Groups I,II and III extracellular phospholipases A2: Selective inhibition of group II enzymes by indomethacin but not other NSAIDs

The three types (groups I, II and III) of stable extracellular 14 kDa phospholipase A₂ enzymes differ in their primary amino acid sequences and their properties. It may thus be possible to design low-molecular weight inhibitors targeted to the secretory form of mammalian PLA₂. this enzyme has been implicated in inflammatory disorders. We have studied the inhibition of four distinct PLA₂ enzymes by a range of NSAIDs, using³H-oleate release from prelabelled membranes ofE. coli for assay. The enzymes used were cobra venom PLA₂ (Naja naja, a group I enzyme), bee venom PLA₂ (Apis mellifera, group III), recombinant human synovial PLA₂ (group II) and rat peritoneal PLA₂ (group II). Under the conditions of the³H-oleateE. coli assay, 1 mM concentrations of aspirin, sodium salicylate, paracetamol (acetaminophen), oxphenbutazone, ibuprofen, flurbiprofen and nabumetone failed to inhibit significantly any of the four enzymes. However, indomethacin inhibited all four enzymes, although effects were greatest on the two group II enzymes (rat peritoneal and human synovial PLA₂). Approximate IC₅₀ values were 28 and 35 M, respectively. Inhibition by indomethacin was not time dependent and was greater at micromolar rather than millimolar levels of calcium. We conclude that indomethacin but not the other tested classes of NSAID inhibits the group II PLA₂ enzyme in a selective manner and suggest that this may be relevant both to its clinical spectrum and to the design of novel pharmaceutical leads.     

Flavonoids as Phospholipase A2 Inhibitors: Importance of Their Structure for Selective Inhibition of Group II Phospholipase A2

The inhibitory effect of the plant flavonoid, rutin, on group I phospholipase A₂ (PLA₂-I) from porcine pancreas and Naja naja, and on group II phospholipase A₂ (PLA₂-II) from Vipera russelli and Crotalus atrox was investigated. Rutin efficiently inhibited PLA₂-II from both Vipera russelli and Crotalus atrox but was only a weak inhibitor of PLA₂-I from porcine pancreas and Naja naja. The lack of strong inhibition of pancreatic PLA₂-I was not due to contaminating proteins in the enzyme preparation, since the same weak inhibition was obtained against pancreatic PLA₂ purified to homogeneity as judged by two-dimensional gel electrophoresis. Rutin also efficiently inhibited human PLA₂-II from synovial fluid but was only a weak inhibitor of human PLA₂-I from pancreatic juice, suggesting that rutin is a selective PLA₂-II inhibitor. A number of structurally similar flavonoids were tested for their ability to inhibit PLA₂-II from Crotalus atrox and, for comparison, PLA₂-I from porcine pancreas. The results obtained indicate that the hydroxyl group in 5-position as well as the double bond and the double-bonded oxygen in the oxane ring are all important for the overall ability of flavonoids to inhibit PLA₂ activity, and that the hydroxyl groups in 3- and 4-position are required for selective inhibition of PLA₂-II.     

Modulation of phagocytosis and intracellular bactericidal activity of polymorphonuclear and mononuclear cells by cationic proteins from human granulocytes: Alternative pathway of phagocytic enhancement

