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Pulmonary surfactant's complex mixture of phospholipids and proteins reduces the work of breathing by lowering alveolar surface tension during respiration. One mechanism of surfactant damage appears to be the hydrolysis of phospholipid by phospholipases activated in the inflamed lung. Humans have several candidate secretory phospholipase A2(sPLA2) enzymes in lung cells and infiltrating leukocytes that could damage extracellular surfactant. We considered two mechanisms of surfactant disruption by five human sPLA2s, including generation of lysophospholipids and the depletion of specific phospholipids. All five sPLA2s studied ultimately caused surfactant dysfunction. Each enzyme exhibited a different pattern of hydrolysis of surfactant phospholipids. Phosphatidylcholine, the major phospholipid in surfactant and the greatest potential source for generation of lysophospholipids, was susceptible to hydrolysis by group IB, group V, and group X sPLA2s, but not group IIA or IID. Group IIA hydrolyzed both phosphatidylethanolamine and phosphatidylglycerol, whereas group IID was active against only phosphatidylglycerol. Thus, with groups IB and X, the generation of lysophospholipids corresponded with surfactant dysfunction. However, hydrolysis of and depletion of phosphatidylglycerol had a greater correlation with surfactant dysfunction for groups IIA and IID. Surfactant dysfunction caused by group V sPLA2is less clear and may be the combined result of both mechanisms.

In asthma, inflammation-mediated surfactant dysfunction contributes to increased airway resistance, but the mechanisms for dysfunction are not understood. To test mechanisms that alter surfactant function, atopic asthmatics underwent endobronchial antigen challenge and bronchoalveolar lavage (BAL). BAL fluids were sequentially separated into cells, surfactant, and supernatant, and multiple end points were analyzed. Each end point's unique relationship to surfactant dysfunction was determined. Our results demonstrate that minimum surface tension (γmin) of surfactant after antigen challenge was significantly increased with a spectrum of responses that included dysfunction in 6 of 13 asthmatics. Antigen challenge significantly altered the partitioning of surfactant phospholipid measured as a decreased ratio of large surfactant aggregates (LA) to small surfactant aggregates (SA), LA/SA ratio. Phosphatidylglycerol (PG) was significantly reduced in the LA of the dysfunctional asthmatic BALs. There was a corresponding significant increase in the ratio of phosphatidylcholine to PG, which strongly correlated with both increased γminand decreased LA/SA. Altered surfactant phospholipid properties correlated with surfactant dysfunction as well or better than either increased eosinophils or protein. Secretory phospholipase activity, measured in vitro, increased after antigen challenge and may explain the decrease in surfactant PG. In summary, alteration of phospholipids, particularly depletion of PG, in the LA of surfactant may be an important mechanism in asthma-associated surfactant dysfunction.

Hydrolysis of surfactant-associated phospholipids by secretory phospholipases A2is an important potential mechanism for surfactant dysfunction in inflammatory lung diseases. In these conditions, airway secretory phospholipase A2(sPLA2) activity is increased, but the type of sPLA2and its impact on surfactant function are not well understood. We examined in vitro the effect of multiple secretory phospholipases A2on surfactant, including their ability to 1) release free fatty acids, 2) release lysophospholipids, and 3) increase the minimum surface tension (γmin) on a pulsating bubble surfactometer. Natural porcine surfactant and Survanta were exposed to mammalian group I (recombinant porcine pancreatic) and group II (recombinant human) secretory phospholipases A2. Our results demonstrate that mammalian group I sPLA2hydrolyzes phosphatidylcholine (PC), producing free fatty acids and lysophosphatidylcholine, and increases γmin. In contrast, mammalian group II sPLA2demonstrates limited hydrolysis of PC and does not increase γmin. Group I and group II secretory phospholipases A2from snake venom hydrolyze PC and inhibit surfactant function. In summary, mammalian secretory phospholipases A2from groups I and II differ significantly from each other and from snake venom in their ability to hydrolyze surfactant-associated PC.

Emotional events are often remembered better than neutral events, a type of memory prioritization by affective salience that depends on the amygdala. Studies with rats have indicated that direct activation of the basolateral complex of the amygdala (BLA) can enhance memory for neutral events, and if the activation is brief and temporally targeted, can do so in a way that benefits memories for specific events. The essential targets of BLA activation in the case of event-specific memory enhancement were unknown, but the hippocampus was known to receive direct projections from the BLA and to support memory for events. In the present study, rats received counterbalanced infusions of either muscimol, a GABAA receptor agonist, or saline into the hippocampus prior to performing a novel object recognition memory task during which initial encounters with some of the objects were immediately followed by brief electrical stimulation to the BLA. When memory was tested 1day later in the saline condition, rats remembered these objects well but showed no memory for objects for which the initial encounter had not been followed by BLA stimulation. In contrast, no benefit to memory of BLA stimulation was observed in the muscimol condition. The results indicated that brief activation of the BLA can prioritize memories for events by enhancing memory for some object encounters but not others and that this benefit to memory depends on interactions between the amygdala and the hippocampus.

Arachidonic acid (AA), the precursor of eicosanoids, is released from the sn-2 position of phospholipids by both secretory (8PLA2) and cytosolic phospholipase A2 (CPLA2). Eicosanoids have been shown to contribute to bronchospasm in asthma. We measured the enzymatic activity of 8PLA2 and CPLA2 in the bronchoalveolar lavage fluid and cells, respectively, in male Hartley guinea pigs sensitized with ovalbumin. sPLA2 activity was also measured from alveolar macrophages (AM) in culture from unsensitized and sensitized animals. There was an increase in 8PLA2 activity and AA content in the lavage fluid following sensitization (18.73 ± 1.33 to 25.74 ± 3.22% hydrolysis and 17.97 ± 12.39 to 44.76 ± 13.37 pmol AA/mL BAL, mean ± SD), which remained elevated but without further increase 4 or 24 h after antigen challenge. AM from unsensitized and sensitized-unchallenged animals did not secrete 8PLA2 activity in culture for 3 h and therefore do not appear to be the cell source of the sPLA2 activity present in the alveolar lavage fluid following OA sensitization. In contrast to the increase in 8PLA2 in lung lavage fluid, Western blotting for CPLA2 from lung lavage cells showed no increase 4 or 24 h after antigen challenge compared with sensitization alone. CPLA2 enzymatic activity of the cytosol fraction of lung lavage cells showed no changes with antigen sensitization or challenge. In summary, intraperitoneal sensitization with ovalbumin in male Hartley guinea pigs caused an increase in both sPLA2 and AA in bronchoalveolar lavage fluid without a need for antigen challenge. The increased 8PLA2 enzymatic activity following sensitization may be responsible for the elevation of AA in the bronchoalveolar lavage fluid observed after antigen sensitization.

