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Human Alveolar Macrophages and Granulocyte-macrophage Colony-stimulating Factor-induced Monocyte-derived Macrophages Are Resistant to H2O2 via Their High Basal and Inducible Levels of Catalase Activity

2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês

10.1074/jbc.m102081200

ISSN

1083-351X

Autores

Iwao Komuro, Naoto Keicho, Aikichi Iwamoto, Kiyoko S. Akagawa,

Tópico(s)

Epigenetics and DNA Methylation

Resumo

Human alveolar macrophages (A-MΦ) and macrophages (MΦ) generated from human monocytes under the influence of granulocyte-macrophage colony-stimulating factors (GM-MΦ) express high levels of catalase activity and are highly resistant to H2O2. In contrast, MΦ generated from monocytes by macrophage colony-stimulating factors (M-MΦ) express low catalase activity and are about 50-fold more sensitive to H2O2 than GM-MΦ or A-MΦ. Both A-MΦ and GM-MΦ but not M-MΦ can induce catalase expression in both protein and mRNA levels when stimulated with H2O2 or zymosan. M-MΦ but not GM-MΦ produce a large amount of H2O2 in response to zymosan or heat-killed Staphylococcus aureus. These findings indicate that GM-MΦ and A-MΦ but not M-MΦ are strong scavengers of H2O2 via the high basal level of catalase activity and a marked ability of catalase induction and that catalase activity of MΦ is regulated by colony-stimulating factors during differentiation. Human alveolar macrophages (A-MΦ) and macrophages (MΦ) generated from human monocytes under the influence of granulocyte-macrophage colony-stimulating factors (GM-MΦ) express high levels of catalase activity and are highly resistant to H2O2. In contrast, MΦ generated from monocytes by macrophage colony-stimulating factors (M-MΦ) express low catalase activity and are about 50-fold more sensitive to H2O2 than GM-MΦ or A-MΦ. Both A-MΦ and GM-MΦ but not M-MΦ can induce catalase expression in both protein and mRNA levels when stimulated with H2O2 or zymosan. M-MΦ but not GM-MΦ produce a large amount of H2O2 in response to zymosan or heat-killed Staphylococcus aureus. These findings indicate that GM-MΦ and A-MΦ but not M-MΦ are strong scavengers of H2O2 via the high basal level of catalase activity and a marked ability of catalase induction and that catalase activity of MΦ is regulated by colony-stimulating factors during differentiation. alveolar macrophages macrophage(s) colony-stimulating factor(s) granulocyte-macrophage colony-stimulating factor, GM-MΦ, GM-CSF-induced macrophages macrophage colony-stimulating factor M-CSF-induced macrophages reactive oxygen species human erythrocyte catalase human immunodeficiency virus, type I Human alveolar macrophages (A-MΦ)1 can survive for a long duration (1Thomas E.D. Ramberg R.E. Sale G.E. Sparkes R.S. Golde D.W. Science. 1976; 192: 1016-1018Crossref PubMed Scopus (262) Google Scholar, 2van oud Alblas A.B. van Furth R. J. Exp. Med. 1979; 149: 1504-1518Crossref PubMed Scopus (224) Google Scholar, 3Marques L.J. Teschler H. Guzman J. Costabel U. Am. J. Respir. Crit. Care Med. 1997; 156: 1700-1702Crossref PubMed Scopus (38) Google Scholar, 4Nakata K. Gotoh H. Watanabe J. Uetake T. Komuro I. Yuasa K. Watanabe S. Ieki R. Sakamaki H. Akiyama H. Kudoh S. Naitoh M. Satoh H. Shimada K. Blood. 