Oscillations in Superoxide Anion Release by Polymorphonuclear Leukocytes and its Inhibition by Human Saliva

The release of superoxide anion (O 2 -) by inflammatory polymorphonuclear leukocytes (PMN), obtained from the intraperitoneal cavity of mice, was investigated during two different times of the day. NaF-stimulated PMN at nigth-phase showed greater release of O 2 - than at day. Addition of human saliva in the culture medium dramatically inhibits O 2 - generation by both day- and night-phase cells. Also, the inhibitory effect of diurnal saliva on the cell activation is higher than the nocturnal saliva.     

Stimulus-specific effects of endotoxin on superoxide production by rabbit polymorphonuclear leukocytes

The release of superoxide (O2-) by polymorphonuclear leukocytes (PMN) is an important function that contributes to microbial death. Controversy exists as to the effect of bacterial endotoxin (lipopolysaccharide, or LPS) on the production of O2-. We have injected rabbits with 25 micrograms Escherichia coli LPS intravenously and studied PMN function 18 to 24 hours later. Relative to PMN from saline-injected controls, PMN from LPS-treated rabbits released markedly greater amounts of O2- in response to 10 ng/ml phorbol myristate acetate (PMA) as measured by nmol cytochrome C reduced in 20 minutes (40.8 +/- 7.8 for LPS-treated PMN versus 10.1 +/- 1.6 for control, p less than 0.01). LPS injection, however, significantly reduced O2- release in response to C (complement) 5a (1.4 +/- 0.6 nmole/20 minutes for LPS-treated PMN versus 5.6 +/- 1.3 nmole/20 minutes for control, p less than 0.01). O2- release in response to a third stimulus, n-formyl-methionyl-leucyl-phenylalanine (10(-7) to 10(-9) M), was not affected by LPS. O2- release in response to PMA was enhanced over a wide range of PMA concentrations (10 to 300 ng/ml). Kinetic studies over 30 minutes indicated that, after a brief initial latency in measurable response, LPS enhanced responsiveness to PMA at all time points observed. The reduced responsiveness to C5a corresponds to a previously reported down regulation of receptors for this ligand after intravenous LPS. The observations indicate that intravenous LPS can alter a critical function of PMN for at least 24 hours in a stimulus-specific manner.     

Phagocytosis of immunoglobulin G and C3-bound human sperm by human polymorphonuclear leukocytes is not associated with the release of oxidative radicals.

Antisperm antibody (ASA)- and complement (C)-mediated immune injury to human sperm is thought to be caused in part by phagocytic neutrophils. To investigate this process, we co-cultured purified human polymorphonuclear leukocytes (PMN) with swim-up sperm in the presence of ASA-positive and ASA-negative sera and assayed for PMN respiratory burst activity, monitored by the release of superoxide anion (O2-) and hydrogen peroxide (H2O2). Phorbol myristate acetate (PMA) and opsonized zymosan were used as positive controls. Phagocytosis of ASA-positive and C-bound sperm by PMN did not enhance O2- production when compared to incubation of sperm with ASA-negative sera. Phagocytosis of ASA-positive and C-bound sperm also resulted in minimal release of H2O2 when compared with ASA-positive and C-negative sperm that were not phagocytosed. In contrast, PMN were maximally stimulated to release O2- in response to either opsonized zymosan or PMA. The kinetics of PMA-induced O2- release was unaffected by the presence of ASA-positive and C-bound sperm. Cytocentrifuge preparations of PMN incubated with ASA-positive and C-bound sperm revealed limited O2- release at the site of PMN/sperm contact. These results indicated that 1) phagocytosis of motile sperm by PMN requires the binding of both ASA and C to the sperm surface; 2) phagocytosis of ASA-positive and C-positive sperm by PMN fails to release reactive oxygen species; and 3) metabolic processes associated with PMN respiratory burst activity may not be coupled to the ingestion of ASA-positive and C-bound sperm.     

Activation of Neutrophils is Daily Inhibited by Saliva

The circadian release of superoxide anion (O 2 ?) by inflammatory neutrophils was investigated with mice kept under a constant light regime. Neutrophils were elicited to the peritoneal cavity by injection of 12% sodium caseinate-PBS solution, collected and stimulated to release O 2 ? to the culture milieu upon the addition of phorbol 12-myristate 13-acetate (PMA). The night phase neutrophils were able to release three fold more O 2 ? than day phase cells. Furthermore, the addition of human saliva in neutrophils cultures dramatically inhibits O 2 ? generation. Diurnal human saliva inhibits more effectively neutrophils activation than the nocturnal saliva. The PMA stimulation induces the synthesis of 13 kDa and 14 kDa molecular mass proteins detected in autoradiograms, and saliva additions decrease their synthesis. These two proteins are well known to be essential to the NAD(P)H oxidase assembly complex and, therefore, making neutrophils able to exert their main function as phagocytic cells.     

