Monocytes lack lactoferrin and have much less myeloperoxidase than neutrophils. They also acquire a potential catalyst for .OH production (tartrate-resistant acid phosphatase) as they differentiate into macrophages. Consequently, the nature of free radicals produced by these cells was examined using the previously developed spin-trapping system. When stimulated with either PMA or OZ neither monocytes nor monocyte-derived macrophages (MDM) exhibited spin trap evidence of .OH formation. Pretreatment with IFN-gamma failed to induce MDM .OH production. When provided with an exogenous Fe+3 catalyst, both stimulated monocytes and MDM, but not PMN, exhibited sustained .OH production, presumably due to the absence of lactoferrin in mononuclear phagocytes. Sustained production of .OH could contribute to the microbicidal activity of mononuclear phagocytes as well as inflammatory tissue damage under in vivo conditions where catalytic Fe+3 may be present

Adelberg, Danielle R.

Britigan, Bradley E.

Coffman, Thomas J.

Cohen, Myron S.

B E Britigan

T J Coffman

D R Adelberg

M S Cohen

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Mononuclear phagocytes have the potential for sustained hydroxyl radical production. Use of spin-trapping techniques to investigate mononuclear phagocyte free radical production.

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In the presence of Fell, 02' and H202 react in vitro to form hydroxyl radical(-OH). Considerable attention has been focused on the role of iron-catalyzed -0Hproduction in phagocyte microbicidal activity and tissue damage. Early studies im-plied that both neutrophils(PMN [reviewed in reference 1]) andmononuclear phago-cytes (2-9) generated -OH in the absence of an exogenous catalyst. However, theexperimental systems used in these studies probably lacked specificity for -OH (1).Using spin trapping, generally considered the most specific technique for -OHion, we did not find evidence for -OH production by human PMN in the ab-sence of an exogenous iron catalyst (10, 11). Even then, -OH production appearsto be inhibited by PMN secretion oflactoferrin and myeloperoxidase (MPO)(11-13).Monocytes do not possess lactoferrin (14) and have less MPOthan PMN (15) . Differen-tiation ofmonocytes to monocyte-derived macrophages (MDM) is associated withthe loss ofMPO(15) and the acquisition oftartrate-resistant acid phosphatase(TRAP)(16) . TRAP is an iron-containing enzyme, similar to uteroferrin, that may act asan -OH catalyst (17) . The current work was undertaken to determine ifthese factorsinfluenced the potential for -OH formation by mononuclear phagocytes .MONONUCLEAR PHAGOCYTES HAVE THE POTENTIALFOR SUSTAINED HYDROXYL RADICAL PRODUCTIONUse of Spin-trapping Techniques to Investigate MononuclearPhagocyte Free Radical ProductionBY BRADLEY E. BRITIGAN,' THOMAS J. COFFMAN,'DANIELLE R. ADELBERG,* AND MYRON S. COHEN,tFrom the *Department of Internal Medicine, Veterans Administration Medical Center andUniversity of Iowa College ofMedicine, Iowa City, Iowa, and the IDepartments ofMedicine and Microbiology and Immunology, University of North Carolina,Chapel Hill, North CarolinaMaterials and MethodsBrie,f Definitive ReportHumanmononuclearleukocytes andPMNwereobtainedby dex-. Monocytes and lymphocytes were separated by placingsterile petri dishes at 37°C, for 2 h. After washing, adherent monocytes werescraped into suspension . ForMDM, monocytes were incubated in medium 199 (Universof Iowa Cancer Center) with 13% autologous serum and gentamicin (50 gg/ml) for 5-7MDM were released with trypsin and EDTA or by gentle scraping. In some cases, MDMwere incubated in IFN-'Y (100 U/ml) for an additional 4 d.This study was supported by the Veterans Administration Research Advisory Group (B . E . Britigan),U.S. Public Health Service grants AI-15036-07 (M . S. Cohen) and RR05372(B. E . Britigan), the Chem-istry of Life Processes Program of the National Science Foundation (award DCB-8616115 ; M . S.Cohen), and was performed in part while B. E. Britigan was a Pfizer Scholar.This workwas presented in part at the annual meeting ofAmerican Federation for Clinical Research,Washington, DC, May 2, 1988 (19) .The Journal of Experimental Medicine - Volume 168	December 1988 2367-2372 23672368	BRITIGAN ET AL . BRIEF DEFINITIVE REPORTTRAPActivity. TRAP activity was assessed using the previously described cytochemicaland paranitrophenyl phosphate colorometric assays (16) . For TRAP release studies, MDMwere exposed to desired stimuli for 30 min, pelleted, and supernatant was removed .Spin Trapping. Sequential EPR spectra of phagocytes (0.25-1 x 107/ml), 5,5-dimethyl-1-pyrroline-l-oxide (DMPO, 0.1 M), DMSO (Me2SO, 0.14 M), stimulus (PMA, 100 ng/ml ;or opsonized zymosan (OZ), 3 mg/ml), and buffer (HBSS containing 0 .1 mM diethylenetri-aminepentaacetic acid [DTPC]) were recorded at 25°C using an EPR spectrometer (modelE-104A ; Varian Associates, Inc ., Palo Alto, CA). Where desired, ferrous ammonium sulfate(0 .1 mM), SOD (10 kg/ml), and catalase (600 U/ml) were included . Exclusion of DTPC didnot qualitatively alter the EPR spectra . Spectrometer microwave power was 20 mW, modula-tion frequency was 100 kHz with an amplitude of 1 .0 G, sweep time was 12.5 G/min, andthe receiver gain was 3.2 x 10' with a response time of 1 s .DMPO reacts with 02- and -OH to yield 2,2-dimethyl-5-hydroperoxy-l-pyrrolidinyloxyl(DMPO-OOH) and 2,2-dimethyl-5-hydroxy-l-pyrrolidinyloxyl (DMPO-OH), respectively(11) . However, DMPO-OH is also a decomposition product of DMPO-OOH, making it un-reliable as evidence for -OH production (1, 10, 11) . -OH reacts with Me2SO to form methylradical, which can be spin trapped as 2,2,5-trimethyl-l-pyrrolidinyloxyl (DMPO-CH3) (refer-ences 10, 11) . When Me2SO is present in excess ofDMPO as in this study, -OH productionis manifested primarily as DMPO-CH3, providing a more specific detection system for -OH.ResultsSpin Trapping ofOxygen-centered Radicals After Monocyte Stimulation . EPR spectra wereobtained after monocyte stimulation with PMA or OZ. PMA spectra comprisedmostly DMPO-OOH and DMPO-OH (Fig . 1) . With OZ, DMPO-OH dominatedwith only small DMPO-OOH peaks detected (Fig. 1) . Small DMPO-CH3 peaksobserved were not in excess of those expected from the small quantity of -OH pro-duced as a direct decomposition product of DMPO-OOH (10) . SOD markedly in-hibited all spin-trapped adducts whereas catalase had no effect (Fig . 1) . No qualita-tive difference was noted in spectra obtained after OZ stimulation of monocytespretreated with cytochalasin B (data not shown), excluding the possibility that themonocyte phagosome prevented detection of -OH . Monocytes stimulated with PMAor OZ in the presence of exogenous Fe+3 to induce -OH production yielded EPRspectra dominated by DMPO-CH3 (Fig . 1).Detection ofOxygen-centeredFree Radical Production by MDM MDMstimulated withPMA or OZ yielded spectra that were a composite of DMPO-OOH and DMPO-OH (Fig . 2) . Minimal DMPO-CH3 was detected . DMPO-OOH peaks were greaterwith PMA whereas DMPO-OH peaks were larger with OZ. With either stimulus,SOD inhibited all adducts, whereas catalase had no effect (Fig . 2) . CytochalasinB did not result in qualitative changes in the spectra (data not shown). MDM pos-sessed TRAP that was released by exposure to either stimulus (Table I) . Stimulationof IFN-y-treated MDM with PMA or OZ (Fig . 2) only increased 02 - -derived spin-trapped adducts (DMPO-OOH and DMPO-OH). Minimal DMPO-CHs was ob-served . IFN-y decreased MDM TRAP (Table I) .Free Radical Production by Monocytes andMDMin the Presence ofExogenous Iron. NextOH production by iron-supplemented mononuclear phagocytes and PMN was com-pared. Monocytes and MDM stimulated with PMA or OZ in the presence of Fe13(Fig . 