1999; 93: 667-673Crossref PubMed Google Scholar) to exposure to not only chemical pollutants and exogenous oxidants but also inflammatory mediators and endogenously generated reactive oxygen species (ROS) and play important roles in phagocytosis-mediated host defense against microbial infection via the airway (5Rossi F. Biochim. Biophys. Acta. 1986; 853: 65-89Crossref PubMed Scopus (587) Google Scholar, 6Sibille Y. Reynolds H.Y. Am. Rev. Respir. Dis. 1990; 141: 471-501Crossref PubMed Scopus (936) Google Scholar). Superoxide dismutase, catalase, and glutathione are the main cellular ROS-degrading enzyme systems; superoxide dismutase converts superoxide radical (O⨪2) into H2O2, which is metabolized by catalase and glutathione peroxidase. Previous studies indicated that these enzymes are abundant in A-MΦ (7Zeidler R.B. Flynn J.A. Arnold J.C. Conley N.S. Inflammation. 1987; 11: 371-379Crossref PubMed Scopus (10) Google Scholar, 8Duran H.A. Giulivi C. Boveris A. De Rey B.M. Cell. Mol. Biol. 1988; 34: 507-515PubMed Google Scholar, 9Pietarinen P. Raivio K. Devlin R.B. Crapo J.D. Chang L.Y. Kinnula V.L. Am. J. Respir. Cell Mol. Biol. 1995; 13: 434-441Crossref PubMed Scopus (54) Google Scholar). However, the mechanism to maintain high antioxidant activities in A-MΦ has not been understood because of its heterogeneity and lack of availability to study. Colony-stimulating factors (CSFs) such as granulocyte-macrophage CSF (GM-CSF) and macrophage-CSF (M-CSF) play important roles in survival and differentiation of monocytes/MΦ. Previously, we reported that CSFs such as GM-CSF and M-CSF stimulate MΦ generation from human monocytes, but GM-CSF-induced MΦ (GM-MΦ) and M-CSF-induced MΦ (M-MΦ), however, are distinct in their morphology, cell surface antigen expression (c-fms, CD14, CD71, and 710F), and sensitivity to human immunodeficiency virus, type I (HIV-I) infection (10Akagawa K.S. Nippon Saikingaku Zasshi. 1994; 49: 385-393Crossref PubMed Scopus (3) Google Scholar, 11Akagawa K. Hum. Cell. 1994; 7: 116-120PubMed Google Scholar, 12Matsuda S. Akagawa K. Honda M. Yokota Y. Takebe Y. Takemori T. AIDS Res. Hum. Retroviruses. 1995; 11: 1031-1038Crossref PubMed Scopus (67) Google Scholar, 13Akagawa K.S. Takasuka N. Nozaki Y. Komuro I. Azuma M. Ueda M. Naito M. Takahashi K. Blood. 1996; 88: 4029-4039Crossref PubMed Google Scholar, 14Akagawa K.S. Jpn. J. Med. Mycol. 1997; 38: 209-214Crossref Scopus (3) Google Scholar). Other studies also demonstrated that human monocyte-derived GM-MΦ and M-MΦ are distinct in their expression of CD14, integrin, and antibody-dependent cellular cytotoxicity activity (15Young A.D. Lowe D.L. Clark C.S. J. Immunol. 1990; 145: 607-615PubMed Google Scholar, 16De Nichilo M.O. Burns G.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2517-2521Crossref PubMed Scopus (100) Google Scholar, 17De Nichilo M.O. Yamada K.M. J. Biol. Chem. 1996; 271: 11016-11022Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 18Keler T. Wallace P.K. Vitale L.A. Russoniello C. Sundarapandiyan K. Graziano R.F. Deo Y.M. J. Immunol. 2000; 164: 5746-5752Crossref PubMed Scopus (42) Google Scholar). Numerous studies show that the phenotype of human A-MΦ closely resembles that of GM-MΦ in morphology (fried egg-like shape) (19Nakata K. Akagawa K.S. Fukayama M. Hayashi Y. Kadokura M. Tokunaga T. J. Immunol. 1991; 147: 1266-1272PubMed Google Scholar), the expression of cell surface antigens (c-fmslow, CD14low, CD71+, and 710F+) (11Akagawa K. Hum. Cell. 1994; 7: 116-120PubMed Google Scholar, 13Akagawa K.S. Takasuka N. Nozaki Y. Komuro I. Azuma M. Ueda M. Naito M. Takahashi K. Blood. 