Rat Peritoneal Polymorphonuclear Leukocytes Research Articles

Isolation of eriocitrin (eriodictyol 7‐O‐rutinoside) as an arachidonate lipoxygenase inhibitor from Lumie fruit (Citrus lumia) and its distribution in Citrus species

AbstractAn inhibitory compound acting against rat platelet 12‐lipoxygenase was isolated from the peel of Lumie fruit (Citrus lumia) by activity‐guided separation. It was identified as eriocitrin (eriodictyol 7‐O‐rutinoside) by spectroscopic analyses. Eriocitrin inhibited 5‐lipoxygenase (IC5029.1 µmol L−1) from rat peritoneal polymorphonuclear leukocytes in addition to 12‐lipoxygenase (IC5022.3 µmol L−1). Its aglycone, eriodictyol (5,7,3′, 4′‐tetrahydroxyflavanone), was a much more potent inhibitor of both 12‐lipoxygenase (IC500.07 µmol L−1) and 5‐lipoxygenase (IC500.20 µmol L−1). It also inhibited the production of leukotriene B4 in intact peritoneal polymorphonuclear leukocytes stimulated with calcium ionophore A23187 (IC5012.7 µmol L−1). The distribution of eriocitrin in 39 citrus fruits was investigated by high‐performance liquid chromatography analysis. Lumie, eureka lemon (Citrus limon), Sambokan (Citrus sulcata), Sudachi (Citrus sudachi) and Koji (Citrus leiocarpa) fruits were found to contain high levels of eriocitrin in both peel and juice vesicles. Copyright © 2006 Society of Chemical Industry

Isolation of two triterpenoids and a biflavanone with anti-Inflammatory activity from Schinus molle fruits.

Three compounds with anti-inflammatory activity were isolated from Schinus molle fruits. Two of the compounds were identified as 3- epi-isomasticadienolalic acid ( 1), isomasticadienonalic acid ( 2) and chamaejasmin ( 3). Triterpenes 1 and 2, and biflavanone 3 were tested on two models of mice paw inflammation: one of acute inflammation, induced by subcutaneous injection of either phospholipase A (2) (PLA (2)) or carrageenan in the paws of mice, and one of chronic inflammation in the form of eczema, provoked by repeated administration of TPA to the ears of mice. On the PLA (2)-induced mouse paw oedema, only 2 was active (30 mg/kg, 66 % inhibition at 60 min), whereas all compounds reduced the chronic model of inflammation (48 to 26 % of swelling reduction), but only triterpenes reduced the leukocyte infiltration, measured as tissue peroxidase activity. In the case of the carrageenan-induced mouse paw oedema, only 3 led to a reduction of the swelling 3 h after challenge (50 mg/kg, 46 % oedema inhibition). In addition, 3 inhibited the LTB (4) production in rat peritoneal polymorphonuclear leukocytes with an IC (50) value of 29.8 microM, while triterpenes showed toxicity against cells at 100 microM.

Luminol-dependent chemiluminescence is related to the extracellularly released reactive oxygen intermediates in the case of rat neutrophils activated by formyl-methionyl-leucyl-phenylalanine.

Luminol is a non-radical-specific amplifying molecule which produces light upon interaction with various reactive oxygen intermediates (ROIs). ROI production of rat peritoneal polymorphonuclear leukocytes (PMNLs) elicited by 2.3 microM formyl-methionyl-leucyl-phenylalanine (fMLP) results in a biphasic luminol-dependent chemiluminescence (LDCL) signal. Whereas ROIs are also produced intracellularly, as judged by flow cytometry, addition of non-membrane-permeable catalase reduces the first and second phases of the LDCL signal to around 3% and less than 3%, respectively. This suggests that in the case of fMLP-stimulated rat PMNLs, the LDCL signal is related to the ROIs in the extracellular medium and hydrogen peroxide has a key role in the formation of the LDCL signal. In the presence of the non-specific myeloperoxidase inhibitor Na-azide, the first phase of the LDCL signal decreases slightly (87+/-8%), while the second phase almost disappears (< 3%), indicating the myeloperoxidase dependence of the second phase. The hydroxyl radical scavenger histidine results in an 84+/-4% and a 71+/-4% decrease in the intensity of the first and second phases, respectively. Based on these data, it is concluded that hydrogen peroxide might be the source of hydroxyl radicals directly oxidizing luminol in the first phase of the LDCL signal, while in the second phase it serves as a substrate of myeloperoxidase in the peroxidation reaction of the luminol.

