Enhancement of macrophage spreading and phagocytosis by peroxidative enzymes

Macrophages stimulated by various substances exhibit altered morphology, metabolism, and enhanced phagocytosis. The present studies were done to show if peroxidative enzymes would affect macrophage spreading and phagocytosis. Resident peritoneal macrophages, collected from C57BI mice were exposed to various concentrations of peroxidases and compared with appropriate controls. Results indicated that 0.01 microM myeloperoxidase (MyPO), 0.09 microM horseradish peroxidase (HRP), 0.16 microM lactoperoxidase (LPO) and 70 microM microperoxidase (MPO) significantly enhanced macrophage spreading. It was also noted that peroxidases were able to stimulate phagocytosis by increasing the number of cells with internalized zymosan at least twofold. Stimulation of these functions suggests a possible role of endogenous peroxidases as natural cell activators.     

Peroxidases enhance macrophage-mediated cytotoxicity via induction of tumor necrosis factor

Tumor necrosis factor (TNF) is a monokine which is involved in macrophage-mediated cytotoxicity (MMC). We have previously reported that peroxidases can activate thioglycollate-induced macrophages to the tumoricidal state in vitro. The present study was undertaken in an attempt to correlate peroxidase-induced MMC with production of TNF. Horseradish peroxidase (HRP) was used as the principal model for these studies. Resident and thioglycollate-induced macrophages exposed to peroxidases were examined for both MMC against 3T12 cells and production of TNF. Thioglycollate-induced macrophages exposed to HRP, bovine lactoperoxidase, or human myeloperoxidase demonstrated enhanced secretion of TNF. When exposed to HRP, both resident and thioglycollate-induced macrophages secreted significant amounts of TNF and acquired the ability to lyse 3T12 cells. However, resident macrophages were considerably less efficient in both their cytotoxic activity and TNF secretion. Macrophage-mediated cytotoxicity was eliminated by the addition of specific antisera to TNF. In addition, replacement of culture supernatants within 24 hr after exposure of the macrophages to HRP increased tumor cell killing in the absence of additional detectable TNF production, suggesting that other factors may be involved in peroxidase-induced MMC. These results indicate that TNF is intimately associated with peroxidase-induced MMC and suggest a possible role for peroxidases as immunomodulators via augmentation of macrophage capacities and functions.     

Differentiation of human peripheral blood monocytes to macrophages is associated with changes in the cellular respiratory burst activity.

When phagocytic leukocytes, e.g. neutrophils, monocytes and macrophages, interact with soluble or particulate stimuli, the cells respond with an increased production of reactive oxygen metabolites. This production can be measured with the luminol-amplified chemiluminescence (CL) technique. In the present study, the CL reaction induced in monocyte-derived macrophages was investigated and compared to the responses of neutrophils and monocytes. In systems without additives the CL response of macrophages to soluble stimuli (FMLP, PMA and ionomycin) was very low. Addition of a peroxidase (HRP) to the reaction mixtures resulted in a pronounced increase in CL activity. The cellular CL response in macrophages is thus limited by the amount of peroxidase available. The macrophage response differs qualitatively from the responses of neutrophils and monocytes, in that the intracellular phase of the response is missing.     

Activation of macrophages by peroxidases

Peritoneal macrophages from C57BL/6 mice were activated in vitro with various peroxidases and their cytotoxic activity toward 3T12 cells was determined. Destruction of 3T12 cells by macrophages stimulated with horseradish peroxidase, lactoperoxidase, and microperoxidase was observed at peroxidase concentrations as low as 9, 1.6, and 200 nM, respectively. A 50% cytotoxic effect was obtained at peroxidase concentrations of 0.9, 1.6, and 1.5 microM, respectively. The macrophage-stimulating activity of horseradish peroxidase was not destroyed by boiling. This, together with the high activity of microperoxidase, indicates that the macrophage-stimulating activity of the peroxidases is probably associated with the heme portion of the enzymes. On a molar basis the peroxidases are much less potent macrophage activators than interferon (alpha + beta) and endotoxin. Nevertheless, our data clearly indicate that peroxidases are a group of enzymes capable of inducing macrophage activation, resulting in cytostatic and/or cytocidal activity.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Horseradish and soybean peroxidases: comparable tools for alternative niches?

