Purification and characterization of a membrane-bound NADPH-cytochrome c reductase capable of catalyzing menadione-dependent O2- formation in guinea pig polymorphonuclear leukocytes

A membrane-bound NADPH-cytochrome c reductase, which is capable of forming the superoxide anion (O2-) in the presence of menadione, was highly purified from membrane fractions of disrupted guinea pig polymorphonuclear leukocytes by solubilization with 0.2% Triton X-100 and chromatographies on Sephacryl S-300 and 2',5'-ADP-agarose. The overall purification from the membrane fraction was over 110-fold, with a yield of about 6%. The purified preparation did not contain two other pyridine nucleotide-oxidizing enzymes: NADH- and NAD(P)H-oxidizing enzymes (J. Biochem. 94, 931-936, 1983). Besides cytochrome c, the purified enzyme was able to reduce menadione, Nitroblue tetrazolium (NBT) and 2,6-dichlorophenolindophenol. The reduction of menadione alone resulted in the formation of O2-. The purified enzyme preparation contained FAD. When assayed by measuring O2--generation in the presence of menadione, the enzyme showed an optimum pH at 7.0-7.4, and Km values for NADPH, NADH, and menadione were 25, 230, and 5.3 microM, respectively. The enzyme activity was not inhibited by NaN3 or dicumarol, but was by N-ethylmaleimide, EDTA, and quercetin; these inhibition profiles agree with those observed for the NADPH oxidase in the membrane fraction of phorbol-myristate acetate-stimulated leukocytes. Furthermore, when compared by means of the NBT-staining method combined with disc gel electrophoresis, the purified enzyme was electrophoretically indistinguishable from the NADPH-NBT reductase in the plasma membrane as well as phagosomes of the leukocytes. These results suggest that the purified NADPH-cytochrome c reductase is the putative flavoprotein of the NADPH oxidase system responsible for the respiratory burst.     

Effect of glutaraldehyde on NADPH oxidase system of guinea pig polymorphonuclear leukocytes

In an attempt to elucidate properties and activation mechanisms of the NADPH oxidase system, which is known to be responsible for the production of superoxide anion (O2-) in cell membranes of polymorphonuclear leukocytes (PMNL), intact guinea pig PMNL were treated with glutaraldehyde, a protein crosslinking reagent, before or after stimulation with phorbol 12-myristate 13-acetate (PMA). Then, PMNL were disrupted and NADPH oxidase activity was measured. After the treatment of resting PMNL with glutaraldehyde, NADPH oxidase was no longer activated by PMA. On the other hand, the NADPH oxidase activity enhanced by PMA in advance was markedly retained by the glutaraldehyde treatment of such PMA-stimulated PMNL as compared to that in untreated cells. Similar retention by glutaraldehyde of the stimulated NADPH oxidase activity was observed in PMNL stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP) and cytochalasin D. Furthermore, the oxidase activity of glutaraldehyde-treated PMNL was stable during incubation at 37 degrees C, the half life of the oxidase activity of the treated PMNL being more than 90 min whereas that of the untreated PMNL is about 15 min. This ability of the glutaraldehyde treatment to retain the activity was also observed against inactivation by high concentrations of NaCl and by positively charged alkylamine.     

NaCN inhibits the stereospecific depletion of LTB4 by guinea pig polymorphonuclear leukocytes

Caseinate elicited suspension of guinea pig peritoneal PMNs synthesized LTB4, 6t-LTB4, 12-epi-6t-LTB4 and 5HETE after incubations with A23187 and arachidonic acid. Concentrations of LTB4 peaked in 3 minutes and were then rapidly depleted. 6t-LTB4 and 12-epi-6t-LTB4 also peaked in concentrations in 3 min but were depleted slower than LTB4. NaCN inhibited the depletion of LTB4 in a dose dependent fashion without dramatically affecting biosynthesis.     

Role of the valence of concanavalin A in the activation of guinea pig polymorphonuclear leukocytes

Stimulation of polymorphonuclear leukocytes (PMN) by tetravalent concanavalin A (alpha-ConA) induces membrane depolarization preceding the onset of superoxide anion (O2-) production. Both divalent and monovalent ConA analogues were studied to evaluate the role of valence. Monovalent ConA (m-ConA) was inactive in stimulating O2- production and divalent derivatives were less active than native alpha-ConA. Similarly, membrane depolarization was dependent on the valency of ConA. m-ConA did not induce a marked change in membrane potential, whereas sustained depolarization occurred with multivalent ConA. The formation of multiple linked interactions between surface receptors may be an important early event in the activation of PMN by ConA.     

