some stimuli but the exact role of PKC in neutrophil oxidative metabolism remains to be defined.

Substrate dependence of human neutrophil protein kinase C.

To determine the contribution of phosphate acceptor substrate to the pattern of activity of calcium-dependent, phospholipid-sensitive protein kinase (protein kinase C, PKC), we assayed cytosolic and particulate PKC activity for histone, troponin, myosin light chain (MLC), and endogenous cellular proteins in human neutrophils stimulated with phorbol myristate acetate (PMA), the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) and synergistic stimulation with both agonists. In general, phosphotransferase activity in neutrophil subfractions toward troponin and endogenous proteins paralleled that toward histone, but MLC was a poor substrate for PKC and the pattern of phosphotransferase activity differed from that seen with the other substrates. Furthermore, the phosphorylation of endogenous neutrophil cytosolic proteins increased significantly after stimulation with FMLP, suggesting an endogenous cytosolic substrate(s) which increased in concentration following stimulation. We conclude that histone is a useful phosphate acceptor for study of PKC activity in human neutrophils, but substrate variability occurs and may influence interpretation of results in assays of PKC activity.

Platelet-activating factor induces protein kinase activity in the particulate fraction of human neutrophils.

Platelet-activating factor (PAF) is a proinflammatory lipid that has both platelet- and phagocyte-stimulating properties. Because several known activators of calcium-, phospholipid-dependent protein kinase (protein kinase c, PKC) also stimulate neutrophil responses and because neutrophil stimuli such as phorbol diesters and the chemotactic peptide f-Met-Leu-Phe are reported to increase protein kinase activity in neutrophil (PMN) particulate fractions, we investigated the effect of PAF on neutrophil protein kinase activities. In neutrophils exposed to 10(-6) mol/L PAF, cytosolic PKC activity was 521 +/- 38 pmol 32P/10(7) PMN/min (mean +/- SEM), which was not significantly lower than cystolic activity in buffer-treated controls (558 +/- 32 pmol 32P/10(7) PMN/min, n = 14). PAF-exposed cells exhibited a concomitant rise in protein kinase activity associated with the particulate fraction with 53 +/- 4 pmol 32P/10(7) PMN/min compared with 32 +/- 2 pmol in control cells (n = 14). Particulate protein kinase activity was independent of the presence of calcium and phospholipid in the assay medium. The specific PKC inhibitor H-7 inhibited particulate protein kinase activity, however, which suggested that the enzyme activity assayed in this fraction may be PKC in a constitutively activated form. The increase in particulate protein kinase activity induced by PAF required the presence of cytochalasin B, was detectable within 5 seconds of exposure to PAF, and was not reversed by washing the cells free of extracellular PAF after initial exposure. Although PAF did not have a direct effect on PKC activity from cytosolic fractions from resting cells, the increase in particulate protein kinase activity induced by PAF was inhibited when the cells were first depleted of calcium by incubation with Quin 2. These results suggest that PAF induces an increase in particulate protein kinase activity in neutrophils by a calcium-dependent mechanism and that the induction of membrane-associated protein kinase activity may be involved in neutrophil-stimulating actions such as superoxide production, which occur at higher concentrations of PAF.

Enhancement of phorbol ester-induced protein kinase activity in human neutrophils by platelet-activating factor.

We have shown that platelet-activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol-to-membrane shift of calcium-activated, phospholipid-dependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA-induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of 32P from gamma-32P-ATP into histone H1 induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 +/- 9 pmoles 32P/10(7)PMN/minute in cells treated with 20 ng/ml PMA compared to 267 +/- 24 pmoles 32P in cells exposed to PMA and 10(-6)M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentrations of 10(-7) M and above. Both PKC and calcium-, phospholipid-independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent-extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA-treated neutrophils. However, the PKC-enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA-induced particulate PKC activity by a calcium-dependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid "primes" neutrophils for augmented oxidative responses to PMA.

Systemic metastases in primary intracranial germinoma. Case report and literature review.

Systemic metastases from central nervous system germinomas are exceedingly rare, and when they occur lead to fatal outcomes. The authors report the case of a 10-year-old girl who presented with metastatic involvement of the rib and pelvis 2.5 years after surgical resection and radiation therapy for a suprasellar dysgerminoma. After combination chemotherapy, the patient remains disease-free 30 months after relapse. This case provides evidence that chemotherapy can be an effective therapeutic alternative to the use of radiation in the treatment of children with extracranial germinomas.

Stimulation of neutrophil oxidative metabolism by indomethacin.

Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O2-) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to soluble oxidative stimuli. To further investigate this phenomenon, O2- generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O2- release was increased from 150 to 300% of controls in neutrophils exposed to 400 micrograms/ml indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 micrograms/ml. Neutrophils exposed to methacin alone also generated more O2- than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacin-treated cells were exposed to zymosan. Neutrophil chemiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 micrograms/ml (1.1 mM) enhancement ranged from 253 to 333% of controls. As was observed with O2- generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug-treated cells were stimulated with zymosan. Phagocytosis of radiolabeled S. aureus by neutrophils incubated with indomethacin was increased 13 +/- 5% over controls (P less than 0.01, n = 5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)