Metabolism of the carcinogen N-hydroxy-N-2-fluorenylacetamide by rat peritoneal neutrophils.

The in vitro metabolism of a locally carcinogenic N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) by rat peritoneal polymorphonuclear leukocytes (PMNL), chiefly neutrophils, elicited with intraperitoneal injections of proteose peptone, was examined. At 10(6) PMNL/ml in media containing halide (X-), 0.14 M Cl- +/- 0.1 mM Br- (without Ca++ and Mg++), addition of 10 nM phorbol myristate acetate (PMA) resulted in generation of superoxide anion and H2O2. Subsequent cetyltrimethylammonium Cl- (Cetac) addition at 0.002% effected myeloperoxidase (MPO) activity release. PMNL treated with PMA and/or Cetac did not metabolize N-OH-2-FAA (30 microM). However, 1-2 pulses of H2O2 (50 microM) after Cetac addition resulted in oxidation of N-OH-2-FAA to N-acetoxy-2-FAA (< 0.5 microM) and 2-nitrosofluorene (2-NOF) (1-2 microM). In the presence of Br- 2-NOF was increased (3-5 microM). The results are consistent with oxidation of N-OH-2-FAA by MPO/H2O2 and MPO/H2O2/X- via two pathways: one electron oxidation leading to N-acetoxy-2-FAA and 2-NOF, and X(-)-dependent oxidation to 2-NOF. N-Acetoxy-2-FAA (10 microM) incubated with PMNL under similar conditions was converted non-enzymatically to 4-OH-2-FAA (< or = 5 microM) and enzymatically to N-OH-2-FAA (< or = 3 microM). In the presence of H2O2, smaller amounts of these products were formed. Formation of N-OH-2-FAA was prevented by paraoxon (0.1 mM) suggesting O-deacetylase activity. However, accountability for N-acetoxy-2-FAA decreased with time, presumably because of binding to cellular macromolecules. With H2O2 addition, 2-NOF (10 microM) was converted to 0.5 or 0.25 microM 2-nitrofluorene by active PMNL or heat-inactivated cell lysates, respectively. Low recoveries of 2-NOF were also attributed to binding. The results suggest that PMNL may be involved in activation of the carcinogenic N-arylhydroxamic acids in vivo.

Induction of nitric oxide synthase in rat peritoneal neutrophils and its inhibition by dexamethasone

Rat peritoneal polymorphonuclear leukocytes (PMN) elicited with oyster glycogen contain a Ca(2+)-independent nitric oxide (NO) synthase which is induced in vivo in a time-dependent manner. When washed PMN containing low levels of enzyme activity were cultured ex vivo further expression of NO synthase was observed. This was inhibited by cycloheximide indicating that de novo synthesis of the enzyme occurred during the ex vivo incubation. Enzyme activity was enhanced by interferon (IFN)-gamma, but not by tumor necrosis factor (TNF)-alpha when added ex vivo. However, IFN-gamma and TNF-alpha synergized to increase further the expression of NO synthase. Treatment of rats with dexamethasone inhibited the induction of NO synthase in elicited PMN. This treatment reduced the accumulation of PMN by approximately 30%, without affecting cell viability. Dexamethasone also inhibited the induction of the NO synthase ex vivo in a concentration-dependent manner. Furthermore, the enhanced enzyme activity following treatment of PMN with cytokines was also inhibited by dexamethasone. Once induced, dexamethasone did not affect enzyme activity. These data indicate that PMN elicited in the rat peritoneum with oyster glycogen express an NO synthase in vivo and ex vivo. The induction of the enzyme can be further stimulated ex vivo with IFN-gamma and TNF-alpha and inhibited by dexamethasone. The inhibition of the induction of NO synthase in the PMN by dexamethasone may contribute to the anti-inflammatory activity of this and other glucocorticoids.

Effect of dietary alpha-linolenate on platelet-activating factor production in rat peritoneal polymorphonuclear leukocytes.

