Vol. 38, No. 1INFECTION AND IMMUNITY, Oct. 1982, p. 292-297001 9-9567/82/1 00292-06$02.00/0Copyright © 1982, American Society for Microbiology

Monocyte-Mediated Damage to Rhizopus oryzae Hyphae InVitro

RICHARD D. DIAMOND,'* CHRISTIAN C. HAUDENSCHILD,2 AND NELSON F. ERICKSON III'

Evans Memorial Department of Clinical Research, and Departunent of Medicine, Univ ersity Hospital, andDepartment of Pathology,2 Boston University S(chool of Medicine, Boston, Massachusetts 02118

Received 12 April 1982/Accepted 24 June 1982

Clinicopathological correlations from human cases and experimental animalstudies suggest that neutrophils are critical components of the host response tomucormycosis but that other cellular defense mechanisms appear to be importantas well. Since our previous studies demonstrated that Rhizoplus olyzae hyphaewhich are too large to be ingested completely can be damaged and probably killedby human neutrophils, we studied the antihyphal activity of human monocytes.As with neutrophils, light and electron microscopic studies indicated that mono-cytes attached to hyphae and appeared to destroy them in the absence of serum.As judged by our previously described assay for the leukocyte-induced inhibitionof [14C]uracil uptake by hyphae, quantitative damage to hyphae by monocyteswas 40.8 ± 2.2% in 54 experiments. Neither attachment to nor damage of hyphaeby monocytes was augmented by the presence of 10% human serum. As withneutrophils, monocyte-mediated damage of R. orvzae was significantly decreasedby some inhibitors of oxidative metabolism and scavengers of the potentiallymicrobicidal oxidative leukocyte products, which included 10-4 M sodium azide,10-3 M sodium cyanide, catalase, 10-3 M histidine, 10-3 M tryptophan, and 10-4M 1,4-diazobicyclo[2.2.2]octane but not superoxide dismutase, 1.4 x 10-2 Mdimethyl sulfoxide, and 4.0 x 10-1 M mannitol. Moreover, monocytes from threepatients with chronic granulomatous disease failed to damage hyphae at all. Incontrast to our previous data for neutrophils, polyanions (10-5 M polyaspartic orpolyglutamic acid) did not inhibit monocyte-mediated hyphal damage. Thus,monocytes can damage and probably kill R. orvlzae hyphae by oxidative mecha-nisms and so may be involved in host defense mechanisms against mucormycosis.

Rhizopus oryzae, the commonest cause ofmucormycosis, when injected into normal rab-bits, provokes a response that is initially neutro-philic and then granulomatous before beingcleared; however, this cellular response is blunt-ed and infections progress in animals madeketoacidotic and diabetic (2, 3, 22). In thisexperimental model, nitrogen mustard-inducedgranulocytopenia is associated with extensiveearly lesions of mucormycosis, but infected ani-mals develop a granulomatous response and in-fections recede after 72 h (4). Thus, neutrophilsappear to be an important component of hostdefense mechanisms against mucormycosis, butother cellular factors are likely to contribute tothe host response. Since we have demonstratedthat human neutrophils can damage and proba-bly kill hyphae of R. oryzae (6), we studied theinteractions between these hyphae and humanmonocytes.

MATERIALS AND METHODS

Organisms. An isolate of R. oryzale originally ob-tained from a patient with mucormycosis was main-

tained on Emmons modified Sabouraud agar for sporeharvesting (6). After filtration through cheesecloth,each sample (105 spores) was placed into a series of 15-ml centrifuge tubes (Corning Glass Works, Corning,N.Y.) containing Sabouraud broth, and the tubes wereincubated at 37°C for 5 to 6 h until -90 to 95% of thespores had germinated to .30 [Lm in length. Thesamples were then stored overnight at 4°C before use(6).