In-situ sparging has been accepted as a method to rapidly remediate groundwater at considerably lower costs compared to remedies based on groundwater recovery alone. The success of in-situ sparging depends on effective mass transfer between air and contaminated media in the subsurface. Factors affecting mass transfer include advective airflow, diffusive transport, interphase chemical partitioning, and chemical and biological reaction rates between sparged gases and subsurface contaminants, minerals, and naturally occurring organic compounds. Understanding these factors can increase the design efficiency of in-situ sparging and assist in developing sparging systems that use gases other than air (i.e., oxygen, ozone, and methane).

This investigation examined the concept that arachidonic acid (AA) serves as a second messenger in stimulation of the respiratory burst and degranulation of polymorphonuclear neutrophils (PMN). The main support for this idea is from observations that reagent AA, added to cell suspensions, stimulates the respiratory burst and degranulation and these events are blocked by PLA2 inhibitors. We verified that exogenously-added AA stimulated release of O2-, myeloperoxidase (MPO), and lysozyme (LZ), but this required amounts of AA which approximated the critical micellar concentration. This suggested that such administration of AA might act as an extracellular agonist, similar to particulate stimuli, rather than acting as a second messenger as might occur following mobilization of AA from cellular membranes. To investigate the role of fatty acids released by hydrolysis of cellular phospholipids, exogenously-added group I, II or III PLA2's were used to mobilize fatty acids from cellular membranes. Mole quantities of cell-associated free fatty acids were measured by negative ion chemical ionization gas chromatography/mass spectrometry. AA mobilization in response to exogenous PLA2 was dose- (0.1 to 10 U/ml PLA2) and time-dependent (peak at 1 to 2 min with a reduction by 4 min). Resting neutrophils contained < 10 pmol free AA/10(7) PMN; the receptor-mediated agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP) alone did not increase these values. Exogenously-added PLA2 generated large quantities of free AA in control and fMLP-treated cells (462 +/- 122 and 2097 +/- 176 pmol/10(7) PMN, respectively); however, this did not induce O2-, nor did it augment the level of O2- stimulated by fMLP. Also, PLA2 caused no degranulation and did not alter degranulation induced by fMLP. PLA2 also did not alter O2- or degranulation responses in primed PMN. The data indicate that mobilization of AA from cellular phospholipids neither stimulates nor modulates the respiratory burst or degranulation of PMN.

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.

The NADPH oxidase is a multicomponent enzyme system that produces the reduced oxygen species essential for bacterial killing by polymorphonuclear leukocytes (PMN). Study of the oxidase has typically been carried out in cell-free systems in which Km values of 20-150 microM NADPH have been reported. However, when compared with affinities reported for other flavoprotein dehydrogenases and when considering the cellular concentration of NADPH/NADP+ of approximately 35 microM, the reported affinity of the oxidase for NADPH appears low. To investigate this apparent discrepancy we have studied the kinetics of NADPH oxidase activation in situ in human PMN permeabilized with Staphylococcus aureus alpha-toxin. alpha-Toxin permeabilization of human PMN did not initiate NADPH oxidase activation at physiologic concentrations of NADPH. If permeabilized cells were stimulated with 1 microM formyl-methionyl-leucyl-phenylalanine, 10 microM guanosine 5'-O-(3-thiotriphosphate), 0.5 mM Ca2+, 5 micrograms/ml cytochalasin B in the presence of varying concentrations of NADPH, we were able to demonstrate activation of the oxidase complex as shown by superoxide dismutase-inhibitable reduction of cytochrome c. In this system we determined that the Km for oxidase activation was 4-7 microM NADPH, a 4-10-fold decrease from reported values. The oxidase was the enzyme being studied as shown by the absence of enzymatic activity in patients with chronic granulomatous disease. In addition, if the enzyme was initially activated in permeabilized cells, the cells homogenized, and the Km for the oxidase determined in a cell-free system, the observed Km reverted to previously reported values (36 microM). These results indicate that NADPH oxidase, studied in situ, has a significantly higher substrate affinity than that observed in isolated membranes and, moreover, indicate that substrate affinity is optimal for catalysis at reported concentrations of cytosolic NADPH.

Addition of 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine (alkenyl-lyso-GPE) to human neutrophil membrane preparations containing 1-O-[3H]hexadecyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (1-O-[3H]alkyl-2-arachidonoyl-GPC) resulted in rapid deacylation of the 1-O-[3H]alkyl-2-arachidonoyl-GPC to 1-O-[3H]alkyl-2-lyso-GPC (lyso-platelet-activating factor, lyso-PAF). When acetyl-CoA was included in the incubation mixture, the [3H]lyso-PAF was converted to [3H]PAF. Studies of [3H]arachidonate-labeled neutrophils permeabilized with Staphlococcus aureus alpha-toxin revealed a major shift of labeled [3H]arachidonate from the choline to the ethanolamine-containing phosphoglycerides upon addition of alkenyl-lyso-GPE. The studies indicated that lyso-PAF is formed in the system by the transfer of arachidonate from 1-O-alkyl-2-arachidonoyl-GPC to the alkenyl-lyso-GPE by a CoA-independent transacylase reaction. Mass measurements revealed a rapid loss of arachidonate from 1-radyl-2-acyl-GPE and a concomitant increase in alkenyl-lyso-GPE upon stimulation of the neutrophils by ionophore A23187. Based on these and other findings, a pathway is proposed that may play a significant, if not obligatory, role in the synthesis of PAF in intact stimulated neutrophils. It has been widely accepted that phospholipase A2 acts directly on 1-O-alkyl-2-arachidonoyl-GPC as the first step in the synthesis of PAF via formation of lyso-PAF. In the proposed scheme, phospholipase A2, upon stimulation, acts rapidly on ethanolamine plasmalogen selectively releasing arachidonic acid and generating alkenyl-lyso-GPE. The CoA-independent transacylase then selectively transfers arachidonate from 1-radyl-2-arachidonoyl-GPC to the alkenyl-lyso-GPE generating lyso-PAF, which is then acetylated to form PAF. The interactions outlined can account for the synthesis of 1-acyl-2-acetyl-GPC, 1-O-alk-1'-enyl-2-acetyl-GPE, and eicosanoids, in parallel with PAF.

Tumor necrosis factor alpha (TNF) primes human neutrophils (PMN) for enhanced superoxide (O2-) production if cells are subsequently stimulated with the chemotactic peptide, n-formyl-Met-Leu-Phe (fMLP). fMLP activates phospholipase D to form phosphatidic acid (PA), and a correlation may exist between PA production and O2- generation in PMN. Therefore, we assessed the ability of TNF to prime phospholipase D activation in PMN stimulated with fMLP. TNF (100 units/ml) pretreatment primed enhanced PA production in PMN challenged with 1 microM fMLP, in the absence of cytochalasin B, as demonstrated by increased production of tritiated PA from PMN label with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine ([3H]LPAF) and by increased PA mass. PA was formed via activation of phospholipase D and occurred with minimal production of diglycerides. Production of O2- was also enhanced in identically treated cells, and we demonstrated a direct correlation between enhanced PA formation and O2- production. Conversely, ethanol inhibition of PA formation led to a comparable reduction in O2- generation. This report of priming of phospholipase D by physiological agonists is the only natural system where enhanced PA formation has been dissociated from diglyceride formation. Our results suggest a link between PA production and NADPH oxidase activation in human PMN.

