Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.

Alkalinizing the intralysosomal pH inhibits degranulation of human neutrophils.

Degranulation of lysosomes is one of the consequences of neutrophil activation. Regulatory mechanisms of lysosomal secretion are thought to be localized largely in the plasma membrane and cytosol, with the lysosome playing a passive role in secretion. Recent evidence indicates that the intralysosomal pH is highly acidic (pH congruent to 5.5) and is maintained by active transport of H+. We investigated whether changes in the intralysosomal pH altered the availability of lysosomes for exocytosis. Intralysosomal pH in intact neutrophils was monitored with the weakly basic fluorescent probe, 9-aminoacridine (9AA). The weak bases, methylamine, chloroquine, clindamycin, propanolol, and ammonium chloride (0.1-50 mM), caused an alkalinization of the intralysosomal pH as determined by reversal of quenching of 9AA fluorescence. Similarly, each of the weak bases, including ammonium chloride, methylamine, chloroquine, ethylamine, propylamine, propanolol, clindamycin, and dansylcadaverine, inhibited neutrophil degranulation in response to the calcium ionophore A23187, phorbol myristate acetate, or the chemotactic peptide, formyl-methionine-leucine-phenylalanine plus cytochalasin B. These studies indicate that an acid intralysosomal pH is important to the neutrophil secretory response and suggest that the lysosome may play an active part in control of degranulation.

Human leukocytic pyrogen induces release of specific granule contents from human neutrophils.

The ability of highly purified human leukocytic pyrogen (LP) to induce neutrophil lysosomal protein release is described. Human peripheral blood neutrophils isolated by Ficoll-Hypaque and dextran sedimentation were exposed to purified human LP. The specific granule-associated proteins, lysozyme and lactoferrin were selectively released, whereas primary granule (beta-glucuronidase) and cytoplasmic (lactic dehydrogenase) enzyme markers were not. Optimum release was observed after 45 min in the presence of Ca++ and Mg++. Cytochalasin B (5 microgram/ml) had no effect on LP-induced lysosomal enzyme release. Since the pyrogenicity of LP is dependent on prostaglandin synthesis, the effect of two potent inhibitors of prostaglandin synthesis on lysozyme release was studied. Both indomethacin and naproxen failed to inhibit specific granule protein release. These observations suggest that the concommitance of fever, elevated serum or urine lysozyme and hypoferremia may, in part, be explained by the interaction of LP and peripheral blood neutrophils.

Stimulation of neutrophil oxygen-dependent metabolism by human leukocytic pyrogen.

The ability of highly purified human leukocytic pyrogen (LP) to stimulate neutrophil oxygen-dependent metabolism was studied. Human peripheral blood neutrophils exposed to leukocytic pyrogen in vitro demonstrated an increase in the percentage of neutrophils reducing nitroblue tetrazolium (NBT) dye and a marked stimulation of superoxide dismutase inhibitable reduction of ferricytochrome c. LP stimulation of neutrophil oxygen-dependent metabolism was dose and time dependent. Procedures that destroyed the pyrogenicity of LP also abolished the effects on neutrophil metabolism. Neutrophil hexose monophosphate shunt activity was also stimulated by LP. In a rabbit model, the effect of in vivo LP on neutrophil superoxide generation was also studied. There was a consistent increase in the percent and absolute number of NBT positive neutrophils. Peak stimulation of neutrophil metabolism occurred after defervescence suggesting several possible mechanisms. The observations reported here may, in part, explain the nonspecificity of the NBT test in febrile, noninfected patients and provide further understanding of neutrophil physiology during acute inflammation.

Identification of a human neutrophil angiotension II-generating protease as cathepsin G.

A human neutrophil protease, initially termed neutral peptide-generating protease, has been shown to cleave angiotensin II directly from angiotensinogen and has been identified as leukocyte cathepsin G. When purified neutrophils were disrupted by nitrogen cavitation and fractionated by differential centrifugation, 44 and 24% of the angiotensin II-generating activity was in the lysosomal and undisrupted cell fractions, respectively. Cytochalasin B-treated human neutrophils stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine released beta-glucuronidase, lysozyme, and angiotensin II-generating protease in a dose-dependent fashion, consistent with localization of this protease to the neutrophil granule. Individually purified angiotensin II-generating protease and cathepsin G had similar proteolytic and esterolytic activity for angiotensinogen and N-benzoyl-L-tyrosine ethyl ester on a weight basis, exhibited identical mobilities by SDS-gradient polyacrylamide gel electrophoresis and pH 4.3 disc-gel electrophoresis, and gave precipitin lines of antigenic identity on Ouchterlony analysis with goat antibody to the angiotensin II-generating protease. Thus, the angiotensin II-generating protease of human neutrophils has been identified as cathepsin G on the basis of subcellular localization, substrate specificity, physicochemical characteristics, and antigenic identity.

Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp(-) Escherichia coli.

Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.

Characterization of the surface enhanced raman scattering (SERS) of bacteria.

The surface enhanced Raman scattering (SERS) of a number of species and strains of bacteria obtained on novel gold nanoparticle (approximately 80 nm) covered SiO(2) substrates excited at 785 nm is reported. Raman cross-section enhancements of >10(4) per bacterium are found for both Gram-positive and Gram-negative bacteria on these SERS active substrates. The SERS spectra of bacteria are spectrally less congested and exhibit greater species differentiation than their corresponding non-SERS (bulk) Raman spectra at this excitation wavelength. Fluorescence observed in the bulk Raman emission of Bacillus species is not apparent in the corresponding SERS spectra. Despite the field enhancement effects arising from the nanostructured metal surface, this fluorescence component appears "quenched" due to an energy transfer process which does not diminish the Raman emission. The surface enhancement effect allows the observation of Raman spectra of single bacterial cells excited at low incident powers and short data acquisition times. SERS spectra of B. anthracis Sterne illustrate this single cell level capability. Comparison with previous SERS studies reveals how the SERS vibrational signatures are strongly dependent on the morphology and nature of the SERS active substrates. The potential of SERS for detection and identification of bacterial pathogens with species and strain specificity on these gold particle covered glassy substrates is demonstrated by these results.

Phase I trial of interleukin 2 in combination with the soluble tumor necrosis factor receptor p75 IgG chimera.

Our purpose was to determine the effective biological dose and/or maximum tolerated dose of recombinant human tumor necrosis factor receptor:IgG chimera (rhuTNFR:Fc; Immunex, Seattle, WA) in combination with interleukin 2 (IL-2) with regard to reduction in IL-2 toxicity and modulation of biological effects of high-dose IL-2 administration. Twenty-four patients with metastatic cancer were treated with escalating doses of rhuTNFR:Fc at 1, 1, 5, 10, and 20 mg/m2 i.v. on days 1 and 15 (dose levels 1-5) or 10, 20, and 30 mg/m2 days 1 and 15 plus 50% dose on days 3, 5, 17, and 19 (dose levels 6-8) prior to IL-2 at doses of 300,000 IU/kg (dose level 1) and 600,000 IU/kg (dose levels 2-8) i.v. every 8 h on days 1-5 and 15-19. The t1/2 of rhuTNFR in patients receiving IL-2 was 72 h. The median number of IL-2 doses was 24, and central nervous system, skin, and cardiac arrhythmias were the major dose-limiting toxicities. TNF bioactivity was inhibited, and the polymorphonuclear leukocyte chemotactic defect normally seen with IL-2 was not observed. Increases in C-reactive protein, IL-6, IL-8, and IL-1 receptor antagonist levels were partially suppressed relative to historical controls, whereas peripheral blood mononuclear cell phenotypes, urinary nitrate, endothelial adhesion molecule expression in skin biopsies, and cellular infiltrates in tumor biopsies were consistent with findings in patients treated with IL-2 alone. Four patients developed thyroid dysfunction. There were five responses: two complete responses (both melanoma) and three partial responses (response rate, 21%). rhuTNFR:Fc may modulate the toxicity and some of the biological effects of IL-2 while preserving antitumor activity. Dose level 6 (10 mg/m2 on days 1 and 15, and 5 mg/m2 on days 3, 5, 17, and 19) has been chosen for a randomized, double-blind, placebo-controlled trial of IL-2 with and without rhuTNFR:Fc.

An abnormal calcium uptake pump in Chediak-Higashi neutrophil lysosomes.

Calcium is mobilized from intracellular stores during phagocyte activation and appears to be involved in stimulus-response coupling in these and other cell types. Because the lysosome is a calcium-sequestering organelle in the neutrophil and abnormal lysosome morphology is associated with defective neutrophil function in Chediak-Higashi syndrome (CHS), we examined ATP-dependent calcium uptake in neutrophil lysosomes from the beige mouse model of CHS. We present findings indicating that CHS lysosomes have an enhanced capacity for ATP-dependent calcium uptake relative to control lysosomes. Kinetic analysis showed differences in Vmax and in the Km for both ATP and calcium, suggesting that both the number of lysosomal calcium uptake pumps and their substrate affinity may be altered in CHS. We conclude that a genetically determined abnormality of a subcellular calcium transport system may contribute to the structural and functional defects of CHS cells.

Recombinant human interleukin-11 does not affect functions of purified human neutrophils in vitro.