1996; 88: 4029-4039Crossref PubMed Google Scholar,20Radzun H.J. Kreipe H. Heidorn K. Parwaresch M.R. J. Leukocyte Biol. 1988; 44: 198-204Crossref PubMed Scopus (13) Google Scholar, 21Andreesen R. Brugger W. Scheibenbogen C. Kreutz M. Leser H.G. Rehm A. Lohr G.W. J. Leukocyte Biol. 1990; 47: 490-497Crossref PubMed Scopus (183) Google Scholar, 22Hirata T. Bitterman P.B. Mornex J.F. Crystal R.G. J. Immunol. 1986; 136: 1339-1345PubMed Google Scholar), and function (resistance to MΦ-tropic HIV-I infection) (12Matsuda S. Akagawa K. Honda M. Yokota Y. Takebe Y. Takemori T. AIDS Res. Hum. Retroviruses. 1995; 11: 1031-1038Crossref PubMed Scopus (67) Google Scholar,23Nakata K. Weiden M. Harkin T. Ho D. Rom W.N. Mol. Med. 1995; 1: 744-757Crossref PubMed Google Scholar). In contrast, M-MΦ are elongated and spindle-shaped, express c-fmshigh and CD14high, which are similar to the phenotype of anaerobic peritoneal MΦ (11Akagawa K. Hum. Cell. 1994; 7: 116-120PubMed Google Scholar, 20Radzun H.J. Kreipe H. Heidorn K. Parwaresch M.R. J. Leukocyte Biol. 1988; 44: 198-204Crossref PubMed Scopus (13) Google Scholar, 21Andreesen R. Brugger W. Scheibenbogen C. Kreutz M. Leser H.G. Rehm A. Lohr G.W. J. Leukocyte Biol. 1990; 47: 490-497Crossref PubMed Scopus (183) Google Scholar, 24Hashimoto S. Yamada M. Motoyoshi K. Akagawa K.S. Blood. 1997; 89: 315-321Crossref PubMed Google Scholar), and are sensitive to MΦ-tropic HIV-I infection (12Matsuda S. Akagawa K. Honda M. Yokota Y. Takebe Y. Takemori T. AIDS Res. Hum. Retroviruses. 1995; 11: 1031-1038Crossref PubMed Scopus (67) Google Scholar). These findings suggest that CSF is one of the critical factors in the determination of phenotypical characteristics of tissue MΦ in the human system, and CSF-induced monocyte-derived MΦ are available to analyze tissue MΦ. In the present study, we investigated whether the antioxidant states of GM-MΦ are at similar levels to those of A-MΦ by assessment of H2O2 sensitivity and catalase activity. We found that GM-MΦ express high basal and inducible levels of catalase activity, are highly resistant to H2O2 compared with M-MΦ, and inhibit H2O2 production when stimulated with microbial stimulants. We also observed that catalase activity and sensitivity to H2O2 in GM-MΦ are at similar levels to those in A-MΦ. These findings suggest that GM-CSF but not M-CSF induces a strong antioxidant system in human tissue MΦ during the differentiation. RPMI 1640 medium (Nissui Seiyaku Co., Ltd., Tokyo, Japan) was supplemented with 3 mg/ml glutamine (Sigma), 100 units/ml penicillin G potassium (Banyu Seiyaku Co., Ltd., Tokyo, Japan), 100 μg/ml streptomycin (Meiji Seika Co., Ltd., Tokyo, Japan), 10% of autoclaved NaHCO3, and finally 10% heat-inactivated fetal calf serum (Z. L. Bockneck Laboratories Inc., Ontario, Canada). Fetal calf serum and distilled water were shown to contain 3 pg and less than 1 pg of lipopolysaccaride per ml by the Limullus amebocytelysate test, respectively. Recombinant human GM-CSF (1 × 108 units/mg) and recombinant human M-CSF (2 × 108 units/mg) were kindly provided by Schering-Plough Japan (Osaka, Japan) and Morinaga Milk Industry Co., Ltd. (Tokyo, Japan), respectively. Peripheral blood mononuclear cells were obtained from venous blood drawn from normal healthy volunteers as described previously (12Matsuda S. Akagawa K. Honda M. Yokota Y. Takebe Y. Takemori T. AIDS Res. Hum. Retroviruses. 1995; 11: 1031-1038Crossref PubMed Scopus (67) Google Scholar, 13Akagawa K.S. Takasuka N. Nozaki Y. Komuro I. Azuma M. Ueda M. Naito M. Takahashi K. Blood. 