Lipoteichoic acid-antilipoteichoic acid complexes induce superoxide generation by human neutrophils

Human neutrophils (PMNs) which have been incubated with lipoteichoic acid (LTA) from group A streptococci generated large amounts of Superoxide (O ₂ ^– chemiluminescence and hydrogen peroxide when challenged with anti-LTA antibodies. Cytochalasin B further enhanced O ₂ ^* generation. The onset of Of generation by the LTA-anti-LTA complexes was much faster than that induced by BSA-anti-BSA complexes. LTA-treated PMNs generated much less O ₂ ^* when challenged with BSA complexes, suggesting that LTA might have blocked, nonspecifically, some of the Fc receptors on PMNs. PMNs treated with LTA-anti-LTA complexes further interacted with bystander nonsensitized PMNs resulting in enhanced Of generation, suggesting that small numbers of LTA-sensitized PMNs might recruit additional PMNs to participate in the generation of toxic oxygen species. Protelolytic enzyme treatment of PMNs further enhanced the generation of O ₂ ^– by PMNs treated with LTA-anti-LTA. Superoxide generation could also be induced when PMNs and anti-LTA antibodies interacted with target cells (fibroblasts, epithelial cells) pretreated with LTA. This effect was also further enhanced by pretreatment of the target cells with proteases. PMNs incubated with LTA released lysosomal enzymes following treatment with anti-LTA antibodies. The amounts of phosphatase,-glucoronidase,N-acetylglucosaminidase, mannosidase, and lysozyme release by LTA-anti-LTA complexes were much smaller than those released by antibody or histone-opsonized streptococci, suggesting that opsonized particles are more efficient lysosomal enzyme releasers. However, since the amounts of O ₂ ^– generated by the LTA complexes equaled those generated by the opsonized particles, it is assumed that the signals for triggering a respiratory burst and lysosomal enzyme secretion might be different.Supported by a research grant from Dr. S. M. Robbins of Cleveland Ohio, and by grants HL-288442, HL-31963 and GM-29507, from the National Institutes of Health, Bethesda, Maryland, and by grant IM-432 from the American Cancer Society.     

Influence of neutrophil cationic proteins on generation of superoxide by human polymorphonuclear cells during phagocytosis

The cationic proteins from neutrophyl lysosomes have been shown to modulate phagocytic activity of granulocytes. The present study reports the effects of the cationic protein fractions on the generation of O ₂ ^– by human PMNs during phagocytosis. Human PMNs were reacted win different phagocytic stimuli in the presence and absence of lysosomal cationic proteins and the amount of O ₂ ^– generated was determined by superoxide dismutase inhibitable reduction of cytochromec. Total cationic protein extract from neutrophil lysosomes enhanced O ₂ ^– generated by PMNs during the phagocytosis of IgG-coated latex beads and opsonized zymosan particles. The analysis of the fractions of cationic proteins obtained from a Sephadex G-75 column showed that the O ₂ ^– generation-enhancing activity was associated with the proteins eluted in fractions III and IV. A protein fraction mainly eluted in void volume inhibited the cytochromec reduction by O ₂ ^– formed during phagocytosis. This was due to the presence of superoxide dismutase-like activity since O ₂ ^– generated by the xanthine-xanthine oxidase system was also inhibited by this fraction. The cationic protein fractions III and IV from the Sephadex G-75 column were further subfractionated. Although the O ₂ ^– -enhancing activity was eluted in the same fractions as chymotrypsin activity, there was no quantitative correlation between the amount of O ₂ ^– generation and chymotrypsin activity. Moreover, commercial chymotrypsin did not enhance O ₂ ^– generation. Electrophoretic analysis of the isolated protein fractions suggests that O ₂ ^– generation enhancing protein (SGEP) is different from lysozyme or chymotrypsin and probably represents previously undescribed protein.     