Secretory A(2) phospholipases (sPLA(2)) hydrolyze surfactant phospholipids cause surfactant dysfunction and are elevated in lung inflammation. Phospholipase-mediated surfactant hydrolysis may disrupt surfactant function by generation of lysophospholipids and free fatty acids and/or depletion of native phospholipids. In this study, we quantitatively assessed multiple mechanisms of sPLA(2)-mediated surfactant dysfunction using non-enzymatic models including supplementation of surfactants with exogenous lysophospholipids and free fatty acids. Our data demonstrated lysophospholipids at levels >or=10 mol% of total phospholipid (i.e., >or=10% hydrolysis) led to a significant increase in minimum surface tension and increased the time to achieve a normal minimum surface tension. Lysophospholipid inhibition of surfactant function was independent of the lysophospholipid head group or total phospholipid concentration. Free fatty acids (palmitic acid, oleic acid) alone had little effect on minimum surface tension, but did increase the maximum surface tension and the time to achieve normal minimum surface tension. The combined effect of equimolar free fatty acids and lysophospholipids was not different from the effect of lysophospholipids alone for any measurement of surfactant function. Surfactant proteins did not change the percent lysophospholipids required to increase minimum surface tension. As a mechanism that causes surfactant dysfunction, depletion of native phospholipids required much greater change (equivalent to >80% hydrolysis) than generation of lysophospholipids. In summary, generation of lysophospholipids is the principal mechanism of phospholipase-mediated surfactant injury in our non-enzymatic models. These models and findings will assist in understanding more complex in vitro and in vivo studies of phospholipase-mediated surfactant injury.

In most eukaryotic cells, mitochondria use the respiratory chain to produce a proton gradient, which is then harnessed for the synthesis of ATP. Recently, mitochondrial roles in regulation of apoptosis have been discovered in many cell types. Eosinophils (Eos) die by apoptosis, but the presence and function of mitochondria in Eos are unknown. This study found that Eos contain mitochondria in small numbers, as shown by labeling with membrane potential-sensitive dyes andin situPCR for a mitochondrial gene. Eos generate mitochondrial membrane potential from hydrolysis of ATP rather than from respiration, as shown by mitochondrial respiratory inhibitors and mitochondrial uncouplers. The mitochondria provide insignificant respiration but can induce apoptosis, as shown by using the mitochondrial F1F0-ATPase inhibitor oligomycin and translocation of cytochromec.Thus during differentiation of Eos, although respiration is lost, the other central role of mitochondria, the induction of apoptosis, is retained.

We have shown previously that both 1,2-diacylglycerol (AAG) and 1-O-alkyl-2-acylglycerol (EAG) prime neutrophil release of arachidonic acid via uncharacterized phospholipases A2. Therefore, we investigated the actions of EAG and AAG specifically on neutrophil cytosolic (cPLA2) and secretory (sPLA2) phospholipase A2s. We hypothesized that AAG as a protein kinase activator would activate cPLA2 via phosphorylation events. EAG is antagonistic to the AAG activation of PKC, thus it was not expected to act via phosphorylation of cPLA2. Neutrophils were primed with either AAG or EAG and then stimulated with fMLP. When neutrophils were primed with 5-20 microM 1,2-diacylglycerol, a shift was observed in cPLA2 migration on SDS-PAGE gels, consistent with phosphorylation of the protein. This gel shift was not seen after exposure to EAG. AAG also caused a parallel increase in enzymatic activity of cPLA2 that was not seen with EAG. We also investigated whether either diglyceride would cause similar priming or direct secretion of sPLA2. Both AAG and EAG directly caused significant secretion of neutrophil sPLA2. EAG also increased the release of sPLA2 in cells subsequently stimulated with fMLP. Thus, AAG activated cPLA2 and stimulated secretion of sPLA2. In contrast, EAG did not activate cPLA2, but directly activated secretion of sPLA2. We also demonstrated that human synovial fluid sPLA2 increased AA release from resting and fMLP-stimulated neutrophils. Given that diglycerides prime for release of AA, PAF, and LTB4, these current data support the hypothesis that such priming may be mediated by phosphorylation dependent (cPLA2) or phosphorylation independent (e.g. secretion of sPLA2) events.

Cytokines alter neutrophil (PMN) function during inflammation, and Tumor Necrosis Factor (TNF) in vitro primes PMN such that receptor-mediated stimulation causes markedly enhanced release of arachidonic acid. We hypothesized that two Ca 2+ -dependent PLA 2 's in PMN might be activated during priming of the cell, thus affecting arachidonate release. A low molecular weight, secretory PLA 2 was identified by enzymatic activity in the cell free supernates of primed or stimulated PMN, and in PMN disrupted by nitrogen cavitation. The enzymatic activity was calcium-dependent, acid stable, destroyed by dithiothreitol, and blocked by anti-sPLA 2 antibodies. TNF caused secretion of sPLA 2 and also caused an increase in cell-associated sPLA 2 enzymatic activity. Activation and release were maximal with fMLP stimulation of TNF-primed PMN. Neutrophils also contained a cytosolic PLA 2 (cPLA 2 ) characterized by enzymatic activity which was calcium dependent, enhanced by dithiothreitol, and blocked by anti-cPLA 2 antibody. TNF caused a doubling of cPLA 2 enzymatic activity which was associated with phosphorylation of the enzyme as judged by a migration shift on Western blots. Thus, TNF priming of human PMN caused marked increase in fMLP stimulated AA release in parallel to enhanced activity of two different PLA 2 's.

In human neutrophils, the choline-containing phosphoglycerides contain almost equal amounts of alkylacyl- and diacyl-linked subclasses. In contrast to phosphatidylinositol hydrolysis which yields diacylglycerol, hydrolysis of choline-containing phosphoglycerides by phospholipase D coupled with phosphohydrolase yields both alkylacyl- and diacylglycerol. While diacylglycerol activates protein kinase C, alkylacylglycerol does not, and its role is unclear. Yet previous studies have shown that exogenous alkylacyl- and diacylglycerols can prime for the release of radiolabeled arachidonic acid (AA) in intact neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine. We have now examined the effects of both diacylglycerol (1-oleoyl-2-acetylglycerol; OAG) and alkylacylglycerol (1-O-hexadecyl-2-acetylglycerol; EAG) on the activation of mitogen-activated protein (MAP) kinase and the 85-kDa cytosolic phospholipase A2 (cPLA2) in human neutrophils. We observed that while OAG could effectively activate p42 and p44 MAP kinases along with cPLA2 in a time- and concentration-dependent manner, EAG could not. A novel p40 MAP kinase isoform is also present and activated in response to OAG treatment; the behavior of this MAP kinase isoform is discussed. The activation of cPLA2 and MAP kinase by 20 microM OAG could be inhibited by pretreatment with 1 microM GF-109203X, a selective inhibitor of protein kinase C. Although only OAG activated cPLA2, both OAG and EAG primed for the release of AA mass as determined by gas chromatography/mass spectrometry. The priming of AA release by OAG may be explained by the phosphorylation of cPLA2 through the activation of protein kinase C linked to MAP kinase. However, priming by EAG appears to involve a separate mechanism that is dependent on a different PLA2. Our results support a role for phospholipase D-derived products modulating the activation of cPLA2, further supporting the idea of cross-talk among various phospholipases.