1996; 88: 4029-4039Crossref PubMed Google Scholar). Briefly, peripheral blood mononuclear cells were isolated by centrifugation on a Ficoll-Metrizoate density gradient (Lymphoprep; Nycomed, Oslo, Norway) and then placed into monocyte-isolating plates (MSP plates; Japan Immunoresearch Laboratories, Co., Ltd., Takasaki, Japan) for 2 h at 37 °C in a humidified 5% CO2 atmosphere (CO2 incubator). More than 97% of the recovered cells were judged to be monocytes based on morphology, nonspecific esterase staining (cells were stained using a kit for α-naphthyl butyrate esterase), CD14 positivity, and their ability to phagocytize latex particles. Monocytes (2.5 × 105 per ml or 5 × 105 per 2 ml in 12- or 6-well tissue culture plates, respectively) were then cultured with a optimal concentration of GM-CSF (500 units/ml) or M-CSF (104 units/ml) for 7 days at 37 °C in a CO2 incubator. During the culture, monocytes underwent morphologic changes characteristic of monocytes to MΦ differentiation such as an increase in their size and adherence. Human A-MΦ (2.5 × 105 per ml or 5 × 105 per 2 ml in 12- or 6-well tissue culture plates, respectively) were obtained from healthy volunteers (non-smokers without pathogenesis) by bronchoalveolar lavage (4Nakata K. Gotoh H. Watanabe J. Uetake T. Komuro I. Yuasa K. Watanabe S. Ieki R. Sakamaki H. Akiyama H. Kudoh S. Naitoh M. Satoh H. Shimada K. Blood. 1999; 93: 667-673Crossref PubMed Google Scholar, 25Goto H. Yuasa K. Sakamaki H. Nakata K. Komuro I. Iguchi M. Okamura T. Ieki R. Tanikawa S. Akiyama H. Onozawa Y. Mochida Y. Bone Marrow Transplant. 1996; 17: 855-860PubMed Google Scholar). All volunteers agreed with a document to permit the use of A-MΦ in part of this study, as informed consent. A-MΦ were incubated in plastic dishes for 1 h at 37 °C in a CO2 incubator, and non-adherent cells were removed by repeated washing. Cell viability was assessed by trypan blue dye exclusion. The number of adherent monocytes and monocyte-derived MΦs was determined by the method described previously by Nakagawara and Nathan (26Nakagawara A. Nathan C.F. J. Immunol. Methods. 1983; 56: 261-268Crossref PubMed Scopus (131) Google Scholar). Briefly, cultures were depleted of medium by gentle aspiration and then replenished with 1% (w/v) cetyltrimethyl ammonium bromide (Cetavlon; Wako Pure Chemical Industries, Ltd., Osaka, Japan) in 0.1 m citric acid with 0.05% (w/v) naphthol blue black (Sigma) at room temperature for 3 min. This treatment readily lysed the adherent cells and liberated stained intact nuclei, which were then counted using a TATAI hemocytometer (American Optical). Intracellular and extracellular catalase activity was measured according to the method described by Aebi (27Aebi H. Methods Enzymol. 1984; 105: 121-126Crossref PubMed Scopus (18933) Google Scholar). Briefly, MΦ were cultured in the phenol red-free medium (Life Technologies, Inc.) supplemented with the indicated concentrations of M-CSF or GM-CSF. Culture supernatants were harvested at 48 h for the measurement of extracellular catalase. To measure the intracellular catalase, cell lysates were prepared with a specific lysis buffer (10 mmol/liter EDTA, 2% Triton-X, 0.05% deoxycholic acid in phosphate-buffered saline, pH 7.4) and then diluted with 50 mmol/liter phosphate buffer (pH 7.0). 