Stabilizing effect of glutaraldehyde on the respiratory burst NADPH oxidase of guinea pig polymorphonuclear leukocytes

The membrane fraction of guinea pig polymorphonuclear leukocytes stimulated with phorbol myristate acetate exhibits the respiratory burst NADPH oxidase activity. This activity is markedly unstable at 37 degrees C, disappearing with a half-life of 11.0 min. When the membrane fraction was pretreated with 0.1% glutaraldehyde, the NADPH oxidase was found to become more stable; its half-life increased about sixfold without any enhancement of the initial activity. The glutaraldehyde treatment of the membrane fraction also protected the NADPH oxidase against inactivation with 0.1-0.2% Triton X-100. These stabilizing effects of glutaraldehyde on the NADPH oxidase seem to be due to its protein cross-linking ability, since its monovalent analogue, butyraldehyde, did not show any effect on the NADPH oxidase activity. In fact, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that glutaraldehyde cross-linked many proteins constituting the membrane.     

Separation of granule subpopulations in human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes were isolated, disrupted by sonification and the nuclei and unbroken cells removed by centrifugation. The supernatant was applied on top of an optimised discontinuous Percoll gradient. After centrifugation we found nine gradient bands of distinct density. Both the nine bands and the whole fractionated gradient material were assayed for granule marker enzymes. Granule fractions of distinct density, enclosing different enzyme concentrations demonstrated the existence of granule subpopulations. There were three subpopulations of azurophil granules, about four subpopulations of specific granules, one granule fraction perhaps representing the C-particles, and a fraction of plasma membrane vesicles.     

Stimulatory effects of a short chain phosphatidate on superoxide anion production in guinea pig polymorphonuclear leukocytes

Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with a phosphatidate containing short-chain fatty acids, 1,2-didecanoyl-3-sn-phosphatidate (PA10), induced substantial superoxide anion (O2-) production in a dose-dependent manner, whereas phosphatidates prepared from egg lecithin and 1,2-dioleoyl-3-sn-phosphatidate (PA18:1) had no such effect. Calcium was not involved in PA10-induced O2- production, since the production was also observed in the case of addition of EGTA prior to PA10 or pretreatment of PMNL with quin-2 and EGTA to eliminate contributions of both extracellular and intracellular calcium. We have reported in previous papers that the phosphorylation of 46K protein(s), which was commonly observed in parallel with an activation of NADPH oxidase in PMNL, was increased by treatment with 10 microM 1-oleoyl-2-acetylglycerol (OAG) with little change in the O2- production (Okamura et al. (1984) Arch. Biochem. Biophys. 228, 270-277; Ohtsuka et al. (1988) Arch. Biochem. Biophys. 260, 226-231). Treatment of PMNL with a combination of PA10, which slightly increased 46K protein phosphorylation, and such a low concentration of OAG induced a marked increase in the O2- production with the increase in 46K protein phosphorylation, which was probably due to OAG action. Thus, it is likely that this protein phosphorylation plays a significant role in the stimulation of the O2- production by phosphatidate in PMNL.     

Involvement of membrane charges in constituting the active form of NADPH oxidase in guinea pig polymorphonuclear leukocytes

NADPH oxidase activity in a membrane fraction prepared from phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig polymorphonuclear leukocytes (PMNL) was inhibited by positively charged myristylamine. The inhibitory effect of myristylamine was significantly suppressed by simultaneous addition of a negatively charged fatty acid, such as myristic acid. However, the suppression by myristylamine was not sufficiently restored when myristic acid was added later. On the other hand, pretreatment of PMA-stimulated PMNL with glutaraldehyde, a protein crosslinking reagent, stabilized NADPH oxidase activity against inhibition by myristylamine, but not against that by p-chloromercuribenzenesulfonic acid. In a cell-free system of reconstituted plasma membrane and cytosolic fractions prepared from unstimulated PMNL, arachidonic acid-stimulated NADPH oxidase activity was also inhibited by myristylamine. During the activation of NADPH oxidase by PMA in intact PMNL and by arachidonic acid in the cell-free system, cytosolic activation factor(s) translocated to plasma membranes. The bound cytosolic activation factor(s) was released from the membranes by myristylamine, accompanied by a loss of NADPH oxidase activity. It is plausible from these results that the inhibitory effect of alkylamine on NADPH oxidase is due to induction of the decoupling and/or dissociation of the cytosolic activation component(s) from the activated NADPH oxidase complex by increments of positive charges in the membranes, and that the glutaraldehyde treatment prevents the dissociation of component(s).     

