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.).     

Polyl-histidine

Poly-l-histidine (PHSTD) of molecular weight 26,000 induced the generation of large amounts of Superoxide (O ₂ ^– ) and hydrogen peroxide (H₂O₂) in human neutrophils (PMNs). Despite its low solubility at neutral pH, PHSTD was bound very rapidly to the PMN surfaces. Maximal generation of O ₂ ^– took place with 4–5 ×10^–6 M of PHSTD, starting after a lag of about 25 sec and proceeding for 15–17 min at a rate of 150 nmol/10⁷ PMNs/min, suggesting that this polycation is one of the most potent stimulators of O ₂ ^– generation known, PHSTD was found to be non-toxic for PMNs even at millimolar concentrations. Generation of O ₂ ^– by PHSTD depended on extracellular calcium; it was inhibited by calcium channel blockers and by trifluoperazine, and it triggered a sharp rise in intracellular calcium as determined by the Quin 2 fluorescence technique. The generation of both O ₂ ^– and H₂O₂ by PHSTD was partially inhibited by cytochalasin B or (CYB, CYE). On the other hand, CYB markedly enhanced the generation of both O ₂ ^– and H₂O₂ following stimulation of PMNs either by PHSTD, polyarginine, histone, or by antibody-opsonized group A streptococci. Electron microscopic analysis and NBT reduction tests revealed that both PHSTD and PHSTD-opsonized streptococci were avidly phagocytosed by PMNs. Since CYB totally inhibited internalization of both PHSTD and the PHSTD-opsonized streptococci, it was suggested that these agents stimulated oxygen radical generation mainly on the leukocyte surfaces. Complexes (CX) formed between PHSTD and polyanethole sulfonate (a strong polyanion) or between histone and the polyanion mimicked immune CX in their ability to trigger the generation of large amounts of O ₂ ^– which were inhibited by CYB. Generation of O ₂ ^– and chemiluminescence either by PHSTD or by PHSTD-opsonized streptococci were markedly inhibited by poly-l-glutamate, suggesting that PHSTD acted as a cationic agent which interacted via electrostatic forces with some negatively charged sites in the leukocyte membrane. Generation of H₂O₂ by PHSTD was also markedly inhibited by deoxyglucose, KCN, DASA, as well as by the lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, and propylgallate. On the other hand, cyclooxygenase inhibitors such as aspirin, indomethacin, and piroxicam were inactive, suggesting that arachidonic acid metabolism via lipoxygenase pathway might have been involved in the activation by PHSTD of the NADPH oxidase in PMNs. PHSTD may mimic the effects of antibodies both as an opsonin and as a potent stimulator of the respiratory burst in PMNs and may thus serve as a model for further study of leukocyte-bacteria interactions in infectious and inflammatory sites and of the pathogenicity of immune complexes.Supported by a research grant from Dr. S. M. Robbins of Cleveland, Ohio.     

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.     

Characteristics ofN-formyl-methionyl-leucyl-phenylalanine as an inducer of lysosomal enzyme release from human neutrophils

N-Formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated a timeand concentration-dependent release of granule associated-glucuronidase and lysozyme but not cytoplasmic lactate dehydrogenase from human neutrophils. Maximum discharge of lysosomal enzymes occurred two minutes after cell contact with FMLP. The percent of total enzyme activity released is insignificant when cells are not preincubated with cytochalasin B prior to being exposed to FMLP (10^–10-10^–7 M); although 11.2 ± 1.3 and 12.4 ±1.1% of total activity for-glucuronidase and lysozyme, respectively, is secreted from neutrophils with 10^–4 M FMLP in the absence of cytochalasin B. FMLP-stimulated release of lysosomal enzymes occurs but is significantly curtailed in the absence of extracellular calcium. Incubation of neutrophils with EGTA in calcium-free medium, however, had no effect on FMLP-elicited lysosomal enzyme extrusion. 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), a purported antagonist of intracellular calcium, demonstrated a dose-dependent inhibition of FMLP-induced release of-glucuronidase and lysozyme from cytochalasin B-treated neutrophils in the absence of extracellular calcium. This effect of TMB-8 could be reversed with the addition of calcium to the extracellular medium. These studies indicate that TMB-8 may be useful in elucidating the role of calcium in the mechanism of lysosomal enzyme release.     