The effects of dietary alpha-linolenate (18:3, n-3) and linoleate (18:2, n-6) on platelet-activating factor (PAF) production were examined. Rats were fed an alpha-linolenic acid-rich (perilla oil) diet or a linoleic acid-rich (safflower oil) diet for 6 wk, and polymorphonuclear leukocytes (PMN) were elicited by peritoneal injection of casein. The overall phospholipid content and composition as well as the subclass distribution of choline and ethanolamine glycerophospholipids in PMN were not altered by these diets. However, with the perilla oil diet their content of a putative precursor of PAF, 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine was approximately 50% of that with safflower oil diet. On exposure to various concentrations of FMLP, PAF formation by PMN in the perilla oil group was less than 50% of that by PMN in the safflower oil group. A larger difference in PAF productions by PMN in the two dietary groups was observed on their stimulation with calcium ionophore A23187. These results demonstrate that PAF production is modulated in some as yet unknown way by changing the alpha-linolenate/linoleate balance of the diet.

Inhibition of leukotriene B4 formation in rat peritoneal neutrophils by an ethanolic extract of the gum resin exudate of Boswellia serrata.

Suspensions of rat peritoneal polymorphonuclear leukocytes (PMNL) elicited with glycogen were stimulated by calcium and ionophore to produce leukotrienes and 5-HETE from endogenous arachidonic acid (AA). We investigated the effect of ethanolic extracts of the gum resin exudate of Boswellia serrata. A concentration-dependent inhibition of LTB4 and 5-HETE production by different charges of exudate extracts were found. All products of the 5-lipoxygenase (5-LOx) from endogenous arachidonic acid (AA) in PMNL were reduced to the same extent by the extracts tested. The ethanolic extract of the gum resin also decreased 5-LOx mediated metabolisation of exogenously added AA to LTB4 and 5-HETE. Since steroidal-type anti-inflammatory drugs do not exert an immediate effect in the test system used, we conclude that the activity of the 5-LOx itself represents the side of inhibition by the gum resin extract. Therefore, an inhibition of 5-LOx catalysed mediator synthesis might be involved in the previously reported anti-inflammatory activity in vivo.

High affinity receptor binding of platelet-activating factor in rat peritoneal polymorphonuclear leukocytes

Rat peritoneal polymorphonuclear leukocytes (PMNs) showed a single class of high affinity binding sites for platelet-activating factor (PAF) with an equilibrium dissociation constant (K D) of 4.74 (±2.59) nM. Each cell contained 2.79 (±1.40) × l0 4 receptors. In the isolated membranes at pH 7.0 in 10 mM MgCl 2, 10 mM Tris and 0.25% bovine serum albumin, the K D value was 0.61(±0.1) nM, which is roughly identical to the K D values reported previously for various membrane systems under identical ionic conditions. The receptors were highly specific for PAF. Several receptor antagonists that are reported to inhibit the binding of [ 3H]PAF and the PAF-induced function in platelets could fully displace the binding. The biologically inactive enantiomer (enantio-C 16-PAF), a PAF analog, azido-PAF, and an indene derivative of the PAF receptor antagonist, L-651,142, had different potencies to inhibit [ 3H]PAF binding to rat and human PMN membranes. L-652,731, a tetrahydrofuran analog of the PAF receptor antagonist was about 10 times more potent to inhibit the binding in rat liver tissues than in rat PMNs. These results suggest that PAF receptors on human and rat PMNs are not identical and that PAF receptor subtypes may exist in rat liver and PMNs.

Oxidative Metabolism of Polymorphonuclear Leukocytes: Modulation by Adhesive Stimuli

Different agents such as phorbol myristate acetate (PMA), N-formyl-methionyl-leucylphenylalanine (fMet-Leu-Phe), or opsonized zymosan induced an oxidative burst in rat peritoneal polymorphonuclear leukocytes (PMNs) elicited by casein. Plastic adhesion of PMNs down-regulated superoxide (O2) release stimulated by PMA or fMet-Leu-Phe but had no effect on zymosan-induced O2 generation, indicating that the O2 forming enzyme, the NADPH oxidase, was not affected by modulation and that a common step of the transductional events induced by PMA or fMet-Leu-Phe might be involved in this regulation. We demonstrated that a differential translocation of protein kinase C (PKC) was not responsible for that modulation. PMA-induced secretion of granule content (vitamin B12-binding protein) was not susceptible to modulation, suggesting that the transductional pathways leading to O2 generation and granule secretion are partly separated. The adhesion of PMNs to different substrates (glass, plastic, albumin-, laminin-, fibronectin-, poly-lysine-, or concanavalin A-coated plastic) down-regulated to different extent superoxide release. Whether the nature of the biochemical signal induced by the diverse adhesive stimuli or a physical parameter such as binding strength was involved in this differential behavior remains to be elucidated. Since adhesiveness was dependent on the state of the cytoskeleton and O2 inducers were reported to stimulate actin polymerization, we studied the F-actin content and distribution of PMNs by using the specific fluorescent probe NBD-phallacidin and an original methodology allowing a quantitative analysis of fluorescence on both adherent and suspended cells. PMA induced a polarization of F-actin on suspended PMNs but had no effect on the intracellular distribution of F-actin in adherent PMNs. Thus, we suggest that the adhesion of PMNs induced an immobilization of F-actin, possibly correlated to the down-regulation of one of the transductional pathways involved in the NADPH oxidase activation.