Recombinant human interleukin-11 (rHu-IL-11) is a multifunctional cytokine with thrombopoietic activity and demonstrated clinical efficacy in treating chemotherapy-induced thrombocytopenia. rHu-IL-11 also exhibits anti-inflammatory activity and is currently in clinical trials for the treatment of several inflammatory diseases. As neutrophils are involved in both innate immunity and an acute inflammatory response, the effect of rHU-IL-11 on the function of human peripheral blood neutrophils in vitro was examined. rHu-IL-11 was not cytotoxic and did not induce superoxide anion production or the release of granular enzymes from resting neutrophils. Phagocytosis and chemotaxis were unaffected. rHu-IL-11 treatment did not block the response of neutrophils to stimulation. Pretreatment with rHu-IL-11 did not reduce production of IL-8 following activation with lipopolysaccharide (LPS) or zymosan A particles. Pretreatment with rHu-IL-11 did not affect the release of lysozyme and beta-glucuronidase in response to A23187 or PMA-stimulated production of superoxide anion. These results indicate that rHu-IL-11 does not directly modulate key functions of neutrophils in vitro.

Central nervous system disease in systemic lupus erythematosus. Therapy and prognosis.

The effect of corticosteroid therapy in 28 patients with 52 episodes of neuropyciatric disease in systemic lupus erythematosus (SLE) was elevated. Categories of organic central nervous system disease were seizures (eight patients), organic brain syndromes (nine patients), aseptic meningitis (four patients) and a variety of focal neurologic findings (seven patinets). Fourteen pateints had 15 episodes of functional psychosis without other evidence of neurologic disease. Although there was a general correlation between clinical and serologic evidnce of active SLE and the development of organic neurolgic disease, there was no evidence that therapy with very large doses of corticosteroids was beneficial. Of the deaths in this series, two were due to probable active SLE involving the central nervous system wheras five were attributable to complications of therapy. The long-term morbidity, likewise, was high in the patients who recieved large doses of corticosteroids. In all, 12 patients had major complications of corticosteroid therapy. Functional psychosis was usually preciptated by corticsoteroid therapy and respond to a reduction in steroid dosage and administration of psychotropic drugs.

Lysosomotropic behavior of adrenergic antagonists in interactions with human neutrophils.

Autonomic neurohormones affect the secretory activity of neutrophils by modulating release of lysosomal enzymes in response to immunologic stimuli. Autonomic agents are also weak bases which might modify cell function by accumulating in the acidic interior of the lysosome, in addition to their receptor-mediated activity. We examined the association of the beta-adrenergic antagonist [3H]dihydroalprenolol with human neutrophils and lysosome and membrane fractions derived from neutrophils, and the subcellular distribution of the photoaffinity-labeled beta-adrenergic ligand [3H]azidobenzylcarazolol after incubation with intact cells. Isolated neutrophil lysosomes accumulated significantly more [3H]dihydroalprenolol than isolated membrane preparations. Decreasing the transmembrane pH gradient markedly reduced [3H]dihydroalprenolol accumulation by intact cells or lysosomes but not by membranes. Since [3H]dihydroalprenolol accumulated by intact cells remained rapidly exchangeable, the photoaffinity ligand [3H]azidobenzylcarazolol was used to assess ligand distribution after association with whole cells. After cell disruption, 18.5 +/- 1.3% of this ligand appeared in the lysosome fraction as compared to 2.2 +/- 0.6% in the membrane fraction. The secretagogue phorbol myristate acetate caused release of the ligand as well as lysosomal enzymes from cells. These findings suggest that there is significant pH-dependent lysosomal accumulation of beta-antagonists. This lysosomotropic interaction may be important both as it affects the sequestration and redistribution of the drugs, and as it might in some circumstances affect host defense functions of the neutrophil.

In vitro evaluation of clindamycin in combination with oxacillin, rifampin, or vancomycin against Staphylococcus aureus.

In a study of antibiotic combinations of clindamycin with rifampin, oxacillin, or vancomycin using the time kill-curve method, the combination of clindamycin and rifampin were sometimes synergistic (5 of 15 times), otherwise indifferent and always enhanced killing of fifteen tested Staphylococcus aureus isolates. In contrast, vancomycin and clindamycin or oxacillin and clindamycin were either indifferent or antagonistic (approximately 50%). Vancomycin alone, however, was generally as effective as the combinations of clindamycin and rifampin.

Effects of pH on killing of Staphylococcus aureus and Escherichia coli by constituents of the neutrophil phagolysosome.

Lysosomotropic weak bases impair in-vitro neutrophil functions including intracellular killing of Staphylococcus aureus strain 502a. To investigate whether prevention of phagosomal acidification could account for impaired microbicidal activity, a model phagosome was formulated with a freeze-thawed granule extract as a source of lysosomal enzymes and H2O2 as a source of toxic oxygen metabolites. The lysosomal extract alone killed Escherichia coli strain S15 efficiently at pH 5.5 and 7.0, but had little activity against S. aureus 502a. Sublethal concentrations of the two agents, when combined, acted synergically against either organism. Each organism was killed more effectively at pH 5.5 than at pH 7.0 by the lysosome extract-H2O2 combination, but the killing of E. coli was more rapid than that of S. aureus in the same conditions. These findings suggest that impairment of neutrophil antistaphylococcal activity by weak bases may be mediated by their ability to raise phagosomal pH, and that persistence of E. coli in similar conditions does not occur because the latter is killed by lysosomal constituents in a non-pH-dependent fashion.