Cationic lysosomal proteins from human polymorphonuclears (PMN) were isolated by column chromatography and divided into five fractions. On acrylamide gel electrophoresis, fraction I had four bands slower than lysozyme (LZM) mobility; fraction II had five or six bands slower than LZM; fraction III had at least seven bands slower and two bands faster than LZM; fraction IV contained LZM, two bands faster and a few faint bands slower than LZM; fraction V was composed of almost pure LZM. Partial characterization of the fractions showed presence of neutral protease in fractions I-IV, chymotrypsin in fraction III, lysozyme in fractions IV and V, and phospholipase A₂ mainly in fractions II and III. Modulatory activity of fractions I-V were tested at concentrations up to 50 g/ml. Enhancement of phagocytosis ofStaphylococcus aureus was observed by fractions I, IV, and V, whereas phagocytic index was enhanced by all but the fraction II. Intracellular bactericidal activity (ICBA) was markedly enhanced by fractions I, II, and V. Addition of DNA or cytochalasine B inhibited or abolished phagocytosis-enhancing activity of cationic fractions. Their influence on ICBA was much less pronounced. Fraction III enhanced phagocytic index and phagocytosis ofE. colt, whereas fractions I and II enhanced intracellular bactericidal activity against this bacteria. Enhancement of phagocytic activity of monocytes has also been observed. The data suggest that some cationic lysosomal fractions from human PMNs enhance phagocytosis and phagocytic index by human PMNs and monocytes and intracellular bactericidal activity of human PMNs. This alternative pathway of phagocytic enhancement is unrelated to the previously described enhancers of phagocytosis and may play a role in defense mechanisms against infection.Supported by grants-in-aid from the Medical Reseach Council of Canada and the Wellesley Hospital Research Institute.This study was presented in part at the 9th International RES Congress, Davos, Switzerland, 1982.     

Edema-inducing activity of phospholipase A2 purified from human synovial fluid and inhibition by aristolochic acid

A neutral-active, Ca²⁺-dependent phospholipase A₂ (PLA₂) purified 11,000-fold from human synovial fluid (HSF) induced edema when injected into the mouse foot pad. The edema produced by HSF-PLA₂ was dose-dependent and was positively correlated with the dose-dependent in vitro expression of PLA₂ activity. Maximum edema was achieved within 45 min after the injection and persisted for atleast 6 h. Aristolochic acid [8-methoxy-6-nitrophenanthro(3,4-d)-1,3-dioxole-5-carboxylic acid], a major chemical component derived from various species ofAristolochia plant, produced a dose-dependent inhibition of in vitro phospholipid hydrolysis by HSF-PLA₂, porcine pancreatic PLA₂, snake venom (Naja naja) PLA₂, and PLA₂ isolated from human platelet. The sensitivity of these PLA₂s to inhibition by aristolochic acid varied markedly: HSF-PLA₂>N.naja PLA₂>human platelet PLA₂>porcine pancreatic PLA₂. The inhibition of HSF-PLA₂ by aristolochic acid was independent of substrate concentration (18–144M and Ca²⁺ concentration (0.1–4.0 mM). These observations indicate that inhibition of HSF-PLA₂ by aristolochic acid may result from direct interaction with the enzyme. When aristolochic acid was mixed with HSF-PLA₂ and then injected into the mouse foot pad, edema was inhibited in a dose-dependent manner and was positively correlated with in vitro inhibition of PLA₂ activity. Alkylation of HSF-PLA₂ withp-bromophenacyl bromide concomitantly inhibited both enzyme and edema-inducing activity. These results clearly demonstrate that the neutral-active, Ca²⁺-dependent PLA₂ isolated from human synovial fluid is proinflammatory and that catalytic activity is positively correlated with in vivo proinflammatory effects.     

Comparative study of phagocytosis and intracellular bactericidal activity of human monocytes and polymorphonuclear cells

Simultaneous assessment of the total number of bacteria (TNB) ingested, phagocytosis (Ph), phagocytic index (PI), and intracellular bactericidal activity (ICBA) of human monocytes was done by applying the fluorochrome acridine orange technique. Living bacteria stained orthochromatically green, whereas the dead ones were metachromatically red. The stain of extracellular bacteria was completely quenched by crystal violet counterstain. Using the Hypaque-Ficoll separation method combined with glass adherence, the yield of monocytes was 84±11%, the purity 90±8%, and the viability 99±1%. After 60 min of incubation of monocytes withStaphylococcus aureus, phagocytosis was 94±4%, PI 10.0±0.5, ICBA 76 ±5%, and TNB ingested 946±67/100 cells.E. coli B₄ was equally ingested by PMNs and monocytes and killed intracellularly more efficiently by the latter type of cells. Over the ratios of bacteria to cells of 51 to 201, phagocytic activity of monocytes was equal or superior to that of PMNs. Phagocytic and bactericidal activities were enhanced by AB serum, more by the fresh one than by inactivated. Phagocytic activity of monocytes was markedly influenced by temperature of incubation. Room temperature (24°C) significantly suppressed phagocytosis. Contrary to the previous beliefs no significant quantitative differences were found between phagocytic and bactericidal functions of monocytes as compared to polymorphonuclear phagocytes. The acridine orange-crystal violet method is simple, reliable, reproducible, and can be used for assessment of functional capacity of human phagocytes.Supported in part by grant-in-aid of the Medical Research Council of Canada.     