Fluoride activation of neutrophils

Fluoride induced degranulation of both primary and specific granules from neutrophils pretreated with cytochalasin B. There was a similarity in the dependency on extracellular Ca²⁺ for fluoride- and for FMLP-stimulated O₂ ^– generation and degranulation. Pertussis toxin, but not cholera toxin, inhibited FMLP and fluoride activation of neutrophils, while neither toxin affected PMA activation of these cells. These results suggest that fluoride and FMLP activate neutrophils through a common Ca²⁺-dependent and pertussis toxin-sensitive pathway.This work was supported by the Oregon Medical Research Foundation.     

Separation and function of neutrophil karyogranuloplasts and comparison with cytoplasts and intact cells

Since neutrophjl cytoplasts lacking nucleus and granules were first prepared by centrifuging neutrophils over a discontinuous Ficoll gradient containing cytochalasin B, several functional deficits have been reported in these cytoplasts. Although these functional deficits have been considered to originate from the absence of organelles, cell damage during preparation could not be excluded. Therefore, in the following experiments the Ficoll gradient was modified to isolate both cytoplasts and karyogranuloplasts, which have a nucleus and granules and represent the cell after loss of the cytoplast. Electron microscopy and analysis of marker proteins and cell volume showed that karyogranuloplasts were distinct from neutrophils. The phorbol myristate acetate (PMA) orN-formylmethionylleucylphenylalanine (FMLP) -induced O ₂ ^– release, corrected by surface area, was in the following order: neutrophils > cytoplasts > karyogranuloplasts. Both aggregation and membrane potential depolarization were maximal in neutrophils, intermediate in karyogranuloplasts, and lowest in cytoplasts when either PMA or FMLP was used as a stimulant. Extracellular release of the granule contents (degranulation) was triggered by FMLP in both neutrophils and karyogranuloplasts. Cytochalasin B pretreatment greatly enhanced FMLP-induced O ₂ ^– release, degranulation, aggregation, and depolarization of membrane potential in neutrophils and karyogranuloplasts, but not in cytoplasts. The ability of cytochalasin B to potentiate FMLP-triggered cell function probably depends on granules or cell organelles which are depleted in cytoplasts. Chemokinesis and chemotaxis were impaired in both karyogranuloplasts and cytoplasts. Specific FML[³H]P binding was greater in karyogranuloplasts than in cytoplasts. Cellular actin content, measured by the DNase I inhibition assay, was abundant in cytoplasts and was extremely low in karyogranuloplasts. Karyogranuloplasts retain various neutrophil functions, except for chemotaxis, and provide an important control when studying the role of cell organelles in cytoplast function.     

Modulation of rabbit neutrophil chemotaxis by cytochalasin a

Chemotaxis and stimulated locomotion by rabbit peritoneal neutrophils are inhibited by cytochalasin A; inhibition is complete with 5 × 10^–7 M cytochalasin A. Under the same conditions exocytosis and activation of the metabolic burst are stimulated maximally with 5 × 10^–7 M cytochalasin A. Inhibition of these function occurs at concentrations higher than 10^–6 M, inhibition being complete with 5 × 10^–6 M cytochalasin A. The sulfhydryl compound glutathione can prevent the inhibitory effect of cytochalasin A on chemotaxis. Inactivation of the sulfhydryl groups on the outer surface of the plasma membrane does not affect the inhibitory effect of cytochalasin A. It is concluded that the cellular target of cytochalasin A has a different significance for chemotaxis as compared with exocytosis and metabolic burst. The possibility that cytochalasin A reacts with sulfhydryl groups, located on a structure closely connected with the inner surface of the plasma membrane, is discussed.     

Priming of human neutrophil functions by tumor necrosis factor: Enhancement of superoxide anion generation,degranulation, and chemotaxis to chemoattractants C5a and f-Met-Leu-Phe