Comparison of granulocyte-macrophage colony stimulating factor and interleukin-1 production from human peripheral blood mononuclear cells as measured by specific radioimmunoassays.

Attention has focused on cytokine networks in which gene and protein expression of some cytokines is under the influence of other cytokines. In the present studies, we addressed the relationship between the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-1 (IL-1) in human peripheral blood mononuclear cells (PBMC) stimulated with mitogens. Since bioassays for cytokines are sensitive to more than one of these factors, it was necessary to measure the amounts of IL-1 and GM-CSF independent of bioassays. A specific and sensitive (40 pg/ml) radioimmunoassay (RIA) was developed for human GM-CSF. The sensitivity of the RIA was greater when lysine residues were iodinated with Bolton-Hunter reagent than tyrosine residues using chloramine T. After stimulating PBMC with concanavalin A (Con A), the biological activity of GM-CSF was determined in bone marrow cultures and compared to immunoreactive GM-CSF; GM-CSF levels detected by bioassays and RIAs were highly correlated in two separate sets of experiments (r2 = 0.95 and 0.43). Incubation with Con A for 48 h induced more GM-CSF than stimulation by phytohemagglutinin (PHA) despite the fact that PHA stimulates large amounts of IL-1 alpha; indomethacin had no effect on Con A stimulated synthesis of GM-CSF or IL-1 alpha. In two separate studies, PBMC from 14 donors and a second group of 12 donors were incubated with Con A for 48 h and the total amount of immunoreactive IL-1 alpha and GM-CSF was determined in the same cell cultures. There was no correlation of the amount of either cytokines in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro effects of omega-3 fatty acids on neutrophil intracellular calcium homeostasis and receptor expression for FMLP and LTB4.

Diets enriched in omega-3 fatty acids exert antiinflammatory properties by suppressing some neutrophil (PMN) functions. Changes in cytosolic Ca2+ concentration, [Ca2+]i, are important for PMN activation and are in part regulated by membrane Ca2+ ATPases. Since membrane proteins are influenced by their lipid environment, we investigated the in vitro effects of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the [Ca2+]i of PMNs in response to f-Met-Leu-Phe (FMLP), leukotriene B4 (LTB4), and ionomycin. The resting [Ca2+]i of PMNs (in high Ca2+ environment) was increased after pretreatment (37 degrees C, 2 h) with DHA, but not with EPA, or the other fatty acids, oleic acid (OA), or linolenic acid (LA). The stimulated [Ca2+]i by either FMLP or LTB4 was suppressed in a high Ca2+ environment after pretreatment with either EPA or DHA but not with OA or LA. The stimulated [Ca2+]i rise by ionomycin was augmented after pretreatment with DHA but not with EPA, OA, or LA. Pretreatment of PMNs with either EPA or DHA reduced the receptor expression for both FMLP and LTB4. Since omega-3 fatty acids inhibit the expression of receptors for two activators of PMNs, FMLP and LTB4, as well as the [Ca2+]i rise in response to those two stimuli, we propose that the antiinflammatory properties of EPA and DHA may be attributed, at least in part, to alteration in membrane activation of phagocytes.

Effects of leukocyte inhibitory factor (LIF) on human neutrophil function.

The effects of the lymphokine, leukocyte inhibitory factor (LIF), on human neutrophil function were studied. This soluble mediator, which is defined by its specific inhibition of neutrophil locomotion, does not interfere with chemotactic factor binding and does not affect basal or stimulated superoxide generation by neutrophils. In contrast, phagocytosis of opsonized Staphylococcus aureus is markedly inhibited by LIF, and degranulation is stimulated by this lymphokine. The possible mechanisms of LIF action on neutrophils are discussed.

Comparison of prostaglandins E2 and D2 as inhibitors of respiratory burst in neutrophils from atopic and nonatopic subjects.

Neutrophils from atopic and nonatopic donors were treated with prostaglandins D2 and E2 before stimulation of the respiratory burst. Both agents inhibited neutrophil response to formyl-methionyl-leucyl-phenylalanine, but superoxide production was inhibited much more profoundly by D2 than by E2. Inhibition was similar in atopics and nonatopics. Phorbol myristate acetate stimulation of superoxide production was not significantly altered by prostaglandins. These findings suggest that minor alterations in pathways of prostaglandin synthesis may have major effects on modulation of neutrophil function, and exploration of the mechanism of stimulus-specific inhibition may further elucidate the role of neutrophils in the inflammatory response.