Phospholipases A2 (PLA2) hydrolyze phospholipids resulting in the release of fatty acids including arachidonic acid (AA) and lysophospholipids. AA, in turn, serves as a substrate for the synthesis of leukotrienes which can cause bronchoconstriction and airways edema and appear to be important mediators of clinical asthma. Further, lysophospholipids may be cytotoxic and/or impair the function of surfactant. We examined the release of secretory PLA2 (sPLA2) and AA into the airways after antigen challenge in 16 subjects with allergic asthma. Asthmatic subjects underwent bronchoscopy with bronchoalveolar lavage (BAL) before and after inhaled antigen challenge; in addition, a single BAL, without inhaled antigen, was performed in 10 control subjects. BAL was obtained at 4 h (n = 7), the time of the late asthmatic response (LAR) (n = 5), or 24 h (n = 4) after challenge. There was no difference between normal and asthmatic subjects in either BAL fluid (BALF) sPLA2 activity or AA concentration at baseline. Both sPLA2 and AA increased after antigen challenge (p0.01 and 0.05, respectively). These changes were most marked 4 h after challenge (p0.03 for both). sPLA2 may play an important role in the generation of AA in patients with asthma.

Neutrophils (PMN) contain two types of phospholipase A2 (PLA2), a 14 kDa ‘secretory’ Type II PLA2 (sPLA2) and an 85 kDa ‘cytosolic’ PLA2 (cPLA2), that differ in a number of key characteristics: (1) cPLA2 prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA2 is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA2 is active at nM calcium (Ca2+) concentrations; sPLA2 requires μM Ca2+ levels. (3) cPLA2 activity is regulated by phosphorylation; sPLA2 lacks phosphorylation sites. (4) cPLA2 is insensitive to reduction; sPLA2 is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus α-toxin to determine whether one or both forms of PLA2 were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [3H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to asses oleate and linoleate mass. A combination of 500 nM Ca2+, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [3H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA2 activation. Dithiothreitol treatment had little affect on [3H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA2 activity and the cytosolic buffer utilized did not support activity of recombinant sPLA2. These results strongly suggested that cPLA2 was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA2 in PMN.

BIRM 270 was developed as a potent and enantioselective inhibitor of LTB4 biosynthesis by human neutrophils, and was also found to inhibit LTC4 production by human eosinophils and lung mast cells. BIRM 270 inhibited LTB4 synthesis in neutrophils by preventing arachidonate release from membrane phospholipids, and over the same concentration range, inhibited PAF biosynthesis. BIRM 270 did not directly inhibit acylhydrolases which have been implicated in eicosanoid and PAF biosynthesis, suggesting an indirect mode of action.

One aspect of human neutrophil (PMN) function during inflammation is formation of platelet-activating factor (PAF), leukotriene B4 (LTB4), and 5-hydroxyeicosatetraenoic acid (5-HETE), but production of these lipid mediators is limited if PMN are directly stimulated with soluble, physiologic agonists. In vitro, PMN activities can be enhanced by the process of primed-stimulation where cells are sequentially treated with non-stimulatory concentrations of different agonists. Many agents that prime PMN also induce production of 1,2-diacyl- and 1-O-alkyl-2-acylglycerols. Therefore, we investigated whether diglycerides were involved in priming PMN for production of lipid mediators. We previously described the ability of the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), and its alkylacylglycerol analog, 1-O-octadecenyl-2-acetylglycerol (EAG), to prime phospholipase A2 (PLA2) for subsequent activation by a second stimulus. However, while OAG also primed 5-lipoxygenase activity (LTB4 and 5-HETE production), EAG priming inhibited LTB4 and 5-HETE formation. We now report the effects of diglyceride priming on acetyltransferase activation (PAF formation). PMN, prelabeled with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine, were primed with OAG or EAG before stimulation. Neither OAG nor EAG induced formation of labeled PAF. Treatment of PMN with the chemotactic peptide, N-formyl-met-leu-phe (FMLP), induced low but significant production of PAF; PAF formation doubled in PMN primed with 20 microM OAG before FMLP stimulation while priming with 20 microM EAG more than tripled the level of PAF. Calcium ionophore strongly induced PAF formation; OAG priming before ionophore challenge had no effect but EAG priming further enhanced PAF formation. These results suggests a role for alkylacylglycerols in modulating the production of lipid mediators of inflammation.

Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway.

