Elizabeth R. Simons

Chronic granulomatous disease: a syndrome of phagocyte oxidase deficiencies.

Chronic granulomatous disease (CGD) is an inherited disorder of host defense due to the inability of the phagocyte to generate toxic oxygen metabolites upon appropriate stimulation. The disorder is heterogeneous even within the confines of a defective respiratory burst oxidase function, and may arise from a biochemical lesion at either the receptor, the activating pathways or the enzyme level. The identification of defects in plasma membrane depolarization, missing cytochrome and abnormal enzymatic function has yielded new insights into the pathophysiologic basis of CGD. A classification of this syndrome based on more precise biochemical criteria is proposed, which defines the disease as distinct from other associated enzymopathies with similar pathology and emphasizes the metabolic basis of the pathophysiologic defect in phagocyte function. Review of the clinical manifestations, pathogenic organisms and natural course of the disease, emphasizes the critical role of the oxidative metabolism of the normal neutrophil and offers a perspective on oxygen free radical biochemistry in the inflammatory response.

Changes in neutrophil functions in astronauts.

Exploration class human spaceflight missions will require astronauts with robust immune systems. Innate immunity will be an essential element for the healthcare maintenance of astronauts during these lengthy expeditions. This study investigated neutrophil phagocytosis, oxidative burst, and degranulation of 25 astronauts after four space shuttle missions and in nine healthy control subjects. Space flight duration ranged from 5 to 11 days. Blood specimens were obtained 10 days before launch, immediately after landing, and 3 days after landing. The number of neutrophils increased by 85% at landing compared to preflight levels. The mean values for phagocytosis of Escherichia coli and oxidative burst capacity in neutrophils from astronauts on the 5-day mission were not significantly different from those observed in neutrophils from the control subjects. Before and after 9- to 11-day missions, however, phagocytosis and oxidative burst capacities were significantly lower than control mean values. No consistent changes in degranulation or expression of surface markers were observed before or after any of the space missions. This study indicates that neutrophil phagocytic and oxidative functions are affected by factors associated with space flight and this relationship may depend on mission duration.

Human Neutrophil-Mediated Nonoxidative Antifungal Activity against Cryptococcus neoformans

ABSTRACT It has long been appreciated that polymorphonuclear leukocytes (PMN) kill Cryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing of C. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation of C. neoformans with these fractions for 18 h resulted in percents inhibition of growth of 67.4 ± 3.4, 84.6 ± 4.4, and 29.2 ± 10.5 (mean ± standard error, n = 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C4 column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules.

A Variant of Chronic Granulomatous Disease: Deficient Oxidative Metabolism Due to a Low-Affinity NADPH Oxidase

OUR defense against bacterial infections depends in part on the action of phagocytic leukocytes, which encounter and kill potentially pathogenic microorganisms. A severe reduction in the number or ...

Flavonoid impairment of neutrophil response

Flavonoids are a class of phenolic plant pigments which impair the oxidative burst of neutrophils to an extent dependent on their hydrophobicity. The distribution of quercetin and of morin in nitrogen-cavitated neutrophils paralleled their respective hydrophobic characteristics and respiratory burst inhibition. While both flavonoids were localized primarily in the specific granule membrane of neutrophils, the amount of quercetin was considerably greater than that of morin. We here demonstrate inhibition of the initial stimulation response, depolarization of the membrane potential as monitored by fluorescence of the membrane probe diS-C3-(5), and of the respiratory burst, monitored by following the destruction of diS-C3-(5), a reaction mediated by the H2O2 produced in the burst. The flavonoids kaempferol, morin, quercetin, or fisetin were preincubated with human neutrophils at a concentration of 100 microM per 2 X 10(6) cells/ml for 2-3 min and subsequently stimulated with 1 microgram/ml of the tumor promoter phorbol myristate acetate (PMA) or with 60 micrograms/ml of immune complex. The effect of each compound differed, i.e. depolarization was enhanced by some and inhibited by others, while H2O2 generation was inhibited by each, supporting our previous findings that membrane potential depolarization and the respiratory burst are dissociable events. Concentration-response experiments, performed at flavonoid concentrations between 12.5 and 500 microM to determine the IC50 values of these compounds for depolarization and burst activation, indicated that none of the flavonoids affected the resting potential, while all perturbed the stimulus-coupled response, the direction and extent of the perturbation depending upon the stimulus, and the function assessed. These data show that the effects of flavonoids on human neutrophils are complex and suggest several sites of action depending upon the flavonoids subcellular distribution and pathway of stimulation.

Effects of amiloride on the response of human platelets to bovine α thrombin

Abstract We have examined the effect of amiloride, a drug known to block the passive movement of Na+ across cell membranes, on the response of the platelet to thrombin stimulation. Preincubation of platelets with 10−4 M or 10−3 M amiloride causes proportional degrees of inhibition of platelet aggregation and release in response to low (≤0.01 U/ml) levels of bovine α-thrombin. The inhibition is overcome by high concentrations of α-thrombin. It can be shown that 10−4 M amiloride inhibits the decrease in platelet trans-membrane potential induced by thrombin and that 10−3 M amiloride leads to an increase in that potential, implying that the depolarization observed to occur in response to thrombin stimulation is due to increased membrane permeability to Na+. We conclude that such a change in membrane permeability plays a role in the platelets response to low levels of thrombin, but not in the response to higher levels. In addition to the above effect we can demonstrate that amiloride inhibits the active transport of serotonin, whose transport is Na+ dependent, into the platelet.