Activation of Neutrophils is Daily Inhibited by Saliva

The circadian release of superoxide anion (O 2 ? ) by inflammatory neutrophils was investigated with mice kept under a constant light regime. Neutrophils were elicited to the peritoneal cavity by injection of 12% sodium caseinate-PBS solution, collected and stimulated to release O 2 fito the culture milieu upon the addition of phorbol 12-myristate 13-acetate (PMA). The night phase neutrophils were able to release three fold more O 2 fithan day phase cells. Furthermore, the addition of human saliva in neutrophils cultures dramatically inhibits O 2 figeneration. Diurnal human saliva inhibits more effectively neutrophils activation than the nocturnal saliva. The PMA stimulation induces the synthesis of 13 kDa and 14 kDa molecular mass proteins detected in autoradiograms, and saliva additions decrease their synthesis. These two proteins are well known to be essential to the NAD(P)H oxidase assembly complex and, therefore, making neutrophils able to exert their main function as phagocytic cells.     

Alteration of human granulocyte functional responses by menadione.

Menadione is a synthetic derivative of the natural vitamins K with antiinflammatory activity among its potentially significant clinical properties. We have found this agent to stimulate the production of superoxide anion (O2-) in human polymorphonuclear leukocytes (PMN) and dimethylsulfoxide-differentiated HL-60 cells in a time-, cell number-, and drug concentration-dependent manner. Conversely, menadione attenuates both O2- production and lysozyme release in cells stimulated by phorbol myristate acetate (PMA), fMet-Leu-Phe, or Ca2+ ionophore. 4-Acetamido-4'-isothiocyano-2-2'-disulfonic acid stilbene and 4,4'-diisothiocyano-2-2'disulfonic acid stilbene, agents which inhibit transmembrane O2-) flux, do not alter menadione's effects on superoxide dismutase (SOD) inhibitable cytochrome c reduction in resting or PMA-stimulated PMN. Likewise, quinone reductase inhibitors, warfarin and dicumarol, known to attenuate vitamin K-dependent responses and enhance quinone-mediated oxidative stress, have no effect upon menadione-stimulated O2- production. Furthermore, menadione-induced suppression of stimulus-mediated lysozyme release is not reversed by cotreatment with oxygen metabolite scavenging enzymes SOD and catalase. Nevertheless, under conditions of restricted oxygen supply, the suppressive effect of menadione on stimulant-induced lysozyme release is greatly diminished. Thus, although pharmacological manipulation suggests otherwise, there appears to exist at least a component of the inhibitory activity of menadione that is oxygen dependent, and may be oxidative stress-related.     

Role of polymorphonuclear cells in Chagas' disease. I. Uptake and mechanisms of destruction of intracellular (amastigote) forms of Trypanosoma cruzi by human neutrophils

The ability of human polymorphonuclear cells (PMN) to take up and destroy intracellular forms of Trypanosoma cruzi (AMA) was investigated as a part of our efforts to elucidate the mechanisms of clearing of these parasites from infected tissues. PMN were found to take up AMA and destroyed parasites were seen after 30 min of cell-parasite interaction. Under our experimental conditions, the rate of uptake of AMA by PMN was maximal during the first 30 min of interaction. AMA were found to be located and destroyed inside the phagolysosomal vacuoles of PMN. The parasite was never found outside these vacuoles despite electron microscopic examination of numerous preparations derived from several experiments. Intracellular destruction of AMA by PMN was visible by electron microscopy and could be monitored by measuring the release of 3H-labeled substances by PMN that had ingested radiolabeled AMA. PMN incubated after removal of unbound parasites destroyed over 90% of the ingested organisms within 3 hr and close to 99% after 12 hr. In cellfree systems, 44% of the AMA were destroyed in the presence of 10(-4) M H2O2 and all of the parasites died at 10(-3) M. Addition of lactoperoxidase and iodide resulted in 100% killing at 10(-5) M H2O2. These mechanisms appeared to be involved in the lysis of AMA by PMN since both H2O2 and peroxidase activity were demonstrated to be present in PMN vacuoles containing the parasite. Addition of NaN3, KCN (inhibitors of myeloperoxidase activity) or catalase (to decompose H2O2) caused a marked reduction in the extent of AMA killing by PMN. Xanthine oxidase was toxic for the AMA in the presence of acetaldehyde. This microbicidal activity was inhibited by catalase but not by heat-inactivated catalase or by reagents that scavenge the intermediate products of reduction of molecular oxygen, O - X 2, X OH, and 1O2. These results suggest that PMN have the potential of clearing AMA liberated in infected chagasic tissues and that parasite killing within the phagolysosomal vacuoles is mediated by myeloperoxidase activity and H2O2.     