Enzymatic activity and immunoreactivity of extracellular phospholipase A2 in inflammatory synovial fluids

Synovial fluid PLA₂ concentration was measured by an ELISA technique using monoclonal antibodies raised against human recombinant synovial-type group II phospholipase A₂. This ELISA was specific for synovial-type PLA₂ and did not detect pancreatic (group I) PLA₂. In all synovial fluids examined, including rheumatoid, osteoarthritic, psoriatic, and gouty fluids, synovial fluid PLA₂ enzyme activity significantly correlated with PLA₂ immunoreactivity (P<0.001). Within the limits of the ELISA technique, there was no evidence for the presence of specific or nonspecific modulation of PLA₂ activity by either putative PLA₂ activating or inhibitory proteins.     

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.     

Selective inhibition of group II phospholipase A2 by quercetin

The influence of quercetin, chlorpromazine, aristolochic acid, and indomethacin on group I phospholipase A₂ (PLA₂) from porcine pancreas and on group II PLA₂ fromVipera russelli was compared. Quercetin and chlorpromazine were found to inhibit PLA₂ activity in lower concentrations (< 100M), while aristolochic acid and indomethacin were inhibitory only in higher concentrations (> 100M). The order of potency againstVipera PLA₂ was: quercetin >chlorpromazin aristolochic acid > indomethacin, while the order of potency against pancreatic PLA₂ was: chlorpromazine > aristolochic acid > indomethacin> quercetin. Thus, quercetin was a potent inhibitor towards group II PLA₂ (IC₅₀=2M), but a very weak inhibitor against group I PLA₂, with maximum 30% inhibition. Aristolochic acid and indomethacin were three to four times more potent towards group II PLA₂ than towards group I PLA₂, while chlorpromazine was equally potent towards the two PLA₂ types. Quercetin and chlorpromazine were also tested against two PLA₂ fractions purified from the plasma of septic shock patients; chlorpromazine was then equally potent towards the two PLA₂ fractions, whereas quercetin was a potent inhibitor of only one of the two PLA₂ fractions (IC₅₀=4M). Together, these results indicate that (1) different PLA₂ inhibitors have different potency depending on which type of PLA₂ they are used against, (2) quercetin selectively inhibits group II PLA₂ and may therefore be used to discriminate between different PLA₂ forms in biological materials, and (3) both PLA₂ of group I and group II are present in septic shock plasma.     

Modulation of functional activity of human polymorphonuclear and mononuclear phagocytes by intravenous gamma globulin

Intravenous-globulin was tested in a range of concentrations compatible with the increments obtained after therapeutic infusions for modulation of phagocytic functions of human polymorphonuclears (PMNs) and monocytes. Intravenous gammaglobulin in concentrations of 3.0 mg/ml or more increased adhesiveness and suppressed chemotaxis of PMNs. There was marked dose-dependent enhancement of opsonization of gram-positive and gram-negative microorganisms. Preincubation of PMNs with intravenous-globuhn caused enhancement of the total bacteria ingested, total bacteria killed, phagocytosis, and phagocytic index, when gram-positive and gram-negative bacteria were tested. During phagocytosis, there was no release of LDH or lysozyme; however, there was release of-glucuronidase. No significant difference in phagocytic enhancement was found when filtered and native intravenous-globulin preparations were compared. There was marked enhancement of the superoxide anion generation by intravenous-globulin above the concentration of 0.01 mg/ml. Intravenous-globulin also markedly enhanced phagocytic activity of monocytes. Therefore, intravenous-globulin modulates not only opsonization-related phenomena, but also exerts a complex influence on other aspects of phagocytic activity.Supported in part by a grant-in-aid from The Wellesley Hospital Research Institute.     