2 ml of the diluted sample was dispensed into a quartz cube, followed by 1 ml of 30 mmol/liter H2O2 in phosphate buffer (pH 7.0). Catalase activity was measured by the consumption of H2O2 at 240 nm in a spectrophotometer (Graphicord UV-240; Shimadzu Co., Kyoto, Japan) at 20 °C. The slope was converted into catalase activity units based on the standard curve of purified human erythrocyte catalase (HEC, 5 × 104units/mg, Lot. number 643793; Calbiochem-Novabiochem). The activity is shown as milliunits/ml per well (2.5 × 105 cells) or units per mg of protein using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Isolation of total RNA and Northern blot analysis were performed as described previously (13Akagawa K.S. Takasuka N. Nozaki Y. Komuro I. Azuma M. Ueda M. Naito M. Takahashi K. Blood. 1996; 88: 4029-4039Crossref PubMed Google Scholar). Briefly, cells were lysed with denaturing solution containing 4 mol/liter guanidine thiocyanate, 25 mmol/liter sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 mol/liter 2-mercaptoethanol. After transfer to a polypropylene tube, total RNA was extracted by sequential addition of 2 mol/liter sodium acetate (pH 4.0), water-saturated phenol, and chloroform-isoamylalcohol (49:1), followed by centrifugation at 10,000 rpm for 20 min at 4 °C and then precipitated with isopropanol and ethanol. Total RNA (10 μg/lane) was size-fractionated by electrophoresis after denaturation with 6% (v/v) deionized glyoxal and 50% (v/v) dimethyl sulfoxide and then transferred to a nylon membrane (Pall BioSupport, East Hills, NY). After cross-linking on the membrane under UV irradiation and boiling in 80 mmol/liter Tris-HCl (pH 8.0) for 5 min, the membrane was prehybridized in prehybridization buffer (5 Prime → 3 Prime, Inc., Boulder, CO) and 50% formamide at 42 °C for 3 h. Hybridization for catalase or β-actin transcript was performed in hybridization buffer containing a human catalase cDNA probe or β-actin cDNA probe as a standard. All probes were labeled using a multiprime DNA labeling system with [α-32P]dCTP (PerkinElmer Life Sciences). Blots were washed at 37 °C in 2× SSC and 0.1% SDS, 45 °C in 2× SSC, 0.2× SSC in 0.1% SDS for 30 min each and then analyzed using a Fuji BAS 2000 bioimage analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan). Cell lysates were prepared with sample buffer containing 4% SDS, 62.5 mmol/liter Tris-HCl (pH 6.8), 10% glycerol, 100 mmol/liter dithiothreitol and 0.005% bromphenol blue. Cell lysates (25 μg protein/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to an Immobilon P membrane (Millipore Corp., Bedford, MA) using a semidry electroblotting system (Bio CRAFT BE300, BIO CRAFT Corp., Tokyo, Japan). The membrane was blocked with non-fat milk (BlockAce; Dainippon Medical Corp., Osaka, Japan) at 4 °C overnight to avoid nonspecific binding and then incubated at 4 °C overnight with 1 μg/ml of rabbit anti-HEC antibody (Athens Research and Technology, Inc., Athens, GA) or normal rabbit IgG. After four washes in Tris-buffered saline (10 mmol/liter Tris-HCl, pH 8.0, 150 mmol/liter NaCl) supplemented with 0.1% Tween 20, the membrane was incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology). After four washes with Tris-buffered saline supplemented with 0.1% Tween 20, the specific bands were visualized with Amersham Pharmacia Biotech ECL reagent on Hyper ECL film (Amersham Pharmacia Biotech). Cellular release of H2O2 was detected by the semiautomated microassay reported by De la Harpe and Nathan (28De la Harpe J. Nathan C.F. J. Immunol. Methods. 