3), exhibited sustained -OH production (stable or increasing DMPO-CH3) .Catalase inhibited DMPO-CH3 90-100% whereas SOD inhibited 20-40% . Underthe same conditions PMN -OH generation terminated after 10-15 min (Fig. 3) .BRITIGAN ET AL .	BRIEF DEFINITIVE REPORT 2369Monocytss 10 Q+ PMA+ PMA + SOD+ OZ+ PMA+ PMA + SODMW (S Cloy) . 10GFIGURE l. Representative (n = 5)EPR spectraobtained immediatelyafter the addition ofPMAto mono-cytes (+PMA) and with the addi-tion of SOD (+SOD) or catalase(+catalase) . Also shown is a repre-sentative (n = 3) ofEPR spectrumobtained 6min after the additionofOZ to monocytes . SOD and cata-lase effects were similar to thosewith PMA. The bottom scan(PMA+ Fe) was obtained after PMAulation of monocytes in thepresence of exogenous Fe' s.tions of high and low fieldcorresponding to each species arenoted.FIGURE 2 . Representative (n = 5)EPR scans obtained immediatelyafter the addition ofPMAto MDM(+PMA) and under the same con-ditions except that SOD (+SOD)or catalase (+catalase) were alsopresent . The fourth scan was ob-tained 6 min after the addition ofOZ to the same concentration ofMDM (n = 3) . Addition of SODtotally ablated this latter spectrumwhereas catalase had no effect (notshown) . The bottom two tracingswere obtained with PMA stimula-tion of IFN-y-treated and controlMDM.2370	BRITIGAN ET AL. BRIEF DEFINITIVE REPORTTABLE IAcid Phosphatase Activity in Mononuclear PhagocytesMean t SEM (n = 8-18) of total acid phosphatase and TRAP activity expressed as mU acidphosphatase/mg cellular protein of monocytes, MDM, and IFN-y-treated MDM (MDM-y).Percentage of TRAP released extracellularly by MDM and MDM-y after stimulation withPMA, OZ, or OZ after preincubation with cytochalasin B (OZ/CB) is also shown (n = 3),as are results of cytochemical stain for TRAP .DiscussionPMN do not possess the endogenous capacity for -OH formation (1, 10, 11) andthe lactoferrin andMPO release limits -OH generation even ifthe cells are providedwith an exogenous iron catalyst (11-13) . Their relative lack of MPO (15), absenceof lactoferrin (14), and presence of TRAP (16) suggested that mononuclear phago-cytes might have a greater propensity than PMN to generate -OH. However, whenmonocytes or MDM were stimulated with either PMA or OZ, only DMPO-OHandDMPO-OOH were observed . Since -OHproduction should have been manifestedas DMPO-CH3 and all spin-trapped adducts were blocked by SOD but not cata-lase, these data indicate only 02- formation . 02- generation manifested as DMPO-OH rather than DMPO-OOH has been noted with PMN andother nonphagocyticcells (1, 10, 11) and appears to result from cell metabolism ofDMPO-OOH to DMPO-OH (1) . The results observed with cytochalasin B-treated cells suggest that failureof the spin trap to reach intraphagosomal sites was not responsible for lack of .OHMW +P"iAA + FePWIN +MA + Fe10G-Ar)"v,rJf 1 ICH,OHOOH OOH OH CH,FIGURE 3. Representative (n= 3) EPR scans obtained be-ginning immediately after(scan1) and -35 min after (scan2) stimulation of MDM withPMA in the presence of Fe+3 .DMPO-CH3 remained theonly detectable adduct, indicat-ing ongoing -OH formation.The bottom two scans are se-quential 6-min scans obtainedimmediately after PMAstimu-lation of PMNin the presenceof exogenous Fe' 3. By the endof the second scan DMPO-OOHwasthe dominant specieswith only a small DMPO-CH3peakdetectable. This indicatestermination of -OH productionin spite of continued 02- for-mation .Acid phosphatase aTRAPRelease cytochemicalPhagocytes Total Tartrate resistant PMA OZ OZ/CB stainMonocytes 21 .5 f 2.6 18 .4 f 2.4 ND ND NDMDM 43.0 t 7.5 36.8 t 6.6 15 25 19MDM-y 9.5 t 2.9 8.3 t 2.5 0 0 0BRITIGAN ET AL .	