Inhibition of human non-pancreatic phospholipases A2 by retinoids and flavonoids. Mechanism of action

The interaction of retinoids and flavonoids with phospholipases A₂ (PLA₂) was studied to assess the mechanism of inhibition. Retinoids, such as retinal, retinol, retinoic acid and retinol acetate, and flavonoids, such as quercetin, rutin, morin and sciadopitysin, inhibit Ca²⁺-dependent PLA₂ activity of human synovial fluid (HSF)in vitro in a dose-dependent fashion; ID₅₀ s ranged from 2–8 M. Retinal inhibited neutral active Ca²⁺-dependent PLA₂s from human platelets, human plasma, human polymorphonuclear leukocytes andNaja mossambica mossambica venom in a dose-dependent manner while quercetin inhibits extracellular PLA₂ activities of human plasma, HSF andN. m. mossambica venon in a dose-dependent manner but not PLA₂ activity derived from human platelets and polymorphomonuclear leukocytes.Inhibition of PLA₂ activity by both flavonoid and retinoids were independent of Ca²⁺ or Na⁺. Increasing substrate concentration (9–144 nmols) relieved the inhibition of HSF-PLA₂ activity by quercetin indicating probable interaction with the substrate. The inhibition by retinal is independent of substrate concentration suggesting that inhibition by retinal is probably due to direct interaction with the enzyme. both retinal and quercetin quenched the relative fluorescent intensity ofN. m. mossambica PLA₂ and in a dose-dependent manner in the same concentration range at which they inhibitin vitro PLA₂ activity. Retinal and quercetin shift the thermotropic phase transition of distearoylphosphatidylethanolamine (DSPE) liposomes. Both compounds broadened the transition peak, shifted theT _m to lower temperature, and decreased enthalpy significantly. These findings indicate that inhibition of non-pancreatic human PLA₂s by retinoids and flavonoids can be mediated by interaction with enzyme and/or substrate.     

Prognostic value of phagocytic activity of neutrophils and monocytes in sepsis. Correlation to CD64 and CD14 antigen expression

The role of the phagocytic function of monocytes and neutrophils in sepsis has been poorly investigated. The present study evaluated the impact of the phagocytic activity of neutrophils and monocytes on the outcome of patients with severe sepsis. Thirty-one patients and 30 healthy individuals were enrolled in the study. The phagocytic activity of monocytes and neutrophils was evaluated during 24 h after admission and the results were correlated to the expression of CD64 on neutrophils and monocytes, CD14 antigen on monocytes, the Simplified Acute Physiology Score II and the patients' survival. A reduced phagocytic activity of neutrophils during the first 24 h after admission was a negative predictor for survival. Increased expression of CD64 antigen on polymorphonuclear cells (PMNs) and monocytes was favourably correlated to the patients' survival. In multivariate analysis the phagocytic activity of PMNs was the only independent predictor factor for survival. Patients with PMN phagocytic activity <37% had lower expression of CD64 on monocytes and PMNs and worse outcome, while those with phagocytic activity >37% had higher expression of CD64 on monocytes and PMNs and better outcome. Reduced phagocytic activity of neutrophils may represent a state of neutrophil inactivation similar to that previously described for monocytes during the compensatory anti-inflammatory response.     

Synergistic effect of azithromycin on the phagocytic killing ofStaphylococcus aureus by human polymorphonuclear leukocytes

Many macrolides have been shown to affect the interaction between bacteria and various immune defense mechanisms, such as chemotaxis, accumulation, and bioactivity within phagocytic cells. The interaction of azithromycin with human polymorphonuclear leukocytes (PMNs) was studied in vitro and compared with the interactions between other macrolides and PMNs. The opsonophagocytic killing ofStaphylococcus aureus was synergistically enhanced by azithromycin at concentrations below and above the minimal inhibitory concentration, with a reduction of up to 2.82 log₁₀ cfu/ml with 2 mg/ml of azithromycin. Other macrolides were effective only at subinhibitory concentrations. The beneficial azithromycin-leukocyte interaction may explain azithromycin's efficacy against intracellular pathogens.     