1985; 78: 323-336Crossref PubMed Scopus (137) Google Scholar). In brief, cultured MΦ (5 × 104 per 100 μl in 96-well flat-bottom tissue culture plates) were rinsed with phosphate-buffered saline, 100 μl of assay mixture (30 mmol/liter scopoletin (Sigma), 1 mmol/liter NaN3, 1 purpurogallin unit/ml horseradish peroxidase (Sigma) in Krebs-Ringer phosphate buffer (145 mmol/liter NaCl, 4.86 mmol/liter KCl, 0.54 mmol/liter CaCl2, 1.22 mmol/liter MgSO4, 5.7 mmol/liter sodium phosphate) with 5.5 mmol/liter glucose) was dispensed into the wells. Immediately, after the addition of stimuli (zymosan or heat-killed Staphylococcus aureus), the plate was placed in a fluorometer (Titertek Fluoroskan II; Flow Laboratories Inc., McLean, VA), and fluorescence was recorded for each well (0–60 min) at 37 °C. H2O2 release was calculated from the loss of fluorescence, using the following formula: H2O2 released (in nmol) = [(E0 − W)/(C0 − W) − (E60− W)/(C60 − W)] × S, where E0 is the initial fluorescence reading for the well, E60 is the fluorescence reading at 60 min, W is the fluorescence recorded in an empty well, C0 and C60 are the mean fluorescence readings in the cell-free control wells at 0 and 60 min, respectively, and S is the amount of scopoletin, 3 nmol, added to each well at the start of the assay. MΦ generated from human monocytes by CSF (M-MΦ and GM-MΦ) and A-MΦ were cultured in the medium containing the indicated concentrations of H2O2 for 48 h and then cell viability was determined. There was a marked difference in their susceptibility to exogenously added H2O2 (Fig.1). When M-MΦ were treated with 10 and 1 mmol/liter H2O2, 100 and 75% of the cells died, respectively, whereas almost 100% of the cells were viable in 0.1 mmol/liter H2O2. In contrast, more than 90% of GM-MΦ were viable even when treated with 10 mmol/liter H2O2. Thus, GM-MΦ were about 50-fold more resistant to H2O2 than M-MΦ. A-MΦ also showed a strong resistance to H2O2, and the level of resistance was similar to that of GM-MΦ (Fig. 1) Because GM-MΦ and A-MΦ were markedly more resistant to H2O2 than M-MΦ, we examined the levels of cell associated- and extracellular catalase activity that catalyze H2O2 to H2O in these MΦs. Extracellular catalase activity in the culture supernatants obtained from GM-MΦ incubated for 48 h was about 4-fold higher than that from M-MΦ (about 160 and 40 milliunits/ml/well in GM-MΦ and M-MΦ, respectively) (Fig. 2 A). Culture supernatants obtained from A-MΦ also contained high extracellular catalase activity (about 160 milliunits/ml/well), and the level was similar to that of GM-MΦ (Fig. 2 A). In accordance with the findings of the enzyme activity, protein levels of extracellular catalase in GM-MΦ and A-MΦ cultures were about 4-fold higher than that in M-MΦ cultures by Western blot analysis using anti-HEC antibody (Fig. 2 B). Similarly, catalase activity in M-MΦ lysates at 24 h was about 1 units/mg protein, whereas those in GM-MΦ- and A-MΦ-lysates were about 5 units/mg protein. (Fig.3 A). In agreement with the enzyme activity, protein levels of catalase among these MΦ lysates were significantly different; catalase protein levels in GM-MΦ and A-MΦ lysates were higher than that in M-MΦ lysate, and the difference was about 5-fold (Fig. 3 B). As the above findings suggest that expression of catalase gene is quite different between M-MΦ and GM-MΦ or A-MΦ, we examined the levels of catalase mRNA among these MΦs at 24 h after their cultivation by Northern