A comparative study of superoxide dismutase activity in polymorphonuclear leukocytes, monocytes, and alveolar macrophages of the guinea pig.

Superoxide dismutase, an enzyme which catalyzes the dismutation of superoxide radical formed during the univalent reduction of oxygen, was quantitated by observing the inhibition of cytochrome C reduction in three cell fractions in guinea pig peritoneal PMNs and monocytes and compared to alveolar macrophages. No differences were found in the 16,000 × g pellets containing mitochondria, membranes, and granules and representing 96% of total SOD activity in PMNs and monocytes but only 48% total SOD activity in alveolar macrophages. The 100,000 × g microsomal pellet of alveolar macrophages contained 8% of total SOD activity and two-five times more activity than the respective fractions from monocytes and PMNs. However, there was 70 times more SOD in the 100,000 × g supernatant from alveolar macrophages containing 44% of total enzyme activity than in the same fraction of PMNs and monocytes containing less than 2% total SOD activity. SOD activity is mainly located in the 16,000 × g particulate fraction of PMN and monocytes but more equally distributed between the particulate fractions and cytosol of alveolar macrophages.     

Evidence for inhibition of leukotriene A4 synthesis by 5,8,11,14-eicosatetraynoic acid in guinea pig polymorphonuclear leukocytes

The sensitivity of the 5-lipoxygenase to inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA) is species- and/or tissue-dependent. Guinea pig peritoneal polymorphonuclear leukocytes prelabeled with [3H]arachidonic acid and stimulated with ionophore A23187 formed 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), as well as several dihydroxy fatty acids, including 5(S),12(R)-dihydroxy-6,8,10-(cis/trans/trans)-14-(cis)-eicosatetraenoic acid. ETYA (40 microM) did not inhibit, but, rather, increased the incorporation of 3H label into 5-HETE. In contrast, ETYA markedly inhibited the formation of radiolabeled dihydroxy acid metabolites by the A23187-stimulated cells. Assay of products from polymorphonuclear leukocytes incubated with exogenous arachidonic acid plus A23187, by reverse phase high performance liquid chromatography combined with ultraviolet absorption, showed a concentration-dependent inhibition of the formation of dihydroxy acid metabolite by ETYA (1-50 microM) and an increase in 5-HETE levels (maximum of 2- to 3-fold). The latter finding was verified by stable isotope dilution assay with deuterated 5-HETE as the internal standard. Another lipoxygenase inhibitor, nordihydroguaiaretic acid, potently inhibited the formation of both 5-HETE and dihydroxy acids, with an IC50 of 2 microM. The data suggest that ETYA can inhibit the enzymatic step whereby 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid is converted to leukotriene A4 in guinea pig polymorphonuclear leukocytes.     

Phagocytosis of polymorphonuclear leukocytes by guinea pig peritoneal macrophages: effects of serum and temperature in vitro

Abstract The regulation of phagocytosis of neutrophils by peritoneal macrophages was studied in vitro. Peritoneal exudate cells (PECs) of guinea pigs were lavaged 15 h after the i.p. injection of thioglycollate medium and were cultured in chamberslides. When PECs were cultured in RPMI 1640 medium in the absence of serum, approximately 20% of the macrophages phagocytized autologous neutrophils during 48–72 h of culture. Addition of guinea pig serum to the culture (2.5–20% v/v) suppressed the extent of the phagocytosis. The suppression was induced by globulin-rich ammonium sulfate fractions of the serum. Sera from rat, mouse, hamster, horse or calf also suppressed the phagocytosis, but fetal bovine serum (FBS) supported the phagocytosis, which was inhibited by globulin-rich Cohn fractions of bovine serum. The rate of neutrophil-phagocytosing macrophages was proportional to the rate of the pyknotic change of neutrophils. At a high temperature (42°C), the autophagocytosis took place at 12 h of culture when fresh, but not heat-inactivated, autologous serum was added, implying that complement components may play a role in the hyperthermia-induced phagocytosis of neutrophils by macrophages. At 42°C, ingested neutrophils did not show the pyknotic changes, indicating that intact neutrophils were ingested by macrophages.     