Role of myeloperoxidase in respiratory burst of human polymorphonuclear leukocytes

A comparative study of the respiratory burst [monitored as superoxide (O₂ ^–) production] of normal and myeloperoxidase (MPO) -deficient poiymorphonuclear leukocytes (PMNs) was carried out on 11 MPO-deficient subjects that represent the largest sample of this kind ever studied. The rate of O₂ ^– production by isolated PMNs and whole blood from normal and MPO-deficient subjects was comparable during the initial 30–40 min of incubation with serum-treated zymosan (STZ). Afterwards, the amount of O₂ ^– produced became progressively higher in MPO-deficient cells at least until 120 min incubation with STZ. On the contrary the rate of O₂ ^– production by both cell types in response to 4--phorbol-12-myristate-13-acetate (PMA) was the same. The PMNs of four MPO-deficient subjects were tested for their ingestion ability by counting the number of ingested particles on toluidine blue-stained sections of epoxy-embedded PMN suspensions. Both cell types ingested STZ particles at a comparable rate at early postphagocytic times, whereas on prolonged incubation MPO-deflcient PMNs ingested more STZ particles than normal PMNs. These results suggest that the ingestion capacity of normal cells may undergo a more rapid deterioration than that of MPO-deficient cells during incubation with STZ. Evidence for a higher deterioration of normal PMNs with respect to MPO-deficient PMNs was obtained also from studies on the effect of storage on O₂ ^– generation. After standing at melting ice temperature for 3 h, normal PMNs produced less O₂ ^– than MPO-deficient PMNs in response to PMA, and the difference in O₂ ^– production by the two cell types in response to STZ was evident at earlier postphagocytic periods than with freshly isolated cells. Taken all together these results suggest that normal PMNs and MPO-deficient PMNs do not intrinsically differ in O₂ ^– generating potential and that the difference in the respiratory burst observed during phagocytosis may be accounted for by a more marked deterioration, in normal PMNs, of one or more functions related to the respiratory burst.     

Ontogeny of inflammatory cell responsiveness

Newborn calves, like human infants, are uniquely susceptible to bacterial infections. Part of this increased susceptibility may be related to defects in newborn polymorphonuclear leukocyte (PMN) defensive functions. It remains unclear whether reported deficits in newborn PMN function represent maturational disorders or are manifestations of some form of perinatal suppression phenomenon. We therefore compared the ability of bovine newborn PMNs (less than 24 h old), newborn PMNs (7–10 days of age), fetal PMNs (210–220 days gestational age), and adult PMNs to generate superoxide anion (O ₂ ^– ) as an indicator of respiratory burst activity. Citrated biood was collected, and PMNs were isolated to greater than 95% purity and 98% viability. O ₂ ^– generation was measured as the superoxide dismutase-inhibitable (10 g/ml) reduction of ferricytochrome c (2 mg/ml) after activation of PMNs with phorbol myristate acetate (PMA, 2 g/ml) to directly stimulate protein kinase C. The reaction kinetics were measured (37°C, 550 nm) using a spectrophotometer and chart recorder for continuous monitoring. O ₂ ^– generation was measured for 5 min after the initial lag period and the total nanomoles of O ₂ ^– generated calculated using the extinction coefficient for ferricytochromec. Newborn PMNs (N=10) generated significantly less O ₂ ^– (5.7 ±0.8 nmol O ₂ ^– /10⁶ cells/5 min,P < 0.01) than did adult PMNs (N=14) (9.6 ±2.1 nmol O ₂ ^– /10¹⁰ cells/5 min) or fetal PMNs (N=4) (10.7 ±0.7 nmol O ₂ ^– /10⁶ cells/5 min). PMNs from 7-to 10-day-old calves (N=9) generated almost identical amounts of O ₂ ^– as newborn PMNs (5.7 ±1.6 nmol O ₂ ^– /10⁶ ceils/5 min). There was no difference in measured lag time period between new-born and adult PMNs, but fetal PMNs had significantly reduced (P < 0.01) mean lag time. The data indicated that bovine newborn PMNs have a decreased ability to generate O ₂ ^– in response to PMA stimulation, which persists for at least 7–10 days, and that this functional decrement may be a manifestation of some form of perinatal PMN suppression phenomenon rather than a developmental abnormality since fetal PMNs produced O ₂ ^– as well as adult PMNs.     