Stimulation of Phagocytic Activity of Leukocytes and Macrophages by Traxanox Sodium in Mice and Rats

Phagocytosis of yeast particles by mouse peritoneal macrophages or rat peritoneal polymorphonuclear leukocytes was enhanced by traxanox sodium in vitro. Traxanox sodium (30 and 100 mg/kg, p.o.) enhanced phagocytosis of yeast particles by leukocytes and macrophages in vivo or ex vivo. On the other hand, prednisolone, isoproterenol and theophylline inhibited phagocytosis by leukocytes and macrophages under the same conditions. Traxanox sodium (30 mg/kg, p.o.) prevented the suppression of phagocytosis by the above drugs. Combined with theophylline, isoproterenol synergistically inhibited phagocytosis by leukocytes in vivo. Traxanox sodium (100 mg/kg, p.o.) administered alone had no influence on carbon clearance in normal mice. However, traxanox sodium (1–30 mg/kg, p.o.) prevented the suppression of carbon clearance by the treatment with carrageenan, but not by the treatment with ethyl palmitate. These results suggest that traxanox sodium stimulates the phagocytic activity of leukocytes or macrophages and prevents the drug-induced suppression of the phagocytic activity of these cells.

Stimulation of phagocytic activity of leukocytes and macrophages by traxanox sodium in mice and rats.

Phagocytosis of yeast particles by mouse peritoneal macrophages or rat peritoneal polymorphonuclear leukocytes was enhanced by traxanox sodium in vitro. Traxanox sodium (30 and 100 mg/kg, p.o.) enhanced phagocytosis of yeast particles by leukocytes and macrophages in vivo or ex vivo. On the other hand, prednisolone, isoproterenol and theophylline inhibited phagocytosis by leukocytes and macrophages under the same conditions. Traxanox sodium (30 mg/kg, p.o.) prevented the suppression of phagocytosis by the above drugs. Combined with theophylline, isoproterenol synergistically inhibited phagocytosis by leukocytes in vivo. Traxanox sodium (100 mg/kg, p.o.) administered alone had no influence on carbon clearance in normal mice. However, traxanox sodium (1-30 mg/kg, p.o.) prevented the suppression of carbon clearance by the treatment with carrageenan, but not by the treatment with ethyl palmitate. These results suggest that traxanox sodium stimulates the phagocytic activity of leukocytes or macrophages and prevents the drug-induced suppression of the phagocytic activity of these cells.

Role of complement C9 and calcium in the generation of arachidonic acid and its metabolites from rat polymorphonuclear leukocytes

We have previously shown that antibody-sensitized mouse peritoneal macrophages release arachidonic acid (C20:4) and its oxygenated derivatives when treated with complement, and that the major part of the release depended on the terminal complement complexes (TCC). To further delineate the process(es) responsible for this release we have extended our studies to rat peritoneal poly-morphonuclear leukocytes (PMNs). Experiments were performed with antibody-sensitized rat PMNs labeled with [ 3H]C20:4 and carrying the TCC, C5b-7, C5b-8 or C5b-9. In contrast to the results of other studies, production of leukotriene B 4 (LTB 4), the major radiolabeled derivative, was strictly dependent on the presence of C9. However, low levels of C20:4 and prostaglandins (PGs) were produced prior to the C5b-9 stage. Kinetic studies demonstrated that release of LTB 4 was rapid; the initial release occurred within 4–6 min and a second rise in release coincided with cell death. Virtually all the LTB 4 produced was released as we found no evidence of retention of intracellular LTB 4 at either the C5b-8 or C5b-9 stages. In the absence of extracellular calcium, the release of LTB 4 was completely abolished and the release of C20:4 and PGs was drastically reduced. [ 3H]C20:4-labeled PMNs carrying C5b-9 did release substantial amounts of radiolabeled material in the presence of EGTA; however, the majority of this lipid was in the form of intact phospholipid and triglyceride. These results indicate that release of C20:4 and its oxygenated derivatives from rat PMNs is (1) dependent on the participation ofC9 in the preexisting C5b-8 complex in the cell membrane, and (2) largely dependent on the presence of calcium.