Both 1,2-diacyl- and 1-O-alkyl-2-acyl-sn-glycerols are released during stimulation of human polymorphonuclear leukocytes (PMNL). 1,2-Diacylglycerols have received intense interest as intracellular "second messengers" due to their ability to activate protein kinase C (Ca2+ phospholipid-dependent enzyme). However, little is known about bioactivities of the alkylacylglycerols. This study compared the ability of 1,2-diacyl- and 1-O-alkyl-2-acylglycerols to modulate the respiratory burst of stimulated PMNL, a response which depends on the activation of an NADPH oxidase to generate bactericidal species of reduced oxygen. Direct stimulation by N-formyl-Met-Leu-Phe caused an abrupt release of H2O2 which ceased within 2.5 min. Preincubation with diacylglycerols (1-oleoyl-2-acetylglycerol,5-30 microM, and 1,2-dioctanoylglycerol,2-5 microM) caused a decrease in lag time, 3-fold increase in initial rate of H2O2 release, and marked prolongation of the response to N-formyl-Met-Leu-Phe (features characteristic of a priming effect). Preincubation with alkylacylglycerols (1-O-delta 9-octadecenyl-2-acetylglycerol, 5-30 microM, and 1-O-octyl-2-octanoylglycerol, 20-50 microM) primed initiation (shortened lag time and increased velocity) but, in contrast to diacylglycerols, did not alter duration of H2O2 release. While low concentrations of diacylglycerols (5-30 microM) primed PMNL, higher concentrations (greater than or equal to 70 microM) stimulated the cells directly. In contrast, higher (70-100 microM) concentrations of alkylacylglycerols did not prime the responses but, in fact, inhibited priming (especially of duration) induced by diacylglycerol. The high concentrations of alkylacylglycerol also inhibited direct stimulation induced by high concentrations of diacylglycerol. Direct stimulation by high concentrations of diacylglycerol probably involves activation of protein kinase C, whereas alkylacylglycerol was found to inhibit activation of protein kinase C by diacylglycerol in vitro. Thus, diacylglycerols are complete priming agonists, altering both rate and duration of the response. In contrast, alkylacylglycerols may have biphasic, concentration-related effects in modulation of functions of PMNL. At low concentrations, they may facilitate initiation of functional events; however, as their concentration increases, they may serve to terminate responses. The distinct priming effects of these diglycerides also reveal that priming can involve at least two distinct events: 1) initiation and 2) prolongation.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute infection is accompanied by a characteristic reduction in circulating eosinophils. This study examined the generally held assumption that the eosinopenia of infection is a manifestation of adrenal stimulation. Trichinosis, Escherichia coli pyelonephritis, and early subcutaneous pneumococcal abscess were used as experimental infections of limited severity. Trichinosis is associated with eosinophilia, but pyelonephritis and pneumococcal infection produce eosinopenia. An assay for serum corticosterone was developed that is sufficiently sensitive to be performed with the small volumes of blood obtained sequentially from individual mice. The corticosterone response to trichinosis fits the sterotyped reaction previously reported for several other bacterial, viral, and rickettsial infections. The peak concentrations of corticosterone in serum from mice with trichinosis was approximately twice normal and occurred at the onset of clinical illness. Serum corticosterone levels gradually declined to the normal range over the next several days. E. coli pyelonephritis produced a similar adrenal response, although the peak serum corticosterone caused by pyelonephritis was less than the serum corticosterone occurring during the first peak of eosinophilia during trichinosis. Infection of a subcutaneous air pouch with penumococci produced eosinopenia within 6 h after inoculation, but there was no rise in serum corticosterone during the first 12 h of the pneumococcal infection. In addition, the eosinopenic response produced by a 12-hpneumococcal abscess occurred mice adrenalectomized 1-4 days before infection with pneumococci. The eosinopenia of acute infection cannot be ascribed to adrenal stimulation.

After a century of study, the functions of eosinophils remain uncertain. The two proposed functions supported by currently available data appear contrasting. First, the eosinophil is viewed as a killer cell, similar to the neutrophil, but having an unusual ability to destroy invasive metazoan parasites. On the other hand, it is viewed as a immunomodulatory cell which serves to minimize the unneeded spread of hypersensitivity reactions. Each of these possibilities can find support in our current knowledge of the biochemistry and metabolism of eosinophils. Eosinophils are capable of a potent oxidative metabolic response which may be directly toxic to some parasites. Eosinophil granules are rich in peroxidase, which could amplify the effect of the metabolic burst by a peroxidase-hydrogen peroxide-halide microbicidal mechanism. Eosinophil cationic proteins may be directly toxic to parasites. Alternately, eosinophils may release a variety of substances which are capable of suppressing immediate hypersensitivity responses. These include supression of basophil degranulation by prostaglandins, inactivation of slow reacting substance of anaphylaxis by eosinophil arylsulphatase, inactivation of histamine by major basic protein. This discussion reviews the current knowledge regarding such distinctive qualities of eosinophil leucocyte biochemistry and metabolism. There are also differences between eosinophils obtained from normal individuals and those obtained from patients with eosinophilia. Moreover, some of these changes could be duplicated by stimulation of normal eosinophils in vitro . Thus, “resting” eosinophils from patients with eosinophilia or normal eosinophils stimulated in vitro , each demonstate a reduced cell surface charge, an enhanced transport of sugars into the cell and an activation of demonstrable acid phosphatase in the specific granules. Also, whereas oxidative metabolism of normal eosinophils is similar to that of normal neutrophils, eosinophils from patients with eosinophilia demonstrate a significant enhancement of oxidative metabolism in both resting cells and cells stimulated during phagocytosis. Thus, eosinophils from patients with eosinophilia appear significantly activated when compared with normal eosinophils.

The ability of several stimuli, all of which have been reported to stimulate eosinophil motility, to augment uptake or extracellular deoxyglucose (DOG), a glucose analogue, was studied. DOG uptake was stimulated in a concentration-dependent manner by the more potent chemotaxins--zymosan-activated serum, partially purified C5a. N-formyl-methionyl-leucyl-phenylalanine, and 5- and 11-hydroxyeicosatetraenoic acids. Less potent stimuli--the eosinophil chemotactic factor of anaphylaxis, histamine, and prostaglandin E2--did not stimulate DOG uptake. However, the correlation between the abilities of the agents to stimulate motility and DOG uptake was not completely uniform. Prostaglandin F2 alpha was a potent stimulant of DOG uptake yet is known to enhance only weakly chemokinesis and to be not chemotactic for eosinophils. The combined data from this and other studies indicate that these stimuli elicit specific and different sets of functional responses from eosinophils.

One distinctive aspect of the response to acute inflammation involves a rapid and persistent decrease in the numbers of circulating eosinophils, yet the mechanisms of this eosinopenia are undefined. One possibility is that the abrupt eosinopenia may be the result of release of small amounts of the chemotactic factors of acute inflammation into the circulation. These studies were designed to examine the numbers of circulating eosinophils after an intravenous injection of zymosan-activated serum, partially purified C5a or the synthetic peptide, N-formyl-methionyl-leucyl-phenylalanine. Each of these factors caused a virtual disappearance of circulating eosinophils within 1 min, a transient return of eosinophils to approximately 50% of control levels after 10-90 min, and a subsequent decrease which persisted for 5 h. In contrast, the numbers of circulating heterophils, although dropping transiently, rapidly returned and rose to elevated levels for 6 h after injection. The response was not caused by adrenal mediation as it occurred normally in adrenalectomized rabbits. Two chemotaxins of allergic inflammation, histamine and the tetrapeptide valine-glycine-serine-glutamic acid, did not cause significant eosinopenia. Circulating granulocytes of patients undergoing hemodialysis, which has been reported to activate complement, demonstrated similar eosinopenic and neutropenic-neutrophilic responses. Thus, in rabbits and in man, intravascular activation or injection of chemotactic factors (C5a or N-formyl-methionyl-leucyl-phenylalanine) causes a brief, nonspecific granulocytopenia followed by a prolonged eosinopenic-neutrophilic response analogous to that seen during acute infection.

The protein C kinase activators 1-0-oleoyl, 2-0-acetylglycerol, 12-0-tetradecanoyl phorbol-13-acetate, and mezerein, stimulated deoxyglucose uptake in human neutrophils. The responses were stimulus specific since no effect was noted with the diether analogues 1-0-hexadecyl-2-0-ethylglycerol, 1-0-palmitoyl-2-0-acetyl or 1-0-palmitoyl-3-0-acetyl diesters of propanediol. or with 1,2-diolein. Stimulation of deoxyglucose uptake had the characteristics of carrier facilitated hexose transport. Stimulated uptake of deoxyglucose was inhibited by trifluoperazine (10–30 μM). Activation of protein kinase C therefore appears to trigger events involved in hexose transport.