Effect of pentoxifylline on the phagocytic activity, cAMP levels, and superoxide anion production by monocytes and polymorphonuclear cells

The effect of pentoxifylline (Trental) on the phagocytic capacity, cAMP levels, and superoxide anion production of human peripheral blood monocytes and polymorphonuclears (PMNs) was studied. The drug inhibited the phagocytosis of latex particles by both monocytes and PMNs in a dose-dependent manner. In addition, superoxide anion production during the phagocytic process was also reduced following incubation of the cells with pentoxifylline. It is suggested that this inhibitory effect is due to the increased intracellular levels of cAMP induced by the drug.     

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.     

Influence of cationic superoxide generation enhancing protein (SGEP) on phagocytic and intracellular bactericidal activity of human polymorphonuclear cells

Cationic fraction III from the lysosomes of normal human peripheral blood polymorphonuclear cells (PMNs) was found to contain superoxide generation enhancing protein (SGEP). Herein, we report on the influence of partially purified SGEP obtained from fraction III (subfractions III-5 and III-6), on various phagocytic functions of human PMNs. SGEP markedly enhanced intracellular bactericidal activity of human peripheral PMNs. The enhancement was time and dose dependent. It also reduced adhesiveness of the PMNs. SGEP did not influence chemotaxis, phagocytosis or phagocytic index. These findings are compatible with our original observation regarding superoxide generation enhancement properties of SGEP.     

Antiflammin-2 (HDMNKVLDL) does not inhibit phospholipase A2 activities

A basic nonapeptide P2 (antiflammin-2, HDMNKVLDL) which is identical to a portion of the amino acid sequence (residues 246–254) of lipocortin I, has been described to have antiinflammatory activity in a rat paw edema model (Nature 335: 726–730 [1988]). P2 (0.05 M) was also reported to inhibit porcine pancreatic phospholipase A₂ (PLA₂). The effect of synthetic P2 (98% pure) on PLA₂ was evaluated in two assay systems. Using porcine pancreatic PLA₂ and phosphatidylcholine/deoxycholate mixed micellar substrate, P2 (0.005–50 M) had no effect on PLA₂ activity, even in the presence of 2-mercaptoethanol to prevent peptide oxidation. In another assay, using human synovial fluid PLA₂ as the enzyme and [¹⁴C]-oleate-labelledE. coli substrate, P2 (0.005–50 M) had no significant effect on PLA₂ activity. A reported PLA₂ inhibitor, manoalide, was a potent inhibitor of PLA₂ in both assay systems. On the basis of these results, we conclude that P2 is devoid of PLA₂ inhibitory activity.     

白藜芦醇苷对低氧大鼠肺血管构型重建的影响

目的:观察白藜芦醇苷对慢性常压低氧性肺动脉高压(HPH)大鼠肺血管构型重建的影响并探讨可能的机理。方法:29只健康SD大鼠随机分为正常对照组、单纯低氧组和低氧加白藜芦醇苷(PD)组。各组内按照肺小血管外径分为I组(30μm-100μm)和II组(101μm-200μm)。右心导管法检测大鼠肺动脉压力(mPAP)、微量滴定法检测血浆和肺匀浆中磷脂酶A₂(PLA₂)活性、光镜下观察肺小血管中膜厚度(MT%)和中膜面积(MA%)的变化。结果:低氧3周后大鼠mPAP、血浆及肺匀浆中PLA₂活性、肺小血管MA%、I组MT%显著高于对照组,II组MT%无显著性改变。低氧加PD预处理后上述改变明显轻于单纯低氧组。结论:PD可有效防治慢性常压低氧性大鼠肺动脉压力的升高,其机理与抑制大鼠肺血管构型重建有关。     