blot analysis. Catalase mRNA in GM-MΦ was about 5-fold higher than that in M-MΦ, which was similar to that in A-MΦ (Fig.3 C).Figure 3Activities and protein levels of intracellular catalase and mRNA expression of the catalase gene in monocyte-derived MΦs and A-MΦ. Enzyme activities (A) and protein levels (B) of cell-associated catalase from MΦ lysates (25 μg protein/lane) at 24 h of cultivation were examined as indicated in the legend for Fig. 2. N.S., not significant. C, mRNA levels of catalase and β-actin genes were examined in total RNA preparations (10 μg/lane) from these MΦs at 3 h of cultivation by Northern blot analysis.kb, kilobase pair.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although there is a significant difference in basal levels of catalase activity between M-MΦ and GM-MΦ or A-MΦ, the findings cannot fully explain their distinct susceptibility to H2O2; the difference in catalase activity was 4–5-fold, whereas the difference in sensitivity to H2O2 was about 50-fold. We therefore examined whether oxidant stress triggers the augmented expression of catalase gene in monocyte-derived MΦs. When these MΦs were treated for 3 h with 0.1 mmol/liter H2O2, catalase mRNA in GM-MΦ was augmented up to about 3-fold, whereas that in M-MΦ did not change significantly (Fig.4 A). Next we examined whether zymosan stimulation induces catalase gene activation in these MΦs. When MΦs were stimulated for 3 h with 0.1 mg/ml zymosan, catalase mRNA in GM-MΦ, but not in M-MΦ, also increased about 3-fold (Fig. 4 A). To confirm that catalase protein was synthesized by induction of the catalase gene via oxidant stress or microbial stimulant, we conducted a Western blot analysis of catalase protein in MΦ lysates (Fig.4 B). The levels of catalase protein in lysates of GM-MΦ stimulated for 24 h with H2O2 or zymosan increased up to about 3-fold, whereas such oxidant-triggered induction of catalase protein was not observed in lysates of M-MΦ (Fig.4 B). Oxidant stress or microbial stimulant-mediated catalase induction in both gene and protein levels was also observed in A-MΦ treated with H2O2 or zymosan, and the induction level was similar to that in GM-MΦ (Fig. 4). These findings indicate that GM-MΦ and A-MΦ have a marked ability to induce catalase expression in both gene and protein levels in response to H2O2 or microbial stimulant, but M-MΦ lacks this ability. As demonstrated above, GM-MΦ and A-MΦ but not M-MΦ express high levels of catalase activity. These findings suggest the possibility that GM-MΦ, but not M-MΦ, has a marked ability to scavenge H2O2. As shown in Fig. 5, when GM-MΦ and A-MΦ were stimulated with 1 mg/ml zymosan for 60 min, M-MΦ and GM-MΦ released 0.8 ± 0.06 nm/ml H2O2 and 0.1 ± 0.02 nm/ml H2O2, respectively. Similar findings were obtained when these MΦs were stimulated with 1 mg/ml heat-killed S. aureus; M-MΦ released 0.5 ± 0.04 nm/ml H2O2 whereas GM-MΦ released 0.1 ± 0.01 nm/ml H2O2. Both MΦs did not produce H2O2 without stimuli. These findings suggest that M-MΦ releases a large amount of H2O2, unlike GM-MΦ, through their distinct regulation of catalase activities. We showed in the present study that GM-MΦ and A-MΦ are highly resistant to H2O2 via the high basal level of catalase activity and a marked ability to express catalase in response to H2O2. About 1–10 mmol/liter H2O2, similar to levels found on expiration in the adult respiratory distress syndrome (29Kietzmann D. Kahl R. Muller M. Burchardi H. Kettler D. Intensive Care Med. 1993; 19: 78-81Crossref PubMed Scopus (132) Google Scholar, 30Heard S.O. Longtine K. Toth I. Puyana J.C. Potenza B. Smyrnios N. Anesth. Analg. 