Horseradish and soybean peroxidases (HRP and SBP, respectively) are useful biotechnological tools. HRP is often termed the classical plant heme peroxidase and although it has been studied for decades, our understanding has deepened since its cloning and subsequent expression, enabling numerous mutational and protein engineering studies. SBP, however, has been neglected until recently, despite offering a real alternative to HRP: SBP actually outperforms HRP in terms of stability and is now used in numerous biotechnological applications, including biosensors. Review of both is timely. This article summarizes and discusses the main insights into the structure and mechanism of HRP, with special emphasis on HRP mutagenesis, and outlines its use in a variety of applications. It also reviews the current knowledge and applications to date of SBP, particularly biosensors. The final paragraphs speculate on the future of plant heme-based peroxidases, with probable trends outlined and explored.     

A comparative study of free and immobilized soybean and horseradish peroxidases for 4-chlorophenol removal: protective effects of immobilization

Horseradish peroxidase (HRP) and soybean peroxidase (SBP) were covalently immobilized onto aldehyde glass through their amine groups. The activity yield and the protein content for the immobilized SBP were higher than for the immobilized HRP. When free and immobilized peroxidases were tested for their ability to remove 4-chlorophenol from aqueous solutions, the removal percentages were higher with immobilized HRP than with free HRP, whereas immobilized SBP needs more enzyme to reach the same conversion than free enzyme. In the present paper the two immobilized derivatives are compared. It was found that at an immobilized enzyme concentration in the reactor of 15 mg l(-1), SBP removed 5% more of 4-chlorophenol than HRP, and that a shorter treatment was necessary. Since immobilized SBP was less susceptible to inactivation than HRP and provided higher 4-chlorophenol elimination, this derivative was chosen for further inactivation studies. The protective effect of the immobilization against the enzyme inactivation by hydrogen peroxide was demonstrated.     

Enzymatic reduction of 5-phenyl-4-pentenyl-hydroperoxide: detection of peroxidases and identification of peroxidase reducing substrates

5-Phenyl-4-pentenyl-hydroperoxide (PPHP) is reduced to 5-phenyl-4-pentenyl-alcohol (PPA) by plant and animal peroxidases in the presence of reducing substrates. PPHP and PPA are rapidly isolated with solid phase extraction, separated by isocratic reverse-phase high-performance liquid chromatography, and quantitated with a fixed-wave-length ultraviolet detector. The procedure described is suitable for detecting peroxide-reducing enzymes, determining the kinetic properties of heme- and non-heme-containing peroxidases, and evaluating oxidizable compounds as reducing substrates for peroxidases. Horseradish peroxidase (HRP) and phenol reduce PPHP with a Km for phenol of 252 microM and a turnover number of 1.05 X 10(4) min-1. Under similar conditions, the Km of HRP for PPHP is 18 microM in the oxidation of guaiacol. A series of 21 compounds was evaluated for the ability to serve as reducing substrates for HRP. The results indicate that the procedure described can not only identify compounds that are reducing substrates but also rank them for relative activity. This may provide a new method with which to identify novel antithrombotic, antimetastatic, or anti-inflammatory drugs as well as to detect and characterize mammalian peroxidases.     

Comparison of the responses of peritoneal macrophages from Japanese flounder (Paralichthys olivaceus) against high virulent and low virulent strains of Edwardsiella tarda

In vivo infection studies in Japanese flounder (Paralichthys olivaceus) demonstrated that the number of viable cells of the virulent strain (NUF251) of Edwardsiella tarda increased gradually in kidney and hepato-pancreas after intraperitoneal injection, but the low virulent strain (NUF194) did not. To gain insight into the virulence factors of E. tarda, in vitro responses of Japanese flounder (P. olivaceus) peritoneal macrophages to these strains were compared in terms of phagocytosis, bactericidal activity, and reactive oxygen species (ROS) generation as measured by chemiluminescence (CL) responses. Microscopic observation revealed that these two strains of E. tarda were phagocytosed by the peritoneal macrophages, and there was no significant difference in the mean numbers of ingested bacteria per macrophage between these strains. A gradual increase in the number of viable cells of the highly virulent strain within macrophages was observed during 9h post-phagocytosis, whereas no significant replication of the low virulent strain within macrophages was detected. These results suggest that the virulent strain of E. tarda has an ability to survive and replicate within macrophages, while the low virulent strain has no such ability. When the peritoneal macrophages were exposed to the opsonized low virulent E. tarda strain, a rapid increase in CL response was induced. However, the highly virulent strain caused only background level of CL response. By the subsequent stimulation with phorbol myristate acetate, the macrophages exposed to the virulent E. tarda strain showed extremely higher CL response than that of the one exposed to the low virulent E. tarda strain. These results suggest that the virulent E. tarda prevents the activation of ROS generation system during phagocytosis, though the system is still capable of responding to other stimulation. The virulent strain significantly reduced the CL response induced by xanthine/xanthine oxidase system, while the low virulent strain had almost no effect. Furthermore, the virulent strain showed greater resistance to H(2)O(2) than the low virulent strain. Our results suggest that the virulent strain of E. tarda is highly resistant to ROS, and such ability might allow the organism to survive and multiply within phagocytes, and may serve to disseminate E. tarda throughout the host during in vivo infection.     