Studies of phagocytosis in chronic granulomatous disease

Abnormal phagocyte function in chronic granulomatous disease (CGD) is associated with decreased bactericidal activity. Ingestion of serum-opsonized organisms is reported to be normal in these patients. We previously showed that in CGD the expression of C3b receptors (CR1) on polymorphonuclear leukocytes (PMNs) is significantly depressed. In this study, we compared the phagocytic activity of the PMNs from normal healthy controls with that of CGD patients and one individual with myeloperoxidase (MPO) deficiency. The ingestion of sheep erythrocytes (E) by PMNs adherent to a glass surface was examined; the E were coated either with excess IgG (E-IgG) or with C3b plus limited IgG (EAC3b-IgG). The PMNs, both in CGD and in MPO deficiency, ingested E-IgG and EAC3b-IgG at levels markedly above normal. C3b-coated erythrocytes were not phagocytosed. Preincubating the PMNs with sodium azide, which blocks MPO, or catalase, a scavenger of H₂O₂, caused a marked increase in phagocytosis by normal PMNs. Azide had a variable effect on PMN activity in CGD and no effect on the activity in the subject with MPO deficiency. Even in the presence of azide, the ingestion of EAC3b-IgG by the PMNs from the CGD patients was significantly greater than that seen in paired normals [mean phagocytic index (PI), 2.13 for CGD vs. 1.48 for normals;P < 0.05 by the paired samplet test]. Similar results were obtained with ingestion of E-IgG. Notably, ingestion of serum-opsonizedCandida organisms (relatively nondegradable particles) was markedly above normal with CGD PMNs and, in normal PMNs, azide treatment also evoked an increase. In addition, rosette formation of the adhered PMNs with E-IgG was enhanced with CGD and the azide-treated normal PMNs. We demonstrated that this increased activity was not the result of increased Fc receptor (FcR) number, as determined from the binding of a monoclonal anti-FcR antibody. Both the E-IgG rosette formation and the ingestion by CGD PMNs were abrogated in the presence of an H₂O₂-generating system. In contrast, the phagocytic activity of MPO-deficient PMNs was not altered by exogenous H₂O₂. These findings suggest that cellular products generated by the H₂O₂-MPO-halide system down-regulate the rosette-forming and phagocytic activity of PMNs from normal healthy individuals, but not that from CGD and MPO-deficient patients.     

Reactivity of active center analogs of Cu2Zn2 superoxide dismutase on activated polymorphonuclear leukocytes

In unseparated human blood the Cu₂Zn₂ superoxide dismutase mimetic reactivity of several differently coordinated low Mr copper chelates on TPA-activated polymorphonuclear leukocytes was evaluated and compared to their apo-chelates, CuSO₄, and the native enzyme. Similar to intact superoxide dismutase, 350–400 nM Cu flexibly complexed in a di-Schiff base mode in CuPu(Py)₂ and CuPu(Im)₂, respectively, was sufficient to inhibit the oxidative burst-dependent superoxide production of human blood phagocytes by 50%. Acetate-or biuret-type copper chelates behaved like CuSO₂. The catalytic superoxide dismuting reactivity of the di-Schiff base active center analogs of SOD was confirmed using isolated porcine PMNs. Even in the presence of 600M albumin as a model for competitive copper chelation in biological fluids CuPu(Py)₂ and CuPu(Im)₂ remained active. The stability during the Cu(I)/Cu(II) redox cycling was demonstrated in the presence of activated PMNs and albumin, taking advantage of the electron paramagnetic properties of CuPu(Py)₂ and CuPu(Im)₂.     

Biochemical characterization of soluble phospholipase A2 from rheumatoid synovial fluid

RadiolabeledE. coli, Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC), were used to characterize the phospholipase A₂ (PLA₂) activity in synovial fluid (SF) from rheumatoid arthritis (RA) patients. Cell-free fractions of SF contain a PLA₂ enzyme that preferentially releases [¹⁴C]oleic acid fromE. coli, requires calcium and is optimally active at neutral pH. Purified PE, but not PC is also readily degraded by the soluble enzyme. A cell-associated PLA₂ present in sonicates of SF mononuclear cells and neutrophils preferentially releases [³H]AA fromE. coli. These studies suggest the presence of at least two different enzymes with activity of PLA₂ in rheumatoid SF.