Preincubation of neutrophils with certain agonists may "prime" the cells to cause increased responses to a second stimulus ("primed stimulation"). We used two approaches to examine the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in priming and stimulation by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and N-formyl-Met-Leu-Phe (fMLP): inhibition of protein kinase C by 1-(5-isoquinolinesulfonyl)-piperazine (C-I) and measurement of protein kinase C translocation induced by priming and stimulatory concentrations of OAG. C-I had little effect on stimulation or primed stimulation by fMLP, suggesting that fMLP invokes events independent of protein kinase C. C-I equally inhibited stimulation and primed stimulation by PMA. Direct stimulation by OAG was inhibited, but priming and primed stimulation by OAG was unaltered by C-I. OAG concentrations greater than or equal to 100 microM caused translocation of protein kinase C, in correlation with direct stimulation of the respiratory burst. Lower OAG concentrations (10-30 microM) primed to stimulation by fMLP and, conversely, stimulated neutrophils primed with fMLP, yet did not cause translocation of protein kinase C. The data are compatible with previous assumptions that PMA and OAG directly stimulate polymorphonuclear neutrophil leukocytes by translocation and activation of protein kinase C. However, priming and primed stimulation by OAG apparently invoke distinct transduction mechanisms other than protein kinase C translocation.

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.

Polymorphonuclear leukocytes (PMNL) from two individuals with congenital Pelger-Huet anomaly (PHA) were examined to determine whether functional or metabolic defects accompanied the known morphological abnormality. No abnormalities of the PHA cells, as compared to normal control cells, were found when tested for quantitative leukocyte enzyme activities, nitroblue tetrazolium reduction, hexose monophosphate shunt activity, superoxide production, generation of chemiluminescence, or iodination. The PHA cells, as compared to normal PMNL, demonstrated normal chemotaxis and random migration, as well as bactericidal activity.

The polymorphonuclear leukocyte (PMNL) depends on glucose as a source of energy for motility, chemotaxis, phagocytosis, and bactericidal activity. Activated complement (C5a) at low concentrations stimulates carrier-mediated carbohydrate transport in PMNLs as measured by the uptake of 2-deoxy-D-[ 3 H]glucose. Human PMNLs were preincubated at 37°C for 15 min with zymosan-activated human serum or various purified preparations of human C5a. A concentration-dependent increase in deoxyglucose transport (>700% of control) into PMNLs occurred with all test substances. Reaction was linear for 30 min, and uptake of deoxyglucose followed saturation kinetics. C5a caused a decrease in the K m for deoxyglucose, from 0.53 to 0.11 mM, without altering the V max (44 nmol/30 min per 5 × 10 6 PMNLs in control and 46.6 with C5a). The optimal concentration of C5a for enhanced carrier-mediated transport of deoxyglucose was similar to that which promoted optimal chemotaxis. Activated serum from C5-deficient mice had little or no effect on deoxyglucose transport whereas that from normal syngeneic mice enhanced deoxyglucose transport. C5a did not enhance deoxyglucose transport into isolated erythrocytes, platelets, or lymphocytes. The deoxyglucose within the cell was primarily in the phosphorylated form, and hexokinase activity was not increased in PMNLs stimulated with C5a, indicating that hexokinase was not rate limiting and that enhanced transport was the mechanism of the C5a activity. Insulin at physiologic concentration (10 ng/ml) had no effect on deoxyglucose transport in PMNL and did not act as a competitive inhibitor of C5a. This insulin-like bioactivity could be detected with the amount of C5a that would be present after activation of 0.1-0.5% of the C5 in 1 ml of serum. This suggests that uptake of [ 3 H]deoxyglucose by PMNLs might serve as a highly sensitive test for activation of the fifth component of complement.

Eosinophil and/or neutrophil leukocytes appear to have important roles in host defense against invasive, migratory helminth infestations, but the mechanisms of larval killing by leukocytes are uncertain. This study examines killing of newborn (migratory phase) larvae of Trichinella spiralis during incubation with granule preparations of human eosinophils or neutrophils and generators of hydrogen peroxide (glucose-glucose oxidase) (G-GO) or superoxide and hydrogen peroxide (xanthine-xanthine oxidase). Larvae were killed by either hydrogen peroxide-generating system in a concentration-dependent manner. Direct enumeration of surviving larvae after incubation in microtiter wells containing the appropriate reagents was used in assess larval killing. Verification of the microplate assay was demonstrated by complete loss of larval ability to incorporate [(3)H]deoxyglucose and loss of infectivity after incubation in comparable concentrations of G-GO. Larvae were highly sensitive to oxidative products; significant killing occurred after incubation with 0.12 mU glucose oxidase and complete killing occurred with 0.5 mU. Comparable killing of bacteria required over 60 mU glucose oxidase. At 5 mU glucose oxidase, killing was complete after 6 h of incubation. Killing by G-GO was inhibited by catalase but not by boiled catalase or superoxide dismutase and was enhanced by azide. Addition of peroxidase in granule pellet preparations of eosinophils or neutrophils did not enhance killing by G-GO. These data indicate a remarkable susceptibility of newborn larvae of T. spiralis to the hydrogen peroxide generated by neutrophil and eosinophil leukocytes.

Early events of stimulus-response coupling in polymorphonuclear leukocytes (PMNL) reportedly include membrane depolarization as a necessary antecedent to oxidative responses. However, depolarization by nonspecific means (ionophores) is insufficient to elicit an oxidative burst. This apparent conflict might be related to whether depolarization is due to membrane receptor-mediated stimulation of PMNL. We used two fluorescent probes and dual laser flow cytometry to monitor both membrane potential and oxidative product formation in individual PMNL, following stimulation by phorbol myristate acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP). Dipentylindocarbocyanine [di-I-C5(3)] is one of a family of dyes that partition between cells and aqueous media as a function of transmembrane potential. The dye appeared stable in the presence of PMNL oxidative products. Oxidation of intracellularly trapped, nonfluorescent dichlorofluorescin (DCFH) to fluorescent dichlorofluorescein (DCF) provided a quantitative assessment of oxidative metabolism (H2O2 production) of stimulated PMNL. Incubating PMNL with both fluorochromes resulted in stable red [di-l-C5(3)] and green (DCF) fluorescence when examined on a Cytofluorograph. Upon stimulation by 0.03 to 0.1 nmol/L PMA, PMNL showed a unimodal apparent depolarization (decrease in di-l-C5(3) fluorescence). Oxidative activity (increased DCF fluorescence) was first seen at a concentration of PMA of 0.17 nmol/L, higher than that required to elicit depolarization. This oxidative burst appeared as a dose-dependent, graded response. Thus, receptor-mediated membrane depolarization, although antecedent to the onset of the oxidative burst, was not in itself sufficient to trigger the oxidative metabolic response. When PMNL were isolated by centrifugation through Ficoll-metrizoate, fMLP caused an apparent depolarization of a variable subpopulation of the cells. However, such purified PMNL appeared relatively unstable and often depolarized spontaneously. PMNL studied without centrifugation through Ficoll-metrizoate were stable. Moreover, fMLP stimulation of such cells did not cause membrane depolarization but did stimulate a two- to six-fold increase in DCFH oxidation. Apparently, membrane depolarization may antecede oxidative responses in PMNL, but appears to depend upon the method of cell preparation and the nature of the stimulus.