Horseradish peroxidase catalyzed degradation of industrially important dyes.

Horseradish peroxidase (HRP) is known to degrade certain recalcitrant organic compounds such as phenol and substituted phenols. Here, for the first time we have shown HRP to be effective in degrading and precipitating industrially important azo dyes. For Remazol blue, the enzyme activity was found to be far better at pH 2.5 than at neutral pH. In addition, Remazol blue acts as a strong competitive inhibitor of HRP at neutral pH. Horseradish peroxidase shows broad substrate specificity toward a variety of azo dyes. Kinetic constants (K(m)(app) and V(max)(app)) for two different dyes have been determined. In addition to providing a systematic analysis of the potential of HRP in degradation of dyes, this study opens up a new area on exploration of commercial dyes as inhibitors of enzymes. 2001 John Wiley & Sons, Inc.     

Plant peroxidases. Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase

The amino acid sequences of the 51% different horseradish peroxidase HRP C and turnip peroxidase TP 7 have previously been completed by us, but the three-dimensional structures are unknown. Recently the amino acid sequence and the crystal structure of yeast cytochrome c peroxidase have appeared. The three known apoperoxidases consist of 300 +/- 8 amino acid residues. The sequences have now been aligned and show 18% and 16% identity only, between the yeast peroxidase and plant peroxidase HRP C and TP 7, respectively. We show that different structural tests all support similar protein folds in plant peroxidases and yeast peroxidase and, therefore, a common evolutionary origin. The following tests support this thesis: (a) predicted helices in the plant peroxidases follow the complex pattern observed in the crystal structure of cytochrome c peroxidase; (b) their hydropathic profiles are similar and agree with observed buried and exposed peptide chain in cytochrome c peroxidase; (c) half-cystines which are distant in the amino acid sequence of plant peroxidases become spatial neighbours when fitted into the cytochrome c peroxidase model; (d) the two-domain structure proposed from limited proteolysis of apoperoxidase HRP C is observed in the crystal structure of cytochrome c peroxidase. The similarities and differences of the plant and yeast peroxidases and the reactive side chains of a plant peroxidase active site are described. The characteristics of Ca2+-binding sequences, derived from several superfamilies, are applied to predict the Ca2+-binding sequences in plant peroxidases.     

The role of the mannose/N-acetylglucosamine receptor in the pinocytosis of horseradish peroxidase by mouse peritoneal macrophages

Saccharomyces cerevisiae mannan inhibits the pinocytosis of horseradish peroxidase (HRP) by resident, thioglycollate-,proteose peptone-, and Corynebacterium parvum-elicited macrophages from 30 to 70% when 1 mg/ml HRP is used, and 65 to 87% when 250 micrograms/ml HRP is used. In contrast, HRP uptake by J774 cells, a macrophage cell line reported to have little mannose receptor activity, is inhibited only about 25% by mannan. HRP uptake by resident and thioglycollate-elicited (thio) macrophages is also inhibited 34 and 66% by addition of EGTA to the medium and 55 and 79% by trypsin treatment of the macrophages, respectively. The inhibitory effect of EGTA can be reversed by 1 mM excess Ca2+. High extracellular concentrations of Ca2+, in the range of 10-20 mM, however, inhibit pinocytosis in resident macrophages by about 50%. Sucrose uptake by resident macrophages is not appreciably affected by mannan. These results support the hypothesis that HRP uptake is mediated by the macrophage mannose/N-acetylglucosamine receptor. PMA stimulates fluid-phase pinocytosis of HRP by thio macrophages but does not affect receptor-mediated uptake of HRP, while the combination of adenosine, homocysteine, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) selectively inhibits bulk-phase uptake by thio macrophages.     