Dissociation of human neutrophil membrane depolarization, respiratory burst stimulation and phospholipid metabolism by quinacrine

Human neutrophils generate a respiratory burst with the elaboration of toxic oxygen metabolites upon appropriate stimulation. Subsequent to receptor—ligand interaction, the activation pathway of this burst is unknown. Here, attempts to correlate phospholipid turnover have demonstrated dissociation of lipid flux and burst activation. Quinacrine inhibited membrane depolarization, superoxide (O− 2) generation, and net phosphatidylserine production with ID 50‐values of 16 μM, 73 μM and >500 μM, respectively. The inhibitory profiles of these neutrophil activation parameters demonstrate a dissociation between membrane depolarization, respiratory burst stimulation, and phospholipid turnover.

Phospholipase D mediates Fc gamma receptor activation of neutrophils and provides specificity between high-valency immune complexes and fMLP signaling pathways.

Neutrophils phagocytize high‐valency immune complexes (HIC) by an Fcγ receptor‐mediated mechanism, activating an oxidative burst and initiating degranulation. In contrast, neutrophils exhibit chemotaxis to N‐formylated peptides [e.g., N‐formylmethionyl‐leucyl‐phenylalanine (fMLP)] and secrete far fewer oxidants or granule contents than neutrophils activated by HIC. However, if neutrophils are treated with cytochalasin B (CB) or permeabilized with streptolysin O, chemoattractant‐induced neutrophil secretion is increased to a level beyond that observed in response to HIC. Because priming neutrophils with CB, or permeabilizing them, also augments activation of phospholipase D (PLD) in response to fMLP, we reasoned that, in intact (i.e., nonpermeabilized) unprimed neutrophils, PLD may participate in a signaling pathway specific to phagocytic stimuli such as HIC and hence may contribute to degranulation control. PLD activity in response to HIC and fMLP correlated closely with stimulus‐induced azurophilic degranulation under a wide variety of experimental conditions, including compounds that abrogated or augmented stimulus‐induced PLD action. PLD activation preceded, and appeared to be necessary for, azurophilic degranulation. These results suggest that PLD may play a central role in controlling azurophilic degranulation and provide signaling specificity between pathways activated by fMLP and HIC in intact neutrophils. J. Leukoc. Biol. 61: 522–528; 1997.

Neutrophil degranulation and phospholipase D activation are enhanced if the Na+/H+ antiport is blocked.

Neutrophils phagocytize high‐valency immune complexes (HIC) by an Fc receptor‐mediated mechanism. Engaging Fc receptors in this manner induces PMN to generate superoxide and release the contents of both their specific and azurophilic granules. Signaling events that precede and accompany PMN secretion include activation of phospholipase D (PLD), as well as changes in cytoplasmic [Ca2+] (δ[Ca2+]in) and pH (δpHin). Although the role of PLD and δ[Ca2+]in in mediating Fc receptor‐mediated PMN secretion has been studied, whether pHin plays a regulatory role has not yet been defined. HIC‐stimulated PMN undergo an intracellular acidification followed by a prolonged Na+/H+ antiport‐mediated alkalinization. To investigate the role of the pH transient in controlling degranulation, the Na+/H+ antiport was inhibited either with 100 μM dimethylamiloride (DMA) or by substituting N‐methyl‐glucamine for extracellular sodium. Blocking the antiport with DMA led to hyper‐acidified PMN, which exhibited an increase in degranulation, but did not affect generation of superoxide. DMA did not alter the ability of neutrophils to phagocytose and oxidize dichlorodihydrofluoresceinated HIC, suggesting the increase in degranulation was not the result of failed phagocytosis. Investigation into whether the observed increase in degranulation when the antiport was blocked was mediated by PLD or δ[Ca2+]in revealed that blocking the antiport increased HIC‐induced PLD activity but had no effect on HIC‐induced δ[Ca2+]in. Blocking the Na+/H+ antiport by ion substitution caused similar effects on PMN signaling and secretion as was seen with DMA. These results indicate that Na+/H+ antiport activity is not necessary for degranulation or superoxide release in HIC‐stimulated PMN and that hyperacidification of the cytoplasm can modulate degranulation. Therefore, pHin, via its effect on PLD, may be a control point of degranulation and may represent one way that neutrophils achieve differential control of their antibacterial products. J. Leukoc. Biol. 64: 98–103; 1998.

A cytosolic calcium transient is not necessary for degranulation or oxidative burst in immune complex‐stimulated neutrophils

Receptor‐mediated activation of neutrophils (PMN) initiates possibly interdependent events, including a rapid transient increase in [Ca2+]i, implicated as a second messenger. To investigate whether this transient is required for eventual degranulation, PMN were incubated with an intracellular Ca2+ chelator (BAPTA), then exposed to chemotactic peptide [N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP)] with or without cytochalasin B (CB) or to high‐valency immune complexes (HIC); δ[Ca2+]i, δpHi, oxidative burst, and elastase release were then evaluated (plus or minus EGTA 15 s before stimulation) after 2 and 15 min incubation in 0.9 mM Ca2+. With either fMLP plus CB or HIC stimulation, BAPTA‐treated cells were unable to achieve a Ca2+ transient with a 2‐min incubation, whereas a 15‐min incubation allowed the BAPTA‐treated cells to recover a portion of the δ[Ca2+]i. Even though BAPTA‐treated cells were unable to mount a δ[Ca2+]i at 2 min, HIC‐stimulated BAPTA‐treated cells were able to elicit an oxidative burst (33% of control) and degranulation (67% of control). Therefore, we conclude that δ[Ca2+]i modulates but is not required for oxidative burst or degranulation. J. Leukoc. Biol. 62: 329–340; 1997.