Summary Chemotactic factors, arachidonic acid and leukocyte-derived lipoxygenase products of arachidonate (5-hydroperoxy-, 5-hydroxy- and 5,12-dihydroxy-eicosatetraenoic acids) stimulated stereospecific uptake of 3 H-2-deoxyglucose by human neutrophils. However, of all stimuli tested, only the effects of 5-hydroxy-eicosatetraenoic acid were not inhibited by 5,8,11,14-eicosatetraynoic acid at concentrations which also inhibit release of arachidonate metabolites from leukocytes. The data suggest that 5-hydroxy-eicosatetraenoic acid mediates the coupling of membrane receptor stimulation to the functional response of hexose uptake in human neutrophils.

We have compared the oxidative metabolism of human eosinophils (80%-90% purity) to that of neutrophils. Hexose monophosphate (HMP) shunt activity of eosinophils was higher than that of neutrophils under either resting or phagocytizing conditions. Eosinophil HMP shunt activity also was stimulated by phorbol myristate acetate, a membrane- active agent. Eosinophils showed a marked incorporation of 125I into trichloroacetic acid-insoluble material under resting conditions, which increased markedly during phagocytosis. Eosinophils likewise showed a greater reduction of nitroblue tetrazolium dye during phagocytosis than did neutrophils. Measurement of other parameters of oxidative metabolism indicated that eosinophils generated superoxide anion following phagocytosis and also elicited a burst of chemiluminescence similar to that observed during phagocytosis by neutrophils. Measurement of NADPH oxidase activity demonstrated that this enzyme was 3–6 times more active in fractions isolated from eosinophils than in corresponding fractions isolated from neutrophils; this was observed over a range of substrate concentrations. The eosinophil enzyme sedimented differently than the neutrophil enzyme with differential centrifugation; neither showed sedimentation characteristics of peroxidase. These data indicate that eosinophils possess a similar, although in some ways more potent, oxidative burst than neutrophils and are consistent with a role for NADPH oxidase in the initiation of that burst.

Eosinophil leukocytes have been reported to have a major role in host defense against invasive, migratory phases of helminth infestations, yet the relative larvicidal abilities of eosinophils and neutrophils have not been thoroughly examined. This study examined the killing of newborn (migratory phase) larvae of Trichinella spiralis during incubation by human granulocytes in vitro. The assay employed cultue of larvae with cells, sera, and reagents in microtiter wells with direct counting of surviving larvae after incubation. Killed larvae appeared to be lysed. Verification of the microplate assay was obtained by demonstrating complete loss of infectivity of larvae incubated with leukocytes and immune serum. In the presence of optimal immune serum concentrations, purified neutrophils or eosinophils achieved >/=95% killing of larvae at cell:larva ratios of 2,000:1 or greater. Fresh normal serum prompted slight (19%) killing by leukocytes at a cell:larva ratio of 9,000:1. Cells plus heat-inactivated normal serum and all sera preparations in the absence of leukocytes killed

Peroxidase, present in both neutrophils and eosinophils, is thought to be involved in the bactericidal activity of leukocytes, through either the decarboxylation of amino acids in the presence of chloride or the iodination of proteins in the presence of iodide. We examined these activities in sonic extracts of purified neutrophils and eosinophils and related them to the ability of the extracts to kill bacteria. In contrast to the neutrophil, the eosinophil cell-free system is unable to decarboxylate L-alanine-1- 14 C. Decarboxylation by the neutrophil peroxidase is dependent on the hydrogen peroxide concentration, with inhibition at high levels. Peroxidase-mediated decarboxylation showed a pH optimum at 4.5 and was dependent on the type of buffer employed, in order of increasing activity, N-(2-acetamido)-iminodiacetic acid (ADA), Tris-maleate, sodium phosphate, and sodium acetate. Iodination, measured as the ability to convert 125I into a trichloroacetic acid-precipitable form, was greater in resting eosinophils than in resting neutrophils. Both leukocyte types show increased iodinating ability upon phagocytosis of zymosan. When iodide was used as the halide, eosinophil and neutrophil cell-free preparations showed bactericidal activity as measured by the reduction in viability of Staphylococcus aureus and Escherichia coli. In contrast, with chloride as the halide only the neutrophil system could effectively kill both organisms. This finding is in agreement with the observation that eosinophilic peroxidase is unable to catalyze the decarboxylation reaction, and it suggests the importance of this mechanism in bactericidal activity.

1-O-Alkyl-2-O-acetyl-sn-glycerol-3-phosphorylcholine (i.e., platelet-activating factor) was prepared and confirmed to possess potent platelet aggregating activity. It was also potent in aggregating and degranulating rabbit and human neutrophils. When injected into rabbits, the lipid induced profound neutropenia, thrombocytopenia, and anaphylactic symptoms. The lyso derivative of this lipid, 1-O-alkyl-sn-glycerol-3-phosphorylcholine, was inactive or several orders of magnitude weaker in inducing these responses. The acetylated lipid appears to be a potent stimulator of both platelets and neutrophils. Its anaphylactic-like toxicity may be related, at least in part, to its ability to aggregate or otherwise stimulate these cells.

The bioflavinoid quercetin was found to exert at least three separate effects on human polymorphonuclear leukocytes. (1) Concentrations of approximately 100 microM inhibited the membrane-associated magnesium adenosine triphosphatase by 60%-80% in either broken cell preparations or intact cells. Lineweaver-Burk plots showed the inhibition to be uncompetitive in nature. (2) Similar concentrations of quercetin inhibited respiratory burst activity of the cells as measured by oxygen consumption, glucose oxidation, or iodination of protein. All inhibitions were dose-dependent and were observed with either opsonized zymosan or phorbol myristate acetate as stimulus. (3) Quercetin likewise inhibited the transport of the nonmetabolizable hexose, 3H-2- deoxyglucose. These observations are most consistent with the hypothesis that quercetin exerts a generalized effect at the level of the cell membrane of the neutrophi.