Stabilization of heat-induced changes in plant peroxidase preparations by ClpX, a bacterial heat shock protein

Peroxidases (PODs) are known to be quite stable at elevated temperatures. Moreover, partially denatured peroxidases are able to regain their catalytic activity during incubation at room temperature. In this paper, we describe the effects of some heat shock proteins on the self-reactivation of plant peroxidase preparations. Horseradish and artichoke peroxidases (HRP and ARP, respectively) were first heated (at 60 °C or 90 °C), then incubated at a slightly elevated temperature (30 °C). The heat-treatment resulted in a considerable loss of activity of both enzymes but the subsequent incubation allowed their reactivation. However, no reactivation could be detected when incubation was carried out in the presence of the molecular chaperone ClpX. Other chaperones that were tested (DnaK, DnaJ and GrpE) did not show the inhibitory effect. Electrophoretic analyses further indicated that the heat-treated horseradish peroxidase, but not the native enzyme, binds to ClpX eliminating the possibility of undesirable protein refolding that would result in aggregation.     

Enhancement of macrophage Fc-dependent phagocytosis by resident thymocytes: effect of a unique heat-stable lymphokine

Lymphocytes, activated by lectins or specific antigens, have been shown to enhance macrophage phagocytosis through the elaboration of a heat-labile soluble factor(s). Recent evidence from our laboratory revealed that resident (nonactivated) murine thymocytes and splenic lymphocytes increase peritoneal macrophage glucose metabolism through the elaboration of a heat-stable soluble factor(s). Therefore, we investigated the effect of resident lymphocyte subpopulations on macrophage Fc-dependent phagocytosis. Thioglycollate-elicited and resident peritoneal macrophages from BALB/c mice were cultured in serum-free media with syngeneic resident thymocytes or splenic T lymphocytes. Macrophage Fc-dependent phagocytosis was assayed by measuring the ingestion of 51CrSHEA. After 4 days in vitro, resident thymocytes produced a mean 160 (+/- 31) and 136% (+/- 22) increase in Fc-dependent phagocytosis by thioglycollate-elicited (thio-macrophages) and resident peritoneal macrophages, respectively. Splenic T lymphocytes increased thio-macrophage phagocytosis by 112% (+/- 41) under similar conditions. Macrophage Fc-dependent phagocytosis was increased after 24 hr of co-culture by supernatant derived from resident thymocytes and could be further enhanced by supernatant from Con A-activated thymocytes. Supernatant from guinea pig embryo fibroblasts did not increase macrophage phagocytosis. The soluble factor(s) was produced by resident thymocytes after 24 hr of preculture. This factor was active despite heating at 100 degrees C for 30 min whereas the effect of Con A-activated thymocyte supernatant was heat-labile. The stimulatory effect of resident thymocyte supernatant was not observed when the macrophages and supernatant were cultured in 2% FCS. In contrast to the factor(s) produced by resident thymocytes, the factor(s) in FCS that increased phagocytosis was heat-labile. These data suggest thymocytes and splenic T lymphocytes promote macrophage Fc-dependent phagocytosis in the absence of antigenic or lectin stimulation. This previously unrecognized effect of resident thymocytes is due to a unique heat-stable soluble factor(s) that is concealed in the presence of serum.     

Substrate specificity of rat sera IgG antibodies with peroxidase and oxidoreductase activities

We have recently shown that intact IgGs from the sera of healthy Wistar rats oxidize 3,3'-diaminobenzidine (DAB) in the presence and in the absence of H(2)O(2) similar to horseradish peroxidase (HRP). Here we demonstrate for the first time that the peroxidase and oxidoreductase activities of IgGs can efficiently oxidize not only DAB but also o-phenylendiamine, phenol, p-dihydroquinone, alpha-naphthol, and NADH but, in contrast to HRP, cannot oxidize adrenalin. In contrast to IgGs, HRP cannot oxidize phenol, p-dihydroquinone, or alpha-naphthol in the absence of H(2)O(2). In contrast to plant and mammalian peroxidases, IgGs were more universal in their metal dependence. The specific wide repertoire of polyclonal peroxidase and oxidoreductase IgGs oxidizing various substances could play an important role in protecting the organism from oxidative stress and serve as an additional natural system destroying different toxic, carcinogenic, and mutagenic compounds.     

Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells

The present study characterized two different internalization mechanisms used by macrophages to engulf apoptotic and necrotic cells. Our in vitro phagocytosis assay used a mouse macrophage cell line, and murine L929sAhFas cells that are induced to die in a necrotic way by TNFR1 and heat shock or in an apoptotic way by Fas stimulation. Scanning electron microscopy (SEM) revealed that apoptotic bodies were taken up by macrophages with formation of tight fitting phagosomes, similar to the 'zipper'-like mechanism of phagocytosis, whereas necrotic cells were internalized by a macropinocytotic mechanism involving formation of multiple ruffles directed towards necrotic debris. Two macropinocytosis markers (Lucifer Yellow (LY) and horseradish peroxidase (HRP)) were excluded from the phagosomes containing apoptotic bodies, but they were present inside the macropinosomes containing necrotic material. Wortmannin (phosphatidylinositol 3'-kinase (PI3K) inhibitor) reduced the uptake of apoptotic cells, but the engulfment of necrotic cells remained unaffected. Our data demonstrate that apoptotic and necrotic cells are internalized differently by macrophages.     