Recombinant human tumor necrosis factor- (rTNF) stimulated increased generation of Superoxide anion (O ₂ ^– ) by human neutrophils in a concentration-dependent fashion. Preincubation of human neutrophils with rTNF (2.2–2200 units/ml) for 10 min enhanced the subsequent generation of O ₂ ^– in response to C5a and f-MetLeu-Phe(FMLP). Recombinant TNF did not enhance O ₂ ^– generation by neutrophils stimulated with phorbol myristate acetate (PMA). Recombinant TNF alone failed to induce release of myeloperoxidase (MPO) and lysozyme by neutrophils. However, it did enhance the release of MPO and lysozyme by neutrophils stimulated with C5a and FMLP, but not with PMA. Although rTNF alone (0.001–50,000 units/ml) was not chemotactic for neutrophils, preincubation of neutrophils with rTNF (0.001–0.1 units/ml) enhanced the chemotactic activity of suboptimal concentrations of C5a (0.1 nM) and FMLP (5 nM). Neutrophils treated with high concentrations of rTNF (100–10,000 units/ml) showed inhibition of random movement and of chemotaxis induced by C5a or FMLP. We conclude from these studies that rTNF primes neutrophils for enhanced responses to subsequent stimuli and thus may augment the inflammatory response by increased oxidant production and lysosomal enzyme release and promote down-regulation of chemotactic movement.     

Effects of trifluoperazine on human neutrophil function.

The interaction of cytochalasin B-treated human neutrophils with the synthetic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) results in a time- and concentration-dependent generation of superoxide anion (O2-) by an extracellular release of granule-associated beta-glucuronidase and lysozyme from these cells. Granule exocytosis was not accompanied by significant cytoplasmic lactate dehydrogenase extrusion. FMLP-stimulated O2- production occurs but is significantly curtailed in the absence of extracellular calcium. Nevertheless, incubation of neutrophils with EGTA in calcium-free medium had no effect on the O2- -generating system. Trifluoperazine (TFP), an inhibitor of calmodulin (a calcium-binding protein), caused a dose-related inhibition of FMLP-elicited degranulation and O2- production in the presence of absence of extracellular calcium. This effect TFP could be reversed by washing the cells before contact with FMLP. These data suggest that TFP represents a useful tool for defining the relevance of calmodulin and calcium to neutrophil function.     

Luminol-independent chemiluminescence by phagocytes is markedly enhanced by dexamethasone,not by other glucocorticosteroids

The effect of several glucocorticosteroids on the generation of reactive oxygen species (ROS) was examined. The ROS assessed were O ₂ ^– , H₂O₂, OH·, and chemiluminescence (CL) (determined in the presence or absence of luminol), generated by both opsonized zymosan-stimulated neutrophils or monocytes and by the xanthine-xanthine oxidase system. Except for luminol-independent CL, only high concentrations (10^–4 M) of steroids could decrease each ROS. In contrast, luminol-independent CL generation in the phagocyte system was increased in a dose-dependent manner by the addition of dexamethasone, but not by any other steroid. Further, in lymphocyte cultures stimulated with Con A for four days, luminol-independent CL generation was demonstrated and enhanced by the addition of dexamethasone, although CL generation was not detected in the absence of dexamethasone. These findings provide evidence that CL does not always represent light specific to ROS, and they suggest the possibility that dexamethasone induces emission of light at sites of inflammation.     

Effect of deuterium oxide on neutrophil oxidative metabolism,phagocytosis, and lysosomal enzyme release

We have previously shown that deuterium oxide (D₂O) enhances the oxidation of methionine, a myeloperoxidase (MPO)-mediated reaction, by human neutrophils during phagocytosis. However, D₂O has no effect on the oxidation of methionine by the purified MPO-H₂O₂-Cl^– system. To explain this observation, we studied the effect of D₂O on the oxidative metabolism, phagocytosis, and lysosomal enzyme release by human neutrophils. D₂O stimulated the hexose monophosphate shunt (HMS) activity of resting neutrophils in a dose-response fashion. In the presence of latex particles or phorbol myristate acetate (PMA), D₂O brought about an exaggerated stimulation of the HMS activity. This enhancement of the HMS activity by D₂O was markedly reduced when neutrophils form two patients with X-linked chronic granulomatous disease (CGD) were used, either in the presence or absence of latex particles or PMA. Superoxide and H₂O₂ production by neutrophils in the presence of latex particles or PMA were also stimulated by D₂O. In contrast, D₂O inhibited the ingestion of latex particles. D₂O enhanced the extracellular release of MPO, but not lactate dehydrogenase, by neutrophils only in the simultaneous presence of cytochalasin B and latex particles. The enhancement of HMS activity and MPO release by D₂O was partially inhibited by colchicine. Our results suggest that enhancement of neutrophil oxidative metabolism by D₂O may in part explain the stimulation of methionine oxidation by phagocytosing neutrophils.     