Production of H2O2 by bovine blood and milk polymorphonuclear leucocytes

The ability of bovine polymorphonuclear leucocytes (PMN) to release H2O2 was investigated. Resting PMN suspended in buffer released only small amounts of H2O2 which was appreciably increased during phagocytosis of heat killed coliforms. However, in the presence of bovine serum (BS), foetal calf serum (FCS) and milk whey (MW) no increase of H2O2 could be detected unless sodium azide (NaN2) was added. It appears that the enzyme content of these fluids (catalase and lactoperoxidase) consumed the released H2O2 and NaN2, which inactivates these enzymes, abolished this interference. Live organisms required BS or MW both for phagocytosis and for H2O2 production. Bovine IgG2 and, to a lesser extent, IgG1 but not SIgA or IgM stimulated the release of H2O2 independently of phagocytosis; this indicates the presence of receptors specific for IgG2 and IgG1 on the cell surface. Ingestion of casein micelles triggered the greatest burst of H2O2 production by cells suspended in buffer. In general, PMN isolated from blood were more active than cells isolated from milk. Since the extracellular release of H2O2 reflects the intracellular level of H2O2, the lower metabolic activity of milk PMN may contribute to the lesser intracellular bactericidal activity of milk leucocytes. The possibility that the release of H2O2 may activate extracellularly the lactoperoxidase system, known to be bactericidal in milk, is discussed.     

Biphasic regulation of leukocyte superoxide generation by nitric oxide and peroxynitrite

Activation of the NADPH oxidase-derived oxidant burst of polymorphonuclear leukocytes (PMNs) is of critical importance in inflammatory disease. PMN-derived superoxide (O(2)) can be scavenged by nitric oxide (NO( small middle dot)) with the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the effects and mechanisms by which NO( small middle dot) and ONOO(-) modulate the PMN oxidative burst. Therefore, we directly measured the dose-dependent effects of NO( small middle dot) and ONOO(-) on O(2) generation from human PMNs stimulated with phorbol 12-myristate 13-acetate using EPR spin trapping. Pretreatment with low physiological (microm) concentrations of NO( small middle dot) from NO( small middle dot) gas had no effect on PMN O(2) generation, whereas high levels (> or =50 microm) exerted inhibition. With ONOO(-) pretreatment, however, a biphasic modulation of O(2) generation was seen with stimulation by microm levels, but inhibition at higher levels. With the NO( small middle dot) donor NOR-1, which provides more sustained release of NO( small middle dot) persisting at the time of O(2) generation, a similar biphasic modulation of O(2) generation was seen, and this was inhibited by ONOO(-) scavengers. The enhancement of O(2) generation by low concentrations of ONOO(-) or NOR-1 was associated with activation of the ERK MAPKs and was blocked by their inhibition. Thus, low physiological levels of NO( small middle dot) present following PMN activation are converted to ONOO(-), which enhances O(2) generation through activation of the ERK MAPK pathway, whereas higher levels of NO( small middle dot) or ONOO(-) feed back and inhibit O(2) generation. This biphasic concentration-dependent regulation of the PMN oxidant burst by NO( small middle dot)-derived ONOO(-) may be of critical importance in regulating the process of inflammation.     

Mechanism of neutrophil activation by an unopsonized inflammatory particulate. Monosodium urate crystals induce pertussis toxin-insensitive hydrolysis of phosphatidylinositol 4,5-bisphosphate.