1999; 89: 353-357PubMed Google Scholar), did not induce cell death of GM-MΦ and A-MΦ. A strong antioxidant mechanism of human A-MΦ supported by high catalase activity may help them to be long survivors in an oxidant-rich environment and contribute to lung homeostasis. In contrast to GM-MΦ and A-MΦ, M-MΦ are sensitive to exogenous H2O2 up to about 50-fold. In accordance with the susceptibility to H2O2, M-MΦs express lower levels of basal catalase activity and lack the ability to induce catalase gene expression in response to H2O2. M-MΦ also produced a large amount of H2O2compared with GM-MΦ in response to microbial stimulants (see Fig. 5and Ref. 24Hashimoto S. Yamada M. Motoyoshi K. Akagawa K.S. Blood. 1997; 89: 315-321Crossref PubMed Google Scholar). These findings suggest the possibility that MΦ induced by M-CSF support oxidant-induced inflammation or H2O2-mediated bactericidal activity. In agreement with the present findings, M-CSF augments anticryptococcal activity of fluconazole in the mouse MΦ mediated by H2O2 production (31Brummer E. Stevens D.A. J. Med. Microbiol. 1989; 28: 173-181Crossref PubMed Scopus (34) Google Scholar, 32Brummer E. Stevens D.A. Clin. Exp. Immunol. 1995; 102: 65-70Crossref PubMed Scopus (38) Google Scholar). In the present study, GM-MΦ and A-MΦ, but not M-MΦ, have a marked ability to induce catalase gene expression by exposure of low levels of H2O2 and zymosan, which can augment their protection against oxidant-rich environments. Analysis of the 5′-flanking region of the catalase gene in human hepatoma cells and bronchoepithelial cells demonstrated that several transcriptional regulation sites in response to oxidant stress exist in the promoter region (33Sato K. Ito K. Kohara H. Yamaguchi Y. Adachi K. Endo H. Mol. Cell. Biol. 1992; 12: 2525-2533Crossref PubMed Scopus (128) Google Scholar, 34Yoo J.H. Erzurum S.C. Hay J.G. Lemarchand P. Crystal R.G. J. Clin. Invest. 1994; 93: 297-302Crossref PubMed Google Scholar). These cells, however, express low levels of catalase activity, and hyperoxia fails to augment catalase transcript but induces transactivation of the heat shock protein 70 gene to support their survival (35Erzurum S.C. Lemarchand P. Rosenfeld M.A. Yoo J.H. Crystal R.G. Nucleic Acids Res. 1993; 21: 1607-1612Crossref PubMed Scopus (99) Google Scholar, 36Erzurum S.C. Danel C. Gillissen A. Chu C.S. Trapnell B.C. Crystal R.G. J. Appl. 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In summary, we present evidence that catalase contributes to protect human tissue MΦ from oxidant-induced cell death and control their respiratory burst, and the activity is regulated at both the protein and mRNA levels by CSF during their differentiation. GM-CSF but not M-CSF plays a critical role in the induction of a strong antioxidant mechanism. The comparison of GM-MΦ and A-MΦ with M-MΦ in response to ROS helps clarify the self-defense mechanism of MΦ against oxidant stress in vivo. We thank Dr. K. Onozaki Faculty of Pharmaceutical Science, Nagoya City University, Nagoya, Japan) for the human catalase cDNA probe. We also thank Professor S. Gordon (Sir William Dunn School of Pathology, University of Oxford) for critical comments and for additional help on the manuscript.

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