A two-fold effect ofL-1-tosylamide-2-phenylethyl chloromethyl ketone on the oxidative metabolism of guinea pig phagocytes

The protease inhibitor,L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK), stimulated the O ₂ ^– production, H₂O₂ generation, oxygen consumption, and the hexose monophosphate shunt of guinea pig peritoneal polymorphs. Other protease inhibitors were not able to stimulate the metabolic burst of these cells. Maximum stimulation was obtained at 100 µM concentration of the compound. No stimulation was seen in human blood polymorphs even at concentrations higher than those effective on guinea pig polymorphs. TPCK also stimulated the oxidative metabolism of guinea pig blood polymorphs and of guinea pig resident peritoneal macrophages. At concentrations which did not stimulate the oxidative metabolism of guinea pig polymorphs, TPCK inhibited the O ₂ ^– production induced in these cells by treatment with phorbol myristate acetate (PMA) or with other soluble stimuli. Other protease inhibitors also inhibited the respiratory burst induced by PMA. It is concluded that TPCK exerts two effects on the metabolism of guinea pig phagocytes, which are probably mediated by different mechanisms. The inhibitory effect on the PMA-stimulated respiratory burst might be related to the antiprotease activity of TPCK, while the stimulation of the respiratory burst seems to be independent of protease inhibition.     

Alterations in complement-induced shape change and stimulus-specific superoxide anion generation by neonatal calf neutrophils

Increased susceptibility of neonates to infection may be related to defects in newborn neutrophil (PMN) functional activities, including altered responses to complement fragments (Cf) and defective microbicidal activity. We therefore compared the kinetics of newborn and adult bovine PMN membrane shape change responses following stimulation with zymosan-activated plasma (ZAP) as a source of Cf. Measurement of PMN membrane shape change was a rapid, sensitive, and reproducible measure of Cf stimulation within a population of PMNs; a maximum of 67–85% of the PMNs exhibited easily detectable membrane ruffling, lamellipodia formation, and polarity within 2 min. Newborn PMNs exhibited significantly increased (P<0.01) membrane shape change at 20, 30, 60, 120, and 300 sec after Cf stimulation. A maximum of 85.8±3.2% of newborn PMNs exhibited such C-finduced shape changes by 120 sec, which was significantly greater (P<0.01) than the maximum stimulation (67.7±4.3%) attained with adult PMNs. These data indicate enhanced kinetics of induced newborn PMN membrane shape change in response to Cf stimulation. We also compared stimulus-specific superoxide anion (O ₂ ^– ) generation as a measure of respiratory burst activity after incubation of new-born and adult PMNs with soluble (phorbol myristate acetate, PMA) and particulate (opsonized zymosan, OZ) stimuli. When PMA was used as the stimulus, newborn PMNs generated significantly less O ₂ ^– (9.3±0.5 nmol O ₂ ^– /10⁶ PMN,P<0.05) than did adult PMNs (12.4±0.3 nmol O ₂ ^– )/10⁶ PMN). This finding was reversed when OZ was used as the stimulus; newborn PMNs generated significantly more O ₂ ^– (7.7±0.4 nmol O ₂ ^– /10⁶ PMN,P <0.05) than did adult PMN (5.5 ±0.5 nmol O ₂ ^– /10⁶ PMN). These findings collectively document biochemical and morphological differences between newborn and adult PMNs as determined by stimulusspecific O ₂ ^– generation and C_f-induced membrane shape change. Such differences may be important to neonatal disease susceptibility.     

Altered function of synovial fluid granulocytes in patients with acute inflammatory arthritis

In rheumatoid arthritis (RA) a chronic inflammatory state exists in which the synovial fluid is periodically filled with large numbers of polymorphonuclear leukocytes (PMNs). Oxygen radicals produced by these cells have been implicated as mediators of tissue damage and may be directly involved in the pathogenesis of RA. We examined the production of oxygen radicals by synovial fluid PMNs (SFPMNs) and peripheral blood PMNs (PB-PMNs) by measuring chemiluminescence (CL) as well as Superoxide anion (O ₂ ^– ) release. Increased spontaneous CL in the presence of luminol and increased CL in response to phorbol myristate acetate (PMA) was observed in SF-PMNs when compared to PB-PMNs. When zymosan was used as the stimulus in the absence of luminol, a slightly lower CL response was observed in SF-PMNs as compared to PB-PMNs. No significant differences were observed in the generation of O ₂ ^– generation with any stimulus. Preincubation of normal PBPMNs in 10% synovial fluid enhanced the luminol-dependent spontaneous and PMA-stimulated CL as well as zymosan-stimulated CL. When O ₂ ^– release from normal PB-PMNs pretreated with 10% synovial fluid was compared to untreated controls, enhancement of spontaneous O ₂ ^– release was observed. PMA- and zymosan-stimulated responses did not differ significantly from controls. Increased spontaneous and PMA-stimulated release of myeloperoxidase (MPO) was also observed in normal PB-PMNs pretreated with synovial fluid. These findings may explain the increased luminol-dependent CL since this type of CL requires the presence of MPO. Our findings suggest that the enhanced chemiluminescence observed in normal PMNs treated with synovial fluids may be related to increases in spontaneous O ₂ ^– generation and myeloperoxidase release. Increased MPO release may account for enhanced CL observed in SF-PMNs.     