Characterization of the Fc receptor for IgG2 on guinea pig polymorphonuclear leukocytes by the use of a monoclonal antibody

Guinea pig polymorphonuclear leukocytes (PMNs) possess two distinct types of Fc gamma receptor (Fc gamma R): Fc gamma 1/gamma 2R for both IgG1 and IgG2, and Fc gamma 2R for IgG2 alone. The Fc gamma 2R was previously shown to differ antigenically from homologous macrophage (M phi) Fc gamma 2R by the use of a monoclonal antibody to M phi Fc gamma 2R (VIIAI IgG1), though the Fc gamma 1/gamma 2R cross-reacts with a monoclonal antibody to homologous M phi Fc gamma 1/gamma 2R (VIA2 IgG1). Recently, we obtained a monoclonal antibody (MP-2) secreted by a hybridoma prepared by fusion of the splenic cells of mice immunized with guinea pig PMNs with a myeloma cell line. This antibody completely inhibited both the Fc gamma 2R-mediated rosette formation of PMNs with IgG2 antibody-sensitized sheep erythrocytes and the Fc gamma 2R-mediated binding of ovalbumin (OA)-complexed IgG2 antibody to PMNs. When the antigen of MP-2 was isolated by affinity chromatography with the antibody-Sepharose, it gave a single band with a molecular weight of 120,000 on SDS-PAGE. The number of antigen molecules per PMN was estimated to be 9 X 10(4) by measuring the binding of 125I-MP-2 Fab. This value was essentially the same as that obtained by measuring the binding of OA-complexed IgG2 antibody to the PMNs treated with the Fab' of VIA2 IgG1. These results strongly suggest that MP-2 is a monoclonal antibody to PMN Fc gamma 2R.     

Protein kinase C-independent pathway for NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes by cytochalasin D

Cytochalasin D (CD) induced production of the superoxide radical (O(2)(-)) in guinea pig polymorphonuclear leukocytes (PMNs). The protein kinase C (PKC) inhibitor GF109203X (GFX) was rarely without effect on CD-induced O(2)(-) production. CD as well as PMA induced the translocation of p47(phox) to the membrane fraction, and this translocation was slightly decreased by GFX. Moreover, the inhibitory effect of a PKCzeta antagonist with sequences based on the endogenous PKCzeta pseudosubstrate region was weaker than the inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced O(2)(-) production. On the other hand, the production of O(2)(-) induced by CD was more strongly suppressed by the PLD inhibitor ethanol and phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin than that induced by fMLP, and the activation of phospholipase D (PLD) by CD was restrained by wortmannin. These findings suggest that NADPH oxidase is activated by CD through a PKC-independent signaling pathway in PMNs, and this pathway involves the activation of PLD through PI3-K.     

The effect of 2-deoxyglucose on guinea pig polymorphonuclear leukocyte phagocytosis

The effects of 2-deoxyglucose (DOG), an inhibitor of glycolysis, on guinea pig polymorphonuclear leukocytes (PMN) obtained from peritoneal exudates was examined. ATP levels in PMN were reduced by 40% by one hour following an incubation with 2-deoxyglucose. When complement (C3) coated ¹⁴C-staphylococcus aureus, C3 coated lipopolysaccharide-paraffin oil droplets (LPSPO), ¹⁴C-pneumococcus opsonized with IgG, or albumin coated paraffin oil droplets opsonized with IgG were added to cell suspensions containing DOG, the phagocytizing rate was 1,310 ± 55 cpm/5 x 10⁶ cells/15 minutes, 6 ± 2 μg paraffin oil (PO)/10⁷ cells/minute, 2,250 ± 175 cpm/1 x 10⁶ cells/20 minutes or 0.037 ± 0.01 mg PO/10⁷ cells/minute compared to control values of 5,970 ± 275 cpm/5 x 10⁶ cells/15 minutes, 35 ± μg PO/10⁷ cells/15 minutes, 4,510 ± 200 cpm/1 x 10⁶ cells/20 minutes and 0.067 ± 0.01 mg PO/10⁷ cells/minute. In parallel studies the phagocytic index for latex was 0.74 ± 0.28 in DOG compared to control of 2.36 ± 1.13 and the phagocytic rate of albumin coated paraffin oil droplets was 0.029 ± 0.01 mg PO/10⁷ PMN/minute in DOG compared to control of 0.048 mg PO/10⁷ cells/minute. When ATP levels were maintained by the simultaneous addition of 5 mM glucose or pyruvate to media containing DOG, latex ingestion was improved to 1.15 ± 0.3 with glucose and 1.59 ± 0.64 with pyruvate and albumin coated particles to 0.045 ± 0.01 mg PO/10⁷ PMN/minute with pyruvate. There was no improvement in the uptake of either the C3 dependent particles or IgG coated Pneumococci in media containing DOG and glucose and/or pyruvate. Following the removal of DOG from the extracellular medium and the addition of pyruvate or glucose, phagocytosis of C3 dependent LPS-PO was restored to normal values. Neither the binding of C3 or IgG coated particles to the PMN nor the lateral movement of glycoprotein utilizing concanavalin A capping was affected by DOG. Thus, the presence of DOG in the PMN containing adequate amounts of ATP will selectively and reversibly inhibit those surface events required for phagocytosis of C3 and IgG bound particles but not latex particles or albumin particles which non-specifically bind to PMN.     