Monosodium urate crystals are believed to trigger acute inflammation via the direct stimulation of leukocytes. Unopsonized urate crystals activate neutrophil (PMN) membrane G proteins in a pertussis toxin (PT)-sensitive manner, but induce PT-insensitive cytosolic [Ca2+]i elevation. Thus, we have further defined the mechanism of PMN responsiveness to urate crystals in this study. Though urate crystals can increase membrane permeability by lytic effects, we observed elevation of PMN cytosolic [Ca2+]i in the absence of extracellular [Ca2+]i. In addition, the early, crystal-induced cytosolic [Ca2+]i transient was buffered in cells loaded with a [Ca2+]i-chelator. This suggested mobilization of internal [Ca2+]i stores, which was supported by demonstrating rapid phosphatidylinositol bisphosphate (PIP2) hydrolysis, and the formation of inositol (1,4,5) trisphosphate (as well as phosphatidic acid) in a PT-insensitive manner. Importantly, PMN activation by urate crystals was discriminatory, as evidenced by the absence of phosphatidylinositol trisphosphate formation, a PT-sensitive event triggered by chemotactic factors. Urate crystal-induced PIP2 hydrolysis was not a nonspecific consequence of the early cytosolic [Ca2+]i transient itself, and it did not require phagocytosis. However, crystal-induced O2- release was markedly inhibited by buffering of the early cytosolic [Ca2+]i transient under conditions where crystal phagocytosis and PMA-induced O2- release were unaffected. We conclude that urate crystals activate PT-insensitive PIP2 hydrolysis, resulting in IP3 generation, and early urate crystal-induced mobilization of cytosolic [Ca2+]i. This pathway appears to modulate crystal-induced O2- release.     

A novel role of growth hormone and insulin-like growth factor-I. Priming neutrophils for superoxide anion secretion.

Growth hormone (GH) and the GH-dependent growth promoting peptide, insulin-like growth factor-I (IGF-I), are both potent signals for priming human and porcine polymorphonuclear neutrophils (PMN) to secrete superoxide anion (O2-). PMA, opsonized-zymosan, or FMLP could all be used as triggering stimuli to demonstrate priming by GH or IGF-I. As positive controls, IFN-gamma and LPS also primed both human and porcine PMN for enhanced O2- release. However, only the LPS-mediated enhancement was inhibited by polymyxin B, which demonstrates that the priming induced by GH, IGF-I, or IFN-gamma was not caused by LPS contamination. Furthermore, a specific antibody to GH abrogated priming induced by this molecule. In contrast to IGF-I, the closely related molecule insulin was unable to prime PMN even at pharmacologic levels. Insulin, at pharmacologic levels, antagonized the priming mediated by IGF-I but had no effect on GH priming. A mAb directed against the human IGF-I receptor blocked the enhanced secretion of O2- by human PMN that was caused by IGF-I, but not GH, indicating that neutrophil priming induced by GH was not mediated by inducing extracellular release of IGF-I. However, priming PMN by both GH and IGF-I required de novo protein synthesis, because cycloheximide completely abrogated enhanced O2- secretion that was caused by these growth factors. These data show that a classic pituitary hormone (GH), as well as its widely recognized growth promoting peptide (IGF-I), are involved in regulating an important functional activity of both porcine and human PMN. Inasmuch as GH and IGF-I have recently been demonstrated to be synthesized by leukocytes, these data support the possibility that both of these proteins could act in a paracrine fashion as cytokines to prime PMN for an enhanced respiratory burst.     

The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.     

Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a Ficoll-Hypaque gradient

Polymorphonuclear neutrophils (PMN) have an important role in the host defence response to infection. These cells produce large amounts of reactive oxygen species (O(2).(-), H(2)O(2) and ONOO(-)) with microbicidal activity. PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density (Ficoll-Hypaque gradient and dextran), yielding a highly homogeneous cellular population. However, some cellular activation due to membrane perturbation is also expected. We studied how the production of reactive oxygen species and release of myeloperoxidase (MPO) from blood PMN are affected by the use of the Ficoll-Hypaque density gradient. PMN isolated by spontaneous sedimentation and total blood were used for comparisons. Lucigenin- and luminol-enhanced chemiluminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli. The release of MPO, estimated by the chemiluminescence of the luminol/H(2)O(2) reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B, was also higher for PMN isolated by a density gradient. In conclusion, it was shown that the PMN isolation procedure affects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results.     