Effect of antiinflammatory agents on neutrophil superoxide production in rheumatoid arthritis

Granulocyte superoxide production by different stimuli was studied in 14 patients suffering from rheumatoid arthritis, and in four cases defective (O ₂ ^– generation was shown. The effect of two chemically related drugs, such as indomethacin and oxamethacin, was also evaluated, since we have previously investigated the action of antiinflammatory agents on cell locomotion. Indomethacin did not affect O ₂ ^– production, whereas oxamethacin reduced significantly superoxide generation in PMNs from all subjects tested. Moreover, the extent of the effect was dependent on the stimulant used, being larger when the activation of O ₂ ^– generating system was induced by opsonized zymosan.     

Lysophosphatides enhance superoxide responses of stimulated human neutrophils

Human neutrophils which are pretreated with subtoxic concentrations of a variety of lysophosphatides (lysophosphatidytcholine, lysophosphatidylcholine oleoyl, lysophosphatidylcholine myrioyl, lysophosphatidylcholine stearoyl, lysophosphatidylcholine gamma-O-hexadecyl, lysophosphatidylinositol, and lysophosphatidylglycerol) act synergistically with neutrophil agonists phorbol myristate acetate, immune complexes, poly-L-histidine, phytohemagglutinin, andN-formyl methionyl-leucyl-phenyalanine to cause enhanced generation of superoxide (O ₂ ^– ). None of the lyso compounds by themselves caused generation of O ₂ ^– . The lyso compounds strongly bound to the neutrophils and could not be washed away. All of the lyso compounds that collaborated with agonists to stimulate O ₂ ^– generation were hemolytic for human red blood cells. On the other hand, lyso compounds that were nonhemolytic for red blood cells (lysophosphatidylcholine caproate, lysophosphatidylcholine decanoyl, lysophosphatidylethanolamine, lysophosphatidylserine) failed to collaborate with agonists to generate synergistic amounts of O ₂ ^– . However, in the presence of cytochalasin B, both lysophosphatidyiethanolamine and lysophosphatidylserine also markedly enhanced O ₂ ^– generation induced by immune complexes. O ₂ ^– generation was also very markedly enhanced when substimulatory amounts of arachidonic acid or eicosapentanoic acid were added to PMNs in the presence of a variety of agonists. On the other hand, neither phospholipase C, streptolysin S (highly hemolytic), phospholipase A₂, phosphatidylcholine, nor phosphatidylcholine dipalmitoyl (all nonhemolytic) had the capacity to synergize with any of the agonists tested to generate enhanced amounts of O ₂ ^– . The data suggest that in addition to long-chain fatty acids, only those lyso compounds that possess fatty acids with more than 10 carbons and that are also highly hemolytic can cause enhanced generation of O ₂ ^– in stimulated PMNs.Supported by a research grant from Dr. Samuel M. Robbins, Cleveland, Ohio; by grant IM-432 from the American Cancer Society; and grants HL-28442-07, HL-31963, and GM-29507 from the National Institutes of Health, Bethesda, Maryland.Dr. Isaac Ginsburg was a visiting professor in the Department of Pathology at The University of Michigan when this research was conducted.     

Chemiluminescence in activated human neutrophils

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of Superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the: result of the interaction among H₂O₂, a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H₂O₂ was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn²⁺, Fe²⁺, Cu²⁺, and Co²⁺ very markedly enhanced CL induced by mixtures of H₂O₂ and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by Superoxide dismutase (SOD) and by deferoxamine. LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H₂O₂ probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.     