Synthesis of 2H8‐leukotriene B4

AbstractA semi‐synthetic method for the preparation of deuterated leukotriene B4 (2H8‐LTB4) from eicosatetraynoic acid (ETYA) is described which involves reduction of ETYA to deuterated arachidonic acid (2H8‐AA) with deuterium gas and then biological conversion of this material to 2H8‐LTB4 by rat peritoneal polymorphonuclear leukocytes (PMNs).

Glutathione peroxidase is neither required nor kinetically competent for conversion of 5-HPETE to 5-HETE in rat PMN lysates

In the 5-lipoxygenase pathway for arachidonic acid metabolism, reduction of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to 5-hydroxyeicosatetraenoic acid (5-HETE) is catalyzed by an activity different from glutathione peroxidase. Glutathione peroxidase here refers to the nonspecific peroxidase that catalyzes the reduction by glutathione of cumeme hydroperoxide and a variety of other peroxides including 5-HPETE. This enzyme is inhibited by mercaptosuccinic acid. Preparations of the 15,000xg supernatant from lysed rat peritoneal polymorphonuclear leukocytes were the source of these activities. Thus, when glutathione peroxidase is inhibited to less than 0.5% of its normal activity by mercaptosuccinic acid, 5-HPETE is reduced as efficiently as in the absence of mercaptosuccinate. In lysate preparations from which endogenous glutathione has been removed, reduction of 5-HPETE is still observed but only in the presence of added reducing agents, e.g., 0.2 mM glutathione. When endogenous glutahione peroxidase is not inhibited, reduction of 5-HPETE occurs at a rate >15-fold faster than can be accounted for by this activity. We conclude, therefore, that the glutathione peroxidase in rat PMNs is not kinetically competent to account for reduction of 5-HPETE. There is a distinct peroxidase that catalyzes this reaction. The 5-HPETE peroxidase can utilize glutathione as reducing agent but is not inhibited by mercaptosuccinate, and additional results indicate that it is inactivated during turnover.

Accumulation of amino acids within intracellular lysosomes of rat polymorphonuclear leukocytes incubated with amino acid methyl esters. Evidence for the internal acidification of azurophilic granules.

Rat peritoneal polymorphonuclear leukocytes accumulate radioactivity when incubated with 17 microM L[4,5-3H]leucine methyl ester; only low levels of accumulation are observed when the cells are incubated with equivalent amounts of labeled D-leucine methyl ester or the free amino acid, L-leucine. More than 98% of the intracellular radioactivity of cells incubated with leucine methyl ester is in the form of leucine. When cells that had been preincubated with L-leucine methyl ester were disrupted by sonication, 72% of the intracellular radioactivity could be trapped on glass fiber filters, indicating that the leucine had accumulated within a particulate intracellular compartment. Granule preparations obtained from the polymorphonuclear leukocytes accumulated leucine in the presence of leucine methyl ester in a manner similar to the intact cells. Separation by density gradient centrifugation of the granules obtained from cells preincubated with labeled leucine methyl ester revealed a nearly exact correspondence between the density distribution of accumulated radioactivity and that for beta-glucuronidase, an enzyme localized with the azurophilic subpopulation of polymorphonuclear leukocyte granules; there was no such correspondence with the activity of alkaline phosphatase, a marker for the specific granules. The results indicate that the azurophilic granules represent the intracellular site of leucine accumulation by the intact cells. This conclusion is supported by the effects of millimolar concentrations of amino acid methyl ester, which cause a dramatic loss of latency for the activities of the azurophilic granule enzymes beta-glucuronidase and myeloperoxidase but have no effect upon the latency of the specific granule enzyme alkaline phosphatase. Leucine accumulation by the intact cells is 80% inhibited by 0.1 mM chloroquine and 50% inhibited by 20 mM NH4Cl and methylamine, lysosomotropic agents that elevate the intralysosomal pH. These observations suggest that the azurophilic granules of these cells, generally considered to be primary lysosomes, are internally acidified.