In vitro effects of certain membrane-acting agents on superoxide and hydrogen peroxide production, protein synthesis and membrane ATPase activity in buffalo PMN cells

Polymorphonuclear (PMN) cells play a key role in innate immunity, due to their ability to produce reactive oxidants such as superoxide (O(2-)) and hydrogen peroxide (H(2)O(2)), and to release antimicrobial proteins and peptides stored in their lysosomal granules. In the present study, the effects of the activation of buffalo PMN cells with various membrane-acting agents were evaluated in terms of O(2-) and H(2)O(2) production, the activities of membrane ATPases, and protein synthesis. Studies involving the incorporation of (35)S-methionine revealed significant protein-synthesising ability in resting PMN cells and in cells treated with lipopolysaccharide (LPS), as well as with opsonised zymosan (OZ). Protein synthesis, as judged by fluorography of the cytosolic fraction, showed more than 12 bands, whilst the cytoskeletal fraction showed 2-3 bands. PMN activation with concanavalin A (ConA), digitonin and sodium nitroprusside (SNP) resulted in increased O(2-) and H(2)O(2) production. However, in the presence of anti-inflammatory agents such as indomethacin and cortisol, the production of O(2-) and H(2)O(2) by these cells was found to decline. Studies pertaining to membrane ATPases revealed that verapamil hydrochloride (VpHCl) significantly increased total ATPase and Na(+)K(+)ATPase activity. ConA treatment yielded only a moderate level of activity. Similarly, digitonin up to 24microM also caused a significant increase in ATPase activity. Our observations indicate that these membrane-acting agents influenced oxygen-dependent and oxygen-independent microbicidal mechanisms in buffalo PMN cells.     

Inhibition by reactive aldehydes of superoxide anion radical production in stimulated human neutrophils

alpha,beta-Unsaturated aldehydes were investigated in vitro for their ability to inhibit superoxide anion radical (O2-.) production in stimulated human polymorphonuclear leukocytes (PMN). The aldehydes investigated were (i) trans-4-hydroxynonenal and malonaldehyde (MDA), two toxic lipid peroxidation products; (ii) acrolein and crotonaldehyde, two air pollutants derived from fossil fuel combustion; (iii) trans,trans-muconaldehyde, a putative hematotoxic benzene metabolite. Preincubation of PMN with reactive aldehydes followed by stimulation with the oxygen burst initiator phorbol myristate acetate (PMA) resulted in a dose-dependent inhibition of O2-. production. The concentration at which 50% inhibition (IC50) was observed was 21 microM for acrolein, 23 microM for trans,trans-muconaldehyde, 27 microM for trans-4-hydroxynonenal and 330 microM for crotonaldehyde. A similar inhibitory effect by these aldehydes was observed in digitonin- and concanavalin A-stimulated PMN. MDA inhibited O2-. production in PMA-stimulated PMN by 100% at 10(-2) M but gave no inhibition at 10(-3) M. The standard aldehyde propionaldehyde did not inhibit O2-. production at 10(-3)-10(-6) M. Preincubation of PMN with acrolein in the presence of cysteine completely protected against the inhibitory effect of this reactive aldehyde. The results indicate that the ability of toxic aldehydes to inhibit O2-. production in stimulated PMN correlates directly with their alkylation potential which is a function of the electrophilicity of the beta carbon.     

Pre-existing glomerular immune complexes induce polymorphonuclear cell recruitment through an Fc receptor-dependent respiratory burst: potential role in the perpetuation of immune nephritis

In immune complex (IC) diseases, FcR are essential molecules facilitating polymorphonuclear cell (PMN) recruitment and effector functions at the IC site. Although FcR-dependent initial tethering and FcR/integrin-dependent PMN accumulation were postulated, their underlying mechanisms remain unclear. We here addressed potential mechanisms involved in PMN recruitment in acute IC glomerulonephritis (nephrotoxic nephritis). Since some renal cells may be recruited from bone marrow (BM) lineages, reconstitution studies with BM chimeras and PMN transfer between wild-type (WT) and FcR-deficient mice (gamma(-/-)) were performed. Severe glomerular damage was induced in WT and W gamma chimeras (BM from WT to irradiated gamma(-/-)), while it was absent in gamma(-/-) and gamma W chimeras (gamma(-/-) BM to WT). Moreover, WT PMN transfer, but not gamma(-/-) PMN, reconstituted the disease in gamma(-/-), indicating that FcR on resident cells is not a prerequisite for PMN recruitment in this disease. Surprisingly, transferred WT PMN were recruited coincidentally with NF-kappa B activation and TNF-alpha overexpression even in glomeruli with preformed IC (nephrotoxic Ab administered 3 days previously), suggesting that PMN can initially be recruited via its own FcR without previous chemoattractant release. Furthermore, H(2)O(2) inhibition by catalase attenuated the acute WT PMN recruitment and the induction of NF-kappa B and TNF-alpha much more than integrin (CD18) blockade, indicating a role for the respiratory burst before integrin-dependent accumulation. In coculture experiments with IC-stimulated PMN and glomeruli, PMN caused acute glomerular TNF-alpha expression predominantly via FcR-mediated H(2)O(2) production. In conclusion, glomerular IC, even preformed, can cause PMN recruitment and injury through PMN FcR-mediated respiratory burst during initial PMN tethering to IC.     