Comparison of the effects of antioxidant non-steroidal anti-inflammatory drugs against myeloperoxidase and hypochlorous acid luminol-enhanced chemiluminescence

The interaction of myeloperoxidase (MPO) with H₂O₂ and Cl^– provides a potent antimicrobial/cytotoxic system for polymorphonuclear leukocytes (PMNs). MPO-related cytotoxicity may be associated with the formation of toxic oxidant MPO intermediates, HOCl, or both. MPO itself is able to oxidize drugs and cellular components. Non-steroidal anti-inflammatory drugs (NSAIDs) able to act as antioxidant free radical scavengers have recently been shown to inhibit luminol-enhanced chemiluminescence (CL) which results from the MPO–H₂O₂–Cl^– reaction. CL is a measure of the activity of this reaction. At that time it was not clear whether the source of CL which these NSAIDs affected was HOCl or components of the initial MPO–H₂O₂–Cl^– reaction. A NSAID antioxidant mechanism could affect MPO oxidant intermediates and HOCl.This study compares the effects of antioxidant NSAIDs, methylprednisone and free radical scavengers against MPO-based and NaOCl-based luminol-enhanced CL. Most NSAIDs which affected both MPO and NaOCl-CL appeared to share similar mechanisms, suggesting that MPO oxidant internediates and HOCl are susceptible to NSAID effects. However, most NSAIDs were more effective against MPO-CL. The effect of these NSAIDs against MPO-CL followed the profile of NSAIDs effective in previous studies against PMN-CL. One exception to this was methylprednisone, which has no effect on PMN or MPO-CL, yet inhibited NaOCl-CL. This and other data suggest that MPO and not HOCl-related reactions are a major source of PMN-CL. Less effective NSAIDs affected NaOCl-CL better than MPO-CL. While both HOCl and MPO oxidant intermediates may be affected by NSAIDs, it appears that MPO oxidant intermediates or MPO itself are the primary target for NSAID antioxidant free radical scavenging mechanisms. These antioxidant effects impair the major killing system of the PMN and may be NSAIDs' primary anti-inflammatory mechanism. Although our data suggests the production of superoxide anion and hydroxyl radical from the MPO–H₂O₂–Cl^– reaction, the actual presence or involvement of these free radical species is not confirmed herein.     

NADPH and “cocktails” containing polyarginine reactivate superoxide generation in leukocytes lysed by membrane-damaging agents

Human blood leukocytes generated large amounts of superoxide O₂ ^– following stimulation by certain cocktails of soluble agents consisting of poly-L-arginine (PARG), phytohemagglutinin, the chemotactic peptide formylmethionyl-leucyl-phenylalanine and polyanethole sulfanote (liquoid). A variety of cytochalasins, which markedly boosted O₂ ^– generation by the soluble cocktails, markedly depressed luminol-dependent chemiluninescence (LDCL) which had been induced either by opsonized streptococci or by soluble agents. Glutathione, which totally reversed the inhibition of LDCL induced by cytochalasin A, failed to reverse the inhibition of LDCL induced by cytochalasin B. Generation of O₂ ^– by all the soluble agents employed, except PMA, was strongly inhibited either by the omission of extracellular calcium and magnesium or by treatment with the calcium blocker TMB-8. Generation of O₂ ^– was enhanced following stimulation of leukocytes with soluble agents if the cells had been exposed to slightly hypotonic buffers. Leukocytes, which had been preincubated for short periods (5 min) with PARG, saponin, digitonin, or lysolecithin(LL) and which lost their viability, and their O₂ ^– and LDCL-generating capacities following stimulation by soluble agents containing cytochalasin B, nevertheless regained these activities by the addition of NADPH. It is suggested that the lytic agents induced the leakage out of NADPH rather than acting as inactivators of the oxidase in the leukocyte membranes. Prolonged incubation of leukocytes with lytic agents failed to allow restoration, by NADPH, of the generation of SOD-inhibitable O₂ ^– generation. Since PARG acted both as a cytolytic agent and as a inducer of O₂ ^– generation, we postulate that lytic agents might also act as primers of the nascent membrane oxidase which could, however, be further potentiated and activated by soluble agents acting in multiple hits, PARG could be totally replaced either by LL or by digitonin in the generation of O₂ ^– provided that both PHA and cytochalasin B were present in the reaction mixtures. We suggest that the various ingredients of the soluble cocktails may help to assemble components of the NADPH oxidase. Such an assembly and regulations are prerequisite for stimulation of the NADPH oxidase and the generation of oxygen radicals in leukocytes.This study was supported in part by a research grant from Dr. S.M. Robbins of Clevlend, Ohio; by grant 993 form the Chief Scientist, Ministry of Health, Government of Israel; by grant DE-05494 from the National Institute of Dental Research, NIH, and by grant AI-14148 from the National Institute of Allergy and Infectious Diseases NIH, Bethesda, Maryland.Isaac Ginsburg was a visiting scientist at the National Jewish Hospital summer of 1983.     