Multiple effects of a new anti-inflammatory agent, timegadine, on arachidonic acid release and metabolism in neutrophils and platelets

Casein-elicited rat peritoneal polymorphonuclear leukocytes (PMNL) and rabbit platelets were prelabelled with [1- 14C]arachidonic acid, and the effect of timegadine, a new anti-inflammatory agent, on the release and metabolism of arachidonic acid induced by A23187 (PMNL) and thrombin (platelets) was studied and compared with the effect of other compounds reported to affect these enzymatic mechanisms. Timegadine inhibited arachidonic acid release from both cells (IC 50 = 2.7 × 10 − M) the lipoxygenase activity in PMNL (IC 50 = 4.1 × 10 −5 M) and the cyclooxygenase activity in platelets (IC 50 = 3.1 × 10 −8 M). By these mechanisms, PMNL leukotriene B 4 formation was inhibited by 50% at 2.0 × 10 −5 M, platelet thromboxane B 2 at 3.2 × 10 −8 M, and platelet 12-HETE at 4.9 × 10 −5 M. These effects might add to the understanding of the anti-inflammatory properties of timegadine.

Neutrophil aggregating and chemokinetic properties of a 5,12,20-trihydroxy-6,8,10,14-eicosatetraenoic acid isolated from human leukocytes

Suspensions of human peripheral blood leukocytes were incubated with arachidonic acid and calcium ionophore A23187. Acidic lipid extracts were purified by silicic acid chromatography and reversed phase high performance liquid chromatography. A polar metabolite with an ultraviolet absorption spectrum identical to that of leukotriene B 4 was identified by gas chromatography and mass spectrometry as a 5,12,20-trihydroxy-6,8,10,14-eicosatetraenoic acid. The data indicated that this compound was the 20-hydroxy metabolite of leukotriene B 4. The compound showed chemokinetic activity towards human polymorphonuclear leukocytes and aggregating activity towards rat peritoneal polymorphonuclear leukocytes. Its activity was greater than that of 5-hydroxy-6,8,11,14-eicosatetraenoic acid but less than that of leukotriene B 4. It is concluded that the 5,12,20-trihydroxyeicosatetraenoic acid may play a role as a mediator of inflammatory reactions.

Identification and measurement of rat eosinophil phospholipase D. Its activity on schistosomula phospholipids

A sensitive assay, using [ 14C]lecithin as a substrate, has been developed for the measurement of phospholipase activity in rat peritoneal polymorphonuclear leukocytes. Cell extracts were found to contain a phospholipase D activity and indirect evidence suggested that eosinophils are responsible for the cleavage of lecithin. Intact peritoneal cells were also able to hydrolyze exogenous [ 14C]lecithin in vitro. When [ 3H]choline-labeled schistosomula were used as targets in antibody-dependent cytotoxicity experiments, the radioactivity of lecithin decreased more rapidly in a complete cytotoxicity system than in controls, suggesting that hydrolysis of schitosomula phosholipids occured during the killing process.

Function of H 2 O 2 , Myeloperoxidase, and Hexose Monophosphate Shunt Enzymes in Phagocytizing Cells from Different Species

The effect of phagocytosis on the H(2)O(2) production and myeloperoxidase (MPO) activities of leukocytes from various species was investigated. The intracellular distribution of MPO, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase (6PGDH) of resting and phagocytizing guinea pig polymorphonuclear leukocytes has also been studied. Phagocytizing cells produce more H(2)O(2) than the corresponding resting cells. This has been found to be true for human peripheral polymorphonuclear leukocytes, mouse peritoneal macrophage, and guinea pig and rat peritoneal polymorphonuclear leukocytes. All of these cells, except rabbit alveolar macrophages, have significant MPO activity. Generally an increased activity is noted with phagocytizing cells. Homogenization and differential centrifugation of guinea pig peritoneal polymorphonuclear leukocytes indicate that the whole homogenate and its fractions from phagocytizing cells have significantly higher MPO and NADPH oxidase activities, when compared to the corresponding fractions from the resting cells. The 27,000 x g supernatant fluid from phagocytizing cells has 6-fold more MPO and 2.5-fold more NADPH oxidase activity than similar supernatant fractions from resting cells. The enzyme 6PGDH was unaffected by phagocytosis. The relationship of these stimulated activities to the intracellular bactericidal function of the phagocytes has been discussed.