Functional activity of enucleated human polymorphonuclear leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.     

Modulation of phagocytosis-associated respiratory burst by human cystatin C: role of the N-terminal tetrapeptide Lys-Pro-Pro-Arg

Cystatin C, a cysteine protease inhibitor, has recently been suggested to be a potent regulator in inflammatory processes. Human cystatin C was isolated from the urine of one patient suffering from tubular disorders and was tested for its effects on two functions of human polymorphonuclear neutrophils (PMN): O2- release and phagocytosis. Slow-form or (des 1-4) cystatin C and fast-form or (des 1-8) cystatin C differed by the presence in (des 1-4) cystatin C only of the N-terminal tetrapeptide Lys-Pro-Pro-Arg. Whereas (des 1-8) cystatin C did not seem to interfere with PMN functions at physiological concentrations, (des 1-4) cystatin C induced an inhibition of PMN phagocytosis-associated respiratory burst in response to opsonized zymosan particles. The inhibition may be attributed to the tetrapeptide Lys-Pro-Pro-Arg which has been synthesized and shown to have the same inhibitor effects, at concentrations similar to those required for (des 1-4) cystatin C. These results support a potential role for cystatin C as a modulator during inflammation.     

Polymorphonuclear leukocyte-mediated, antibody-dependent, cellular cytotoxicity against tumor cells: dependence on oxygen and the respiratory burst.

Experiments were done to determine 1) whether the respiratory burst of superoxide anion (O2-) production in polymorphonuclear leukocytes (PMN) is triggered during antibody-dependent killing of tumor cells and 2) whether O2- production is essential for cytotoxicity. Three parameters of the respiratory burst (1-14C-glucose oxidation, oxygen consumption, and O2- release) were increased 2.5- to 7.3-fold during killing of antibody-primed tumor cells by human PMN. Added catalase and superoxide dismutase did not inhibit lysis, possibly because these enzymes were unable to diffuse into the inter-plasma-membrane space between killer and target cells. Evidence for an O2- requirement for cytotoxicity was the fact that concentrations of amobarbital or phenylbutazone sufficient to inhibit the cyanide-insensitive respiration of PMN also inhibited cytotoxicity. Also, hypoxic conditions inhibited cytotoxicity from 29 to 73%. The requirement for oxygen was most likely related to O2- generation and not mitochondrial respiration since cyanide and azide, which inhibit mitochondrial respiration, increased cytotoxicity.     

Novel synthesis of S-nitrosoglutathione and degradation by human neutrophils.

S-nitrosoglutathione (SNO-GSH), a stable derivative of nitric oxide, is an endothelium-derived relaxation factor, which provokes vasodilation, inhibits platelet aggregation, and inhibits neutrophil (PMN) superoxide anion (O2+) generation. We have established a novel method for synthesis of S-nitrosoglutathione using a column containing S-nitrosothiol covalently attached to agarose. S-nitrosoglutathione was a product as assessed after separation using C-18 reverse-phase HPLC and absorption spectroscopy. We examined the stability of SNO-GSH in the presence or absence of PMN. The half-life (mercuric acid diazotization) of SNO-GSH in Hepes was greater than 60 min. The addition of resting PMN did not affect the T1/2 of SNO-GSH. PMN exposed to N-fMet-Leu-Phe (FMLP, 10(-7) M) reduced measurable SNO-GSH (15 microM) at 5 min (48 +/- 5.0% control, P less than 0.05). Incubation (5 min, 37 degrees C) of PMN with 10 microM tenidap (an anti-inflammatory drug which inhibits PMN activation) before addition of FMLP blocked the PMN-dependent degradation of SNO-GSH (42 +/- 3 vs 78 +/- 1.3% control, P = 0.01). We confirmed the recovery of SNO-GSH through measurements by bioassay (platelet aggregation) and HPLC analysis. The degradation of S-nitrosothiols by activated neutrophils may reverse the inhibitory effect of S-nitrosothiols on PMN functions and contribute to tissue injury at sites of inflammation.