Exogenous phosphatidic acid with saturated short-chain fatty acyl groups induces superoxide anion release from guinea pig peritoneal polymorphonuclear leukocytes by three different mechanisms

Treatment of suspensions of guinea pig peritoneal polymorphonuclear leukocytes (PMN) with four species of phosphatidate (PA) containing short-chain fatty acids induced sustained superoxide anion (O₂⁻) production after a lag time. The rank order of efficiency of these PAs in triggering O₂⁻ production was PA_8:0 [1,2-dioctanoyl-sn-glycerol-3-phosphate (GP)]>PA_10:0 (1,2-didecanoyl-GP)>PA_6:0 (1,2-dicaproyl-GP)≫PA_12:0 (1,2-dilauroyl-GP). The O₂⁻ release from PMN stimulated with PA_10:0 or PA_12:0, but not with PA_6:0 or PA_8:0, was lowered by the addition of 1 mM extracellular Ca²⁺. Studies with various inhibitors showed that the mechanism of multiphasic O₂⁻ production induced by PA_8:0 depended on its concentration: 1 and 3 μM PA_8:0 induced O₂⁻ production constantly after a lag time through a protein kinase-dependent mechanism that was inhibited by 100 nM staurosporine. With concentrations of PA of 10 μM or more, an additional mechanism that was independent of protein kinase became operative and predominant over the protein kinase-dependent one. This protein kinase-independent mechanism was inhibited selectively by 80 μM TMB-8. Concentrations of 30, 60 and 100 μM PA first elicited transient O₂⁻ production via another protein kinase-dependent mechanism that was more sensitive to H-7 than to staurosporine, and then sustained O₂⁻ production, mainly driven by the protein kinase-independent mechanism. Metabolism of exogenously added [¹⁴C]PA_8:0 in intact PMN was examined in the presence and absence of propranolol. Results suggest that PA itself is more important rather than its degradation products such as diacylglycerol, in inducing O₂⁻ production via three different mechanisms described above.     

The NADPH oxidase of guinea pig polymorphonuclear leucocytes

Summary NADPH oxidase from stimulated guinea pig granulocytes was extracted with deoxycholate. The solubilized enzyme was stable in 20% glycerol. Solubilized enzyme was free of myeloperoxidase activity. The properties of the deoxycholate solubilized enzyme indicated that it is a high molecular weight complex with a flavoprotein, calmodulin and cytochrome b possibly forming part of the complex. Maximum activity was between pH 7.0 and 7.5. The K_m value was 15.8 µM for NADPH and 434 µM for NADH indicating that NADPH is the preferential substrate.     

Characterization of glycosaminoglycans in peritoneal leukocytes from guinea pig

The glycosaminoglycans were isolated from polymorphs and monocytes which were induced in peritoneal cavity of guinea pig by the injection of caseinate solution. The glycosaminoglycan contents of polymorphs and monocytes were approximately 25 and 47 μg of glucuronic acid per 100 mg of dry weight, respectively. The compositions of glycosaminoglycans in polymorphs and monocytes were different from each other. Electrophoretic characterization and enzymatic susceptibility demonstrated that the glycosaminoglycans in polymorphs contained hyaluronic acid as the main constituent with minor components of other glycosaminoglycans and that the major glycosaminoglycan in monocytes was dermatan sulfate.Polymorphs and monocytes were incubated in vitro in the presence of labeled precursor. The results indicated that chondroitin sulfate and dermatan sulfate in polymorphs were synthesized by incubation in vitro at the ratio of 60–65 and 30%, respectively, and that monocytes synthesized 20% of chondroitin sulfate and 70% of dermatan sulfate.