Occupancy of adenosine receptors on human neutrophils inhibits respiratory burst stimulated by ingestion of complement-coated particles and occupancy of chemoattractant but not Fc receptors

Chemoattractants are generated at inflammatory loci that not only induce neutrophils (PMNs) to leave the vasculature but also stimulate PMNs to release potentially toxic agents (e.g., H₂O₂, O ₂ ^– or OH). We have recently demonstrated that endothelium releases adenosine which, when bound to a specific receptor on the PMN surface, inhibits release of toxic oxygen metabolites from stimulated PMN. To determine whether occupancy of adenosine receptors modulates generation and release of oxygen metabolites, we have studied the effect of 2-chloroadenosine on O ₂ ^– generation and O₂ consumption in response to opsonized zymosan particles (STZ) and immune complexes (IC). 2-Chloroadenosine inhibits, in a dose-dependent fashion, Of generation by neutrophils that have been exposed to C3b-coated particles (STZ). Inhibition of Of generation is similar in the presence or absence of cytochalasin B (IC₅₀=53 ±19 and 16 ±5nM, respectively,P=NS). Since occupancy of adenosine receptors might inhibit only externalization but not generation of oxygen metabolites, we studied the effect of 2-chloroadenosine on oxygen consumption by activated neutrophils. 2-Chloroadenosine inhibited O₂ consumption stimulated by STZ and the surrogate bacterial chemoattractant FMLP; however, inhibition of O₂ consumption varied with the presence or absence of cytochalasin B. In contrast, when neutrophils were stimulated by immune complexes, 2-chloroadenosine only minimally inhibited O ₂ ^– release and O₂ consumption (10 ± 5 and 5 ± 4% inhibition, respectively). Thus, occupancy of adenosine receptors inhibits O₂ consumption in parallel with inhibition of O ₂ ^– release. These results support the hypothesis that ingestion of complement-opsonized particles stimulates the respiratory burst by a mechanism different from that by which the respiratory burst is stimulated after occupancy of Fc receptors. Moreover, these observations suggest that endothelium, by releasing adenosine, prevent activated neutrophils from damaging the microvasculature at inflammatory loci. In contrast, deposition of immune complexes in vessel walls leads to vascular damage because endothelial cells are incapable of preventing attack by immune complex-stimulated neutrophils.This research was supported by grants from the U.S. Public Health Service (AI-10343 and HL29034) and the Veterans Administration.Dr. Cronstein is the recipient of a Clinical Investigator Award (K11-AR-01490) and was a fellow of the Arthritis Foundation. Dr. Broekman was an Established Investigator of the American Heart Association.     

Dissociation of phagocytosis,metabolic stimulation and lysosomal enzyme release in human leukocytes

In this paper studies are reported concerning the relationship between particle binding to the plasma membrane of human polymorphonuclear leukocytes (PMN's), phagocytosis, generation of oxidative metabolites, and the release of lysosomal enzymes by these cells. Superoxide (O ₂ ^– ) generation by, and lysosomal enzyme release from normal PMN's and cytochalasin B-treated cells were measured. We have found that neither phagocytosis nor lysosomal degranulation are prerequisites for enhanced O ₂ ^– generation. Cytochalasin B-treated PMN's, incapable of ingesting particles but still able to bind particles to membrane receptors, generated enhanced amounts of O ₂ ^– when treated with serum-treated zymosan (STZ), a C3b receptor stimulus, or with aggregated IgG (agg IgG), an Fc receptor stimulus. Moreover, the soluble stimulators complement component C5a, phorbol myristate acetate (PMA), and calcium ions in the presence of the ionophore A23187, also increased the O ₂ ^– production of these cells. In all cases a time and dose-dependent stimulation was found of both the O ₂ ^– generation and the lysosomal enzyme release, but there was no correlation between ability of any stimulus to provoke enzyme release and its ability to stimulate O ₂ ^– generation. When PMN's were preincubated with 5×10^–4 M hydrocortisone-Na-succinate, lysosomal enzyme exocytosis with the immune reactants was inhibited 16–35%. Hydrocortisone also inhibited O ₂ ^– generation, except when STZ was used as the stimulus. Thus, in the case of stimulation of functional processes of PMN's via the C3b receptor, hydrocortisone inhibits membrane fusion without interfering with one of the early biochemical events (O ₂ ^– production).Financially supported by a travel grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Effect of sodium azide on hydrogen peroxide production by zymosan-activated human neutrophils