BRIEF DEFINITIVE REPORT 2311detection . Thus, neither monocytes nor MDM appear to possess the endogenouscapacity to generate -OH, presumably because they do not possess and/or mobilizean appropriate catalyst . MDM possessed TRAP and appeared to release it whenstimulated . It is not clear why TRAP release did not allowMDM -OH production .Previous studies (2-9) reported :OH production by monocytes and/or macrophages .However, the specificity of the assay systems for -OH used in these reports has beenquestioned (1). In addition, the possibility that iron contaminating the buffers al-lowed -OH to be produced was not addressed . To our knowledge no spin-trappingstudies of monocytes have been reported . Using spin trapping we showed that mono-cytic HL-60 cells lack the endogenous capacity for -OH production (18) . Mouse mac-rophages have been studied by EPR (9). DMPO-OH was detected and inhibitedby Me2SO, consistent with -OH production . Unfortunately, no comment was madeas to whether DMPO-CH3 was detected . Endogenous capacity to form -OH maydiffer among macrophages of different species or anatomical sites .Although IFN-y increases MDM microbicidal and tumoricidal activity, we foundno evidence that IFN-y induces human MDM to generate -OH . IFN-y reportedlyincreased murine peritoneal macrophage -OH (ethylene) production 19-fold (4) . Thismay again reflect species differences but more likely relates to differences in thespecificity of the two -OH detection systems (1).Monocytes and MDM, but not PMN, stimulated in the presence of exogenousiron produced sustained -OH. This would be anticipated since monocytes and MDMlack lactoferrin, which terminates PMN -OH production (11, 13).Although we find no evidence for the endogenous capacity for -OH productionby humanphagocytes (PMN or mononuclear), their remains the potential for phago-cytes to induce formation of -OH in vivo under conditions where an appropriatecatalyst is present. The sustained -OH production observed with iron-supplementedmononuclear phagocytes suggests -OH might play a significant role in the cytotox-icity of these cells . However, the contribution of mononuclear phagocyte-derived-OH to inflammatory mechanisms clearly requires additional study.SummaryMonocytes lack lactoferrin andhave much less myeloperoxidase than neutrophils.They also acquire a potential catalyst for -OH production (tartrate-resistant acidphosphatase) as they differentiate into macrophages. Consequently, the nature offree radicals produced by these cells was examined using the previously developedspin-trapping system. When stimulated with either PMA or OZ neither monocytesnor monocyte-derived macrophages (MDM) exhibited spin trap evidence of -011formation . Pretreatment with IFN-y failed to induce MDM -OH production. Whenprovided with an exogenous Fe" catalyst, both stimulated monocytes and MDM,but notPMN, exhibited sustained -OH production, presumably due to the absenceoflactoferrin in mononuclear phagocytes . Sustained production of -OH could con-tribute to the microbicidal activity of mononuclear phagocytes as well as inflamma-tory tissue damage under in vivo conditions where catalytic Fe + 3 may be present.We thank Drs. Gerald Rosen, Robert Clark, and William Nauseef for their helpful com-ments, Dr. Harold Gofffor use ofthe EPR spectrometer, and Linda Shaull for her assistancein manuscript preparation.Recieoed for publication 31 May 1988 and in revisedform 14 September 1988.2372 BRITIGAN ET AL .	BRIEF DEFINITIVE REPORTReferences1 . Cohen, M. S ., B . E . Britigan, D. J . Hasset, and G. M. Rosen . 1988 . Do human neutro-phils make hydroxyl radical? Evaluation of an unresolved controversy. Free Radical Biol.Med. 5 :81 .2 . Weiss, S. J ., G. W King, and A. F. LoBuglio. 1977, Evidence for hydroxyl radical gener-ation by human monocytes . .j Clin. Invest. 60:370,3 . Karnovsky, M. L., and J . A . Badwey. 1983 . Determinants of the production of activeoxygen species by granulocytes and macrophages . ] Clin. Chem. Clin. Biochem. 