The effect of oral inorganic phosphate supplements (1 to 2 Gm. phosphatephosphorus daily) on calcium metabolism during immobilization was assessed in six healthy volunteer subjects who were placed in plaster spica body casts for 40 days. Compared to values during the control phase, those subjects who did not receive phosphate supplements showed an increased urinary excretion of calcium, negative calcium balance (averaging −152 mg./day/subject), and calcium oxalate crystalluria. In contrast, subjects who received the phosphate supplement showed a decreased urinary excretion of calcium, a lesser degree of negative calcium balance (mean −78 mg./day/subject, and no crystalluria. In addition, the hypercalciuria and crystalluria which occurred in subjects who initially did not receive a phosphate supplement were reversed when the supplement was begun. The data suggest that phosphate supplements can prevent or ameliorate the hypercalciuria, crystalluria, and negative calcium balance associated with immobilization.

The hypothesis was tested that men given sodium salicylate would work in the heat at a lower level of temperature regulation. Healthy young volunteers were exposed to a standardized workload consisting of a 100-min walk at 3.5 mph on a level treadmill. Two ambient conditions were studied: a) simulated desert (120 F D.B., 80 F W.B.) and b) simulated jungle (92 F D.B., 87 F W.B.). Rectal temperature (Tr), skin temperature (Ts), sweat rate ( SR), and pulse rate ( PR) were measured in the subjects during work in the heat under three drug regimens: no drug (control), 0.6 g (low dose), and 7.8 g (high dose). It was found that sodium salicylate had no effect on Ts or PR compared with control; this finding applied to both doses in both hot environments. There was, however, a higher Tr and SR in men who received the high dose of the drug in the tropic environment and an increased SR in the desert climate. There was no decrease in Tr in the desert environment during salicylate administration. The results of this investigation do not confirm the original hypothesis; to the contrary, they suggest that high doses of sodium salicylate potentiates the hyperthermia of unacclimatized men working in the heat. body temperature regulation; exercise; simulated desert environment; simulated jungle environment; hyperthermia; rectal temperature, skin temperature, sweat rate, and pulse rate in hot environments; antipyretics Submitted on August 26, 1963

Eosinophil leukocytes obtained from the blood of patients with eosinophilia (EE), are abnormal which compared with eosinophils obtained from normal individuals (NE). Such EE have repeatedly been observed to contain variable numbers of vacuoles, reduced numbers of granules (suggesting some degree of degranulation in vivo) and increased segmentation of nuclear lobes (1). These morphologic abnormalities have suggested that eosinophils might be stimulated by a response to the pathologic process causing the eosinophilia. Such stimulation could lead to augmented (activated) or suppressed (deactivated or exhausted) responses of EE to subsequent stimuli. However, there have been few studies of biochemical, physiologic or functional comparisons between such EE and NE. Indeed, nearly all studies of eosinophil physiology have relied upon EE, as such cells have proven easier to purify in vitro and have provided the quantity of eosinophils necessary for most techniques available for examination of leukocyte physiology and function in vitro.

The influence of extracellular Ca2+ and Mg2+ on the transport of 2-deoxy-[3H]glucose into human polymorphonuclear neutrophils was studied. Omission of these cations from the cell suspensions had little effect on resting hexose uptake. Furthermore, the addition of the bivalent cation chelator, EDTA, depressed uptake only slightly. Similarly, neither cation was essential for the enhanced 2-deoxy-D-[3H]glucose uptake stimulated by two chemotactic factors (C5a and N-formylmethionylleucylphenylalanine) and arachidonic acid: enhanced uptake was only partially depressed by the omission of Ca2+ and Mg2+ from the suspensions and was still prominent in the presence of EDTA. Two other neutrophil stimulants, the ionophores, A23187 and ionomycin, also enhanced hexose uptake but their actions were heavily dependent upon extracellular bivalent cations and were totally abrogated by EDTA. In all instances, extracellular Ca2+, but not Mg2+, supported optimal enhanced hexose transport induced by stimuli. Activation of 2-deoxy-D-[3H]glucose uptake by each of the five stimuli was totally blocked by cytochalasin B (a blocker of carrier-mediated hexose transport) and D-glucose but not by L-glucose. The data indicate, therefore, that a variety of neutrophil stimulants activate carrier-mediated hexose transport. Although this transport can be triggered by the movement of extracellular Ca2+ into the cell (as exemplified by the action of the two ionophores), such Ca2+ movement is not required for the actions of chemotactic factors or arachidonic acid. Other mechanisms, such as a rearrangement of intracellular Ca2+, may be involved in mediating the activation of hexose transport induced by the latter stimuli.

Glutathione peroxidase (GPx) deficiency has been proposed as a cause of some instances of chronic granulomatous disease (CGD). GPx activity varies greatly among species, and specific deficiency of this selenium-dependent enzyme can be produced by dietary selenium deficiency in rats. Bactericidal activity of polymorphonuclear (PMN) leukocytes from normal rats, humans, and guinea pigs (GPx high, intermediate, and nearly absent, respectively), selenium-deficient rats (GPx absent), and a patient with CGD were compared. There was no correlation between natural levels of GPx and bactericidal activity; only CGD was associated with inability to kill a Proteus mirabilis strain in vitro (killing known to be dependent on oxidative mechanisms). Postphagocytic metabolism was examined in normal and GPx-deficient rats. Both demonstrated normal iodination and superoxide production during phagocytosis and gave similar histochemical reduction of nitroblue tetrazolium dye under either resting or endotoxin-stimulation conditions. Postphagocytic hexose monophosphate shunt activity was somewhat lower in PMN from GPx-deficient animals as compared with normal but was substantially (10-fold) higher than that observed in resting cells. Thus, postphagocytic oxidative responses and subsequent bactericidal activity of PMN leukocytes were not compromised by complete absence of GPx, even in the species with the highest natural level of this enzyme. These results are not compatible with the hypothesis that CGD can be caused by a deficiency of GPx.

In order to resolve discrepancies in the literature concerning the subcellular localization of NADPH oxidase, we disrupted human neutrophils by nitrogen cavitation and fractionated the subcellular organelles on a discontinuous sucrose density gradient. The lightest fraction was 20- to 40-fold enriched for plasma membranes as determined by the marker enzymes alkaline phosphatase and phosphodiesterase I as well as by the ratio of lipid phosphorus to protein. There was a significant decrease in the specific activities of the granule markers myeloperoxidase, lysozyme, and beta-glucuronidase. An intermediate fraction was enriched in membrane markers but not to the extent the lightest fraction was enriched. This fraction contained more granular contamination, as shown by the marker enzymes. In contrast, the densest bands of the gradient were enriched for granule markers with little contamination by plasma membrane. Superoxide generation and NADP formation were primarily associated with the two membrane-enriched fractions from polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The NADP formation associated with a dense granule fraction observed previously in our laboratory was probably due to a cyanide-stimulated oxidation of NADPH by myeloperoxidase.