Stimulated neutrophils (PMNs) produce large quantities of superoxide anion, which is the precursor for hydrogen peroxide (H₂O₂). We developed a new fluorimetric assay to measure the H₂O₂ released by zymosan A-activated PMNs utilizing the oxidation ofp-hydroxyphenylacetic acid by H₂O₂ to its fluorescent dimer in the presence of horseradish peroxidase. Zymosan-activated PMNs isolated from nine healthy volunteers and 20 patients with acute hypoxemic respiratory failure (AHRF) released after 90 min 2.3±0.3 and 2.4±1.3 nmol H₂O₂/10⁶ PMNs, respectively. Inhibition of the heme enzymes by 1.0 mM sodium azide (NaN₃) increased the H₂O₂ production to 21.6±4.4 nmol H₂O₂/10⁶ PMNs in the control group (P<0.001), and to 22.5±14.7 nmol H₂O₂/10⁶ PMNs in patients with AHRF (P<0.001). Incubation temperature, room temperature or 37C, did not change the total amount of H₂O₂ produced after 90 min by zymosan-activated PMN. Addition of NaN₃ improved both the sensitivity and reproducibility of the measurement of H₂O₂ and allowed detection of H₂O₂ released by PMNs with coefficients of variation of less than 5% at PMN concentrations as low as 0.1×10⁶ cells/ml. The amount of H₂O₂ released by activated PMNs did not distinguish healthy controls from patients with AHRF.     

Evidence that OH· production by human PMNs is related to prostaglandin metabolism

Recent studies indicate that human granulocytes generate OH· during the phagocytosis of zymosan particles. Several theoretical considerations suggested to us that this OH· production might be related to prostaglandin metabolism, particularly the observation that OH· is generated by the reduction of hydroperoxides in microsomal systems. In our studies, we tested the importance of prostaglandin metabolism in the production of OH· by human granulocytes (PMNs). Indomethacin and aspirin at concentrations known to impair cyclooxygenase activity decreased OH· production by PMNs during the phagocytosis of zymosan particles. Phenol, which is known to alter prostaglandin metabolism, ablated OH· completely. None of these drugs at the concentrations used impaired the generation of O ₂ ^- or H₂O₂ by PMNs, as indicated by their failure to diminish significantly the generation of chemiluminescence. Thus, the decrement in OH· production by these drugs could not be attributed to a nonspecific effect on the production of O ₂ ^- or H₂O₂. These experiments therefore, indicate that the model for OH· production observed during prostaglandin synthesis with microsomal systems applies to human granulocytes.     

Properties of calcium ionophore-induced generation of superoxide anion by human neutrophils

Exposure of human neutrophils to the calcium ionophore, A23187, eventuates in a time- and concentration-dependent generation of Superoxide anion (O ₂ ^– ) in the presence but not absence of extracellular calcium. The selective requirement for calcium is demonstrated by the observation that magnesium caused a dose-related inhibition of A23187-stimulated O ₂ ^– generation. Preincubation of neutrophils with cytochalasin B prior to their interaction with A23187 results in a significant enhancement of O ₂ ^– production. The activity of the O ₂ ^– -generating system was maximum at 37°C and was significantly curtailed at lower and higher temperatures. A23187-induced O ₂ ^– generation was inhibited by the sulfhydryl reagents.N-ethyl maleimide (NEM) and iodoacetic acid (IA), and by the metabolic inhibitor 2-deoxy-D-glucose (2-DG) in the absence of glucose and cytochalasin B. Cyanide was inactive. Therefore, A23187 represents a useful pharmacologic probe for investigating the divalent cation and metabolic requirements of the neutrophil O ₂ ^– -generating system.     