Unique characteristics of superoxide production by human eosinophils in eosinophilic states

Eosinophils from patients with peripheral blood eosinophilia and human neutrophils from normal subjects and patients with neutrophilia produced superoxide anion (O₂ ^–) in vitro at similar rates in the absence of stimulation and exhibited comparably increased rates of O₂ ^– production during the initial 1 h of incubation with opsonized zymosan. In the presence of opsonized zymosan, the rate of O₂ ^– production by eosinophils was constantly high for 3 h, whereas the rate of production by neutrophils fell by more than 65% after 1 h. Consequently, the amount of superoxide produced by phagocytizing leukocytes was twofold higher for eosinophils than for neutrophils at 3 h. O₂ ^– production by cell-free sonicates of zymosan-stimulated eosinophils and neutrophils exhibited the same preference for NADPH over NADH. One mM sodium azide significantly decreased the generation of O₂ ^– by phagocytizing eosinophils, but lacked an effect on neutrophils. The prolonged release of O₂ ^– by eosinophils engaged in phagocytosis may contribute both to their unique microbicidal profile and to the capacity of eosinophils to injure host tissues in some eosinophilic syndromes.     

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.     

Stimulation of neutrophil oxidative metabolism by indomethacin

Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O ₂ ^– ) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to solube oxidative stimuli. To further investigate this phenomenon, O ₂ ^– generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O ₂ ^– release was increased from 150 to 300% of controls in neutrophils exposed to 400 g/ml. indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 /ml. Neutrophils exposed to methacin alone also generated more O ₂ ^– than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacintreated cells were exposed to zymosan. Neutrophil cehmiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 g/ml (1.1 mM), enhancement randed from 253 to 333% of controls. As was observed with O ₂ ^– generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug-treated cells were stimulated with zymosan. Phagocytosis of radiolabeledS. aureus by neutrophils incubated with indomethacin was increased 13±5% over controls (P<0.01,n=5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement. The data are in keeping with the hypothesis that indomethacin has a direct effect on the neutrophil plasma membrane and/or the O ₂ ^– -forming oxidase.     

Effect of benzydamine on exocytosis and respiratory burst in human neutrophils and mononuclear phagocytes

The effect of benzydamine on stimulus-dependent respiratory burst activity and enzyme release was tested in human neutrophils, monocytes and monocyte-derived macrophages. Establihsed anti-inflammatory compounds, indomethacin, phenylbutazone and bufexamac, were tested for comparison. Care was taken to avoid cytotoxic or cytolytic concentrations of the test compounds, and their effect on release of lactate dehydrogenase was also tested.Release of specific and azurophil granules contents were induced in human neutrophils by A23187, PMA and fMLP with and without cytochalasin B pretreatment. Benzydamine inhibited stimulus-dependent release of vitamin B₁₂-binding proteins, a marker for the specific granules, in a concentration-dependent fashion. By contrast, phenylbutazone and bufexamac were practically inactive. The effect of benzydamine on exocytosis of azurophil granules was tested in cytochalasin B-pretreated neutrophils. Benzydamine, again in contrast to the two reference anti-inflammatory compounds, inhibited release concentration-dependently also under these conditions. The concentration of the compound which inhibited exocytosis by 50% was 30–100 M in normal and 3–10 M in cytochalasin B-treated neutrophils.The effect of benzydamine and reference compounds on the respiratory burst was tested by assaying for superoxide formation in neutrophils and H₂O₂ formation in mononuclear phagocytes. Benzydamine was inactive on neutrophils and inhibited slightly the burst response of monocytes and macrophages. Two reference compounds, bufexamac and phenylbutazone, were generally more active. The strongest inhibitory effect was that of phenylbutazone on fMLP-stimulated cells. Benzydamine lacked activity receptor of formylated chemotactic peptides.The profile of activity of benzydamine shown in these experiments on human phagocytes suggest that this compound may act therapeutically by decreasing the release of enzymes and other granule constitutents from stimulated neutrophils.     

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.     