The use of acetylated cytochrome c in detecting superoxide anion production in rabbit alveolar macrophages

Polymorphonuclear phagocytes have been shown to undergo marked alteration in oxidative metabolism during phagocytosis. These alterations, collectively known as the "respiratory burst", include increased glucose oxidation through the hexose monophosphate shunt (1), increased oxygen consumption (1), and increased superoxide (O-2)3 (2) and H2O2 production (3). Similar metabolic events have also been shown to occur in the rabbit alveolar macrophage (AM). There is consistent evidence that the macrophage undergoes increased oxygen consumption (4-6) and hexose monophosphate shunt activity (4-9) upon phagocytosis. There are conflicting data, however, concerning the ability of the macrophage to produce O-2. Some studies suggest that macrophages are incapable of producing measurable amounts of O-2 upon phagocytosis (7, 10-12). Other studies, however, suggest that macrophages are indeed capable of producing substantial amounts of O-2 during phagocytosis (8, 13-15). This study was designed to resolve the discrepancies in the literature concerning O-2 production in macrophages.     

云芝多糖对巨噬细胞GPx基因表达的影响

细胞内存在两种谷胱甘肽过氧化物酶;硒谷胱甘肽过氧化物酶和非硒谷肽甘肽过氧化物酶,它们在保护细胞免受氧化损伤等过程中起重要作用。为揭示云芝多糖作用与细胞抗氧化酶的关系,采用酶活性测定,斑点杂交等方法,探讨云芝多糖对小鼠腹腔巨噬细胞过氧化物酶表达的影响。结果显示,腹腔注射云芝多糖可以提高小鼠腹腔巨噬细胞的两种过氧化物酶活性,并使其ｍＲＮＡ含量增加,应用阻断剂的研究发现,云芝多糖对巨噬细胞ＳｅＧＰｘ及Ｇ     

Comparative studies on lipid and colony-stimulating factor-induced macrophage growth

We previously reported that lipids such as cholesterol esters, triglycerides, and some phospholipids that constitute cell membranes or serum lipoproteins induced growth of mouse peritoneal macrophages in vitro. In this paper, we compared the macrophage growth-stimulating activity of cardiolipin (CL), an active phospholipid with that of CSF-1. Growth kinetics and maximal degree of growth of exudated macrophages induced by CL were similar to those of CSF-1. CL did not stimulate macrophages to release soluble macrophage growth factors. Also, the activity of CL was not blocked as much by anti-CSF-1, suggesting that most of the effect of CL was direct and not mediated by CSF-1 or other protein factors. There was no synergistic effect between CL and CSF-1. CL induced growth of both exudate and resident macrophages, whereas CSF-1 induced very little resident macrophage growth. Furthermore, although the growth-stimulating activities of both substances were inhibited by IFN-gamma and TNF, CL was more resistant to these inhibitory effects. These results suggest that the lipid has some different characters from CSF-1 and may induce the growth of resident macrophages in inflammations or tumors.     

Studies on the Refolding Process of Recombinant Horseradish Peroxidase

Horseradish peroxidase (HRP) is an important heme-containing glyco-enzyme that has been used in many biotechnological fields. Valuable proteins like HRP can be obtained in sufficient amounts using Escherichia coli as an expression system. However, frequently, the expression of recombinant enzyme results in inclusion bodies, and the refolding yield is generally low for proteins such as plant peroxidases. In this study, a recombinant HRP was cloned and expressed in the form of inclusion bodies. Initially, the influence of few additives on HRP refolding was assessed by the one factor at a time method. Subsequently, factors with significant effects including glycerol, GSSG/DTT, and the enzyme concentration were selected for further optimization by means of the central composite design of response surface methodology (RSM). Under the obtained optimal condition, refolding increased about twofold. The refolding process was then monitored by the intrinsic fluorescence intensity under optimal conditions (0.35 mM GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl₂ in 20 mM Tris, pH 8.5) and the reconstitution of heme to the refolded peroxidase was detected by the Soret absorbance. Additionally, samples under unfolding and refolding conditions were analyzed by Zetasizer to determine size distribution in different media.