Differenzierung der Wirkungen von CO2-Druck und Wasserstoffionenkonzentration im Blut auf die Atmung beim Menschen

Summary Data published in the preceding paper on effects of augmented arterial CO₂ tensions on lung ventilation before and after inducing acidosis by NH₄Cl or CaCl₂ or acetazolamide (diamox) were used to differentiate the partial effects of p _H changes at constant CO₂ tension and of CO₂ tension changes at constant p _H in arterial blood on lung ventilation.The total increase of lung ventilation caused by elevation of CO₂ tension was found to be 3.2 l min^–1 Torr^–1. The partial p _H effect was 1.4 l min^–1 Torr^–1 or 43% per cent of the total effect and the partial CO₂ effect was 1.8 l min^–1 Torr^–1 or 57 per cent of the total effect. This is in agreement with the prediction of Gray and the experimental results of Lambertsen and Semple.

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Mit 1 Textabbildung

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Die Ergebnisse wurden teilweise mitgeteilt in Vorträgen vor der Gesellschaft für Morphologie und Physiologie in München und in Oxford im Juli 1959.

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Stipendiat der A. v. Humboldt-Stiftung.     

Endothelin-1 does not prime polymorphonuclear leukocytes for enhanced production of reactive oxygen metabolites

The in vitro effect of endothelin-1 (ET-1) on the capacity of polymorphonuclear leukocytes (PMNLs) to generate reactive oxygen species (ROS) was investigated. Human PMNLs were separated from healthy volunteers and preincubated for 10 min. at 37°C with varying concentrations (10^–7–10^–12 M) of ET-1. After subsequent stimulation with FMLP (10^–7 M) or opsonized zymosan (0.5 mg/ ml) the intra- and extracellular generation of ROS was assessed by luminol-amplified chemiluminescence, superoxide radical (·O ₂ ^– ) and hydrogen peroxide (H₂O₂) production.Results: ET-1 alone failed to stimulate ROS generation. Neither the capacity for extracellular generation of oxygen metabolites nor the production of ROS with an intracellular origin was changed after preincubation of PMNLs with ET-1. ET-1 did not cause a shift of the ·O ₂ ^– /H₂O₂ production ratio after stimulation of PMNLs with FMLP. These findings suggest that ET-1 in vitro does not prime human PMNLs for enhanced production of ROS.     

Modulation of locomotor activity of polymorphonuclear cells by cationic substances and cationic lysosomal fractions from human neutrophils

Seven cationic substances — human and egg-white lysozyme, RNase, protamine, histone, poly-l-lysine and poly-l-arginine; five cationic lysosomal fractions from human polymorphonuclears (PMNs); RNA; poly-l-glutamic acid; DNA; heparin; endotoxin; mastocytotropic agent compound 48/80; and cytochalasin B were tested for the influence on chemotaxis and random migration of human PMNs using under-agarose migration and Boyden chambers with two filters and [⁵¹Cr]PMNs. The above substances were either preincubated with PMNs, added to chemoattractants, or used instead of chemoattractants. In under-agarose migration method chemotaxis was inhibited by 11–35% when egg-white lysozyme, protamine, heparin, endotoxin, or compound 48/80 was added to the cells. High concentration of cytochalasin B inhibited chemotaxis by 73 %. Cationic fractions I and V and low concentration of cytochalasin B enhanced chemotaxis by 11%, 41%, and 30%, respectively. When human and egg-white lysozyme, DNA, or cytochalasin B was added to the chemoattractants, motility of PMNs was inhibited. Cationic fractions II and V from human PMNs, when used as chemoattractants, enhanced cellular motility by 143–167%. Random migration was enhanced by heparin and inhibited by cytochalasin B and by cationic fractions from human PMNs. These findings suggest that various cationic and anionic substances and cationic fractions from human PMNs have heterogeneous influence on random migration and chemotactic activity of human PMN. Analysis relating chemotaxis to phagocytosis and to intracellular bactericidal activity (ICBA) has shown several patterns. Protamine, poly-l-lysine, poly-l-arginine, and agent compound 40/80 all inhibit chemotaxis and enhance phagocytosis and ICBA; cationic fractions II and V enhanced all three functions, whereas cytochalasin B suppressed phagocytosis and ICBA and had concentration-dependent modulatory influence on chemotaxis. It implies diverse mechanisms of action and possible impact on inflammatory reactions.Supported by agrant-in-aid from the Medical Research Council of Canada.