Role of extracellular calcium in neutrophil responsiveness to chemotactic tripeptides

Cellular stores of Ca²⁺, but not extracellular Ca²⁺, are required for effective FMLP stimulation of neutrophil Of production and degranulation. Neutrophils transferred from Ca²⁺-containing to Ca²⁺-free medium gradually lose their responsiveness to FMLP, such that after 40 min in the Ca²⁺-free environment they have lost 60–70% of their initial responsiveness to FMLP, The loss in responsiveness is reflected both in an increase in lag interval and decrease in velocity of O ₂ ^– synthesis. The rate of decline in responsiveness to FMLP is greatly accelerated when neutrophils incubated in the presence of A23187 and Ca²⁺-free medium, while the rate of loss of responsiveness to FMLP is not affected by EGTA but the extent of loss is increased. Gradual recovery of FMLP-induced Of generation occurs when cells are transferred from Ca²⁺-free to Ca²⁺-containing medium. PMA-induced neutrophil of generation is not influenced by the presence or absence of extracellular Ca²⁺. It is our view that the rise or fall of neutrophil responsiveness reflects repletion or depletion of cellular stores of Ca²⁺ essential for stimulus-effector coupling and that the role of extracellular Ca²⁺ is subservient to maintenance of these stores.     

Bradykinin induces a B2 receptor-mediated calcium signal linked to prostanoid formation in human gingival fibroblastsin vitro

The aim of the study was to determine the effect of bradykinin (BK) on the level of cytoplasmic-free Ca²⁺, [Ca²⁺]_i, in human gingival fibroblasts and its relation to BK-induced prostanoid formation. BK, but not des-Arg⁹-BK, induced a significant rapid (within seconds) and transient increase in [Ca²⁺]_i, that was not dependent on extracellular Ca²⁺. The stimulatory effect of BK was seen in concentrations at or above 10^–8 M, with the most pronounced effect at 10^–6 M.d-Arg⁰–Hyp³–Thi^5,8–dPhe⁷-BK, a BK B2 receptor antagonist, but not des-Arg⁹–Leu⁸-BK, a BK B1 receptor antagonist, blocked BK-induced rise in [Ca²⁺]_i. The BK B2 receptor antagonist also significantly reduced BK-induced PGE₂ formation. When extracellular Ca²⁺ in the incubation medium was depleted, either by addition of EGTA or by omission of Ca²⁺ addition, BK still caused a significant stimulation of PGE₂ formation. The calcium ionophores A23187 and ionomycin, similar to BK, caused a burst of PGE₂ formation. The two phorbol esters phorbol 12,13-dibutyrate and 4--phorbol-didecanoate positively amplified calcium ionophore A23187-induced PGE₂ formation. The results indicate that BK-induced PGE₂ formation in gingival fibroblasts is coupled to an increase in [Ca²⁺]_i mediated by the BK B2 receptor, and which is independent of extracellular Ca²⁺.     

Studies of skin-window exudate human neutrophils: Complex patterns of adherence to serum-coated surfaces in dependence on FMLP doses

Human neutrophils were isolated both from peripheral blood (PB) and from aseptic inflammatory exudates obtained by the Senn's skin-window (SW) technique. The respiratory burst (O ₂ ^– release) and the adherence to serum-coated wells of culture microplates was investigated using a simultaneous assay. Unstimulated PB resting neutrophils did not produce a significant amount of O ₂ ^– and were incapable of adhering to serum-coated plastic surfaces, while unstimulated SW neutrophils showed augmented adhesion to serum-coated culture wells. SW neutrophils were primed to enhanced FMLP-dependent O ₂ ^– release in response ton-formyl-methionyl-leucylphenylalanine (FMLP). Adhesion of SW neutrophils was significantly decreased by addition of low doses (10^–10–10^–8M) of FMLP (from 17.1% to 8.4%,P< 0.01, N=12), while fully activating doses (>5×10^–8 M) of FMLP induced a marked increase of the cell adhesion, more pronounced in SW (39.2%) than in PB cells (27.2%). Low (5×10^–9 M) and high (5×10^–7 M) FMLP doses induced morphological changes (polarization) and actin polymerization in the neutrophils from both sources. Biphasic dose-response curves of SW neutrophil adherence were observed using FMLP, but not using concanavalin A or phorbol myristate acetate as stimulatory agents. Therefore, the adherence of SW cells appears to be regulated in a complex fashion, nonlinearly dependent on the chemotactic peptide doses and specifically regulated according to the receptors involved.