21:545 .4 . Ito, M., R . Karmali, and K . Krim. 1985 . Effect of interferon on chemiluminescenceand hydroxyl radical production in murine macrophages stimulated by PMA. Immu-nology. 56:533 .5 . Niwa, Y., T. Sakane, Y. Miyachi, and M. Ozaki . 1984 . Oxygen metabolism in phago-cytes ofleprotic patients: enhanced endogenous superoxide dismutase activity and hydroxylradical generation by clofazamine. J Clin . Microáiol. 20:837 .6 . Speer, C . P, D. R . Ambruso, J . Grimsley, and R. B.Johnson,Jr. 1985 . Oxidative metab-olism in cord blood monocytes and monocyte-derived macrophages. Infect. Immun. 50:919 .7 . Janco, R. L., and D. English. 1983 . Regulation of monocyte oxidative metabolism :chemotactic factor enhancement ofsuperoxide release, hydroxyl radical generation andchemiluminescence . J. Lab Clin . Med. 102:890.8 . Hoidal, J . R., G. D. Beall, and J . R. Repine . 1979 . Production of hydroxyl radical byhuman alveolar macrophages. Infect. Immun . 26:1088 .9 . Hume, D. A., S. Gordon, P J . Thornalley, and J . V. Bannister. 1983 . The productionofoxygen-centered radicals by Bacillus-Calmette-Guerin-activated macrophages: an elec-tron paramagneticresonance study ofthe response to phorbol myristate acetate. Biochim.Biophys. Acta. 763:245 .10 . Britigan, B . E ., G. M. Rosen, Y. Chai, and M. S . Cohen . 1986 . Do human neutrophilsmake hydroxyl radical? Determination of free radicals generated by human neutrophilsactivated with a soluble or particulate stimulus using electron paramagnetic resonancespectrometry. ,j. Biol. Chem, 261:4426 .11 . Britigan, B . E ., G. M. Rosen, B . Y. Thompson, Y. Chai, and M. S. Cohen. 1986. Stimu-lated human neutrophils limit iron-catalyzed hydroxyl radical formation as detected byspin trapping techniques . ,j. Biol. Chem. 261:17026 .12 . Winterbourn, C . C . 1986 . Myeloperoxidase as an effective inhibitor ofhydroxyl radicalproduction : implications for the oxidative reactionsofneutrophils.J. Clin. Invest. 78:545 .13 . Britigan, B. E ., D. J . Hassett, G . M. Rosen, and M. S . Cohen. 1987 . Comparison ofthe impact of myeloperoxidase and lactoferrin release on the potential for neutrophilhydroxyl radical formation using spin trapping techniques. Clin. Res. 35.857A.(Abstr.)14 . Van Snick,J . L ., P L . Masson, andJ, E Heremans . 1974 . The involvement oflactoferrinin the hyposideremia of acute inflammation . j. Exp. Med 140:1068 .nston, Jr., W. D., B. Mei, and Z . A . Cohn. 1977 . The separation, long-term cultiva-and maturation of the human monocyte. J. Exp. Med. 146:1613 .pes, R . G., K . W. Lam, R. C . Dodd, T K. Gray, and M. S . Cohen . 1986, Acid phos-phatase activity in mononuclear phagocytes and the U937 cell line : monocyte-derivedmacrophages express tartrate-resistant acid phosphatase . Blood. 67 :729.17 . Sibille, J . C ., K. Doi, and P Aisen . 1987 . Hydroxyl radical formation and iron-bindingproteins : stimulation by the purple acid phosphatases . ,f. Biol. Chem. 262:59 .18 . Thompson, B . Y., G . Sivarn, B . E . Britigan, and M. S. Cohen. 1988 . Oxygen metabo-lism of the HL-60 cell line : comparison of the effects of monocytoid and neutrophilicdifferentiation, J Leukocyte Biol. 43:140.itigan, B . E,, T J . Coffman, D. R. Adelberg, andM. S . Cohen . 1988 Monocytes andnoncyte-derived macrophages lack the endogenous capacity to form hydroxyl radical asassessed by spin trapping. Clin . Res. 36:452A. (Abstr.)

Mononuclear phagocytes have the potential for sustained hydroxyl radical production. Use of spin-trapping techniques to investigate mononuclear phagocyte free radical production

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Mononuclear phagocytes have the potential for sustained hydroxyl radical production. Use of spin-trapping techniques to investigate mononuclear phagocyte free radical production

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