Monocytes. Mixed mononuclear cells (18 to 42%monocytes with <1.0% neutrophils, 98.5 to 100%viable) were separated from heparinized human pe-ripheral blood on a Ficoll-Hypaque gradient (5). Inselected experiments, monocytes were depleted frompreparations by passage through nylon fiber columns(8) or by successive passages through three sets ofplastic tissue culture dishes. Children with typical,sex-linked, chronic granulomatous disease were iden-tified, and blood was kindly supplied by HarveyCohen (Children's Hospital, Boston, Mass.). For se-lected studies, autologous sera were obtained fromnormal volunteer subjects at the same time as weremonocytes.When necessary, subcellular fractions of monocytes

were separated from disrupted monocytes by centrifu-gation on 0.34 M sucrose gradients as previously

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MONOCYTE-MEDIATED DAMAGE TO RHIZOPUS HYPHAE 293

o with hyphae. Catalase (bovine liver. 6.1 mg/ml. 50.000U/mg) was obtained from Worthington Diagnostics.Freehold, N.J.) and dialyzed against water before use.

00 ,Superoxide dismutase (bovine erythrocyte, lyophi-; / lized powder; 12,300 U/mg; Miles Laboratories, Elk-

o hart, Ind.) was dissolved in water at a concentration of5 mg/ml and stored at -20°C until use. For each

0 n/experiment with catalase or superoxide dismutase.control tubes contained either catalase which had beenheated at 100°C for 15 min or superoxide dismutasewhich had been autoclaved at 121°C for 30 min before

0o s ~ use. Sodium azide. sodium cyanide, histidine. trypto-- 'phan, dimethyl sulfoxide, mannitol. and polymerized

amino acids (polyaspartic and polyglutamic acids)were obtained from Sigma Chemical Co.. St. Louis.Mo. Triethylenediamine, also known as 1.4 diazobicy-

'O - * clo[2.2.2] octane (DABCO), was supplied by Eastman* Organic Chemicals, Rochester, N.Y.

Electron microscopy. After 1 h of incubation togeth-er, the hyphae and the monocytes were fixed in

IIISb suspension as in previous studies (5) by the addition ofo No .. phosphate-buffered glutaraldehyde and phosphate-NoSerum Serum No Serum buffered formaldehyde (final concentrations, 1.0 and

Normal Subjects CGD 4.0%, respectively), spun at 450 x g for 10 min. andsuspended as follows: 1% aqueous osmium tetroxide

6G. 1. Damage by monocytes from normal sub- for 60 mm, 0.1% aqueous uranyl acetate for 30 mm.to hyphae in the presence and absence of 10% graded alcohols, propylene oxide, and Epon 812 (Shell

ilogous serum and by monocytes from patients Chemical Co., Houston, Tex.). Toluidine blue-stainedl chronic granulomatous disease (CGD) in the sections (1 p.m thick) were cut in planes both perpen-nce of serum, determined by monocyte-mediated dicular and parallel to the axis of the g-forces ofbition of ['C]uracil uptake. Each dot represents centrifugation to examine the distribution of cellsocytes from one subject. Results for monocytes within the pellet. Ultrathin sections through the centerd with and without serum simultaneously are of the pellet (including all cells from top to bottom)iected by heavy lines. Means + standard error of were cut with diamond knives. stained with leadm-eans are represented by horizontal lines connect-nyverticanareiesetdbhoznaliscnc- citrate and uranyl acetate, and examined with anty vertical Ines. EM300 microscope at 80 kV (Philips Electronic Instru-

ments, Inc., Mahwah, N.J.).Statistical methods. Means and standard errors of

ribed (5) and incubated with hyphae in place of means for groups of data were compared by the two-:t monocytes. sample, two-tailed Student t test or the paired-sampleisessment of hyphal damage. Damage to hyphae Student t test when appropriate (21).

was quantitated as in previous studies (6). Briefly.hyphae germinated from spores (105 per tube) andmonocytes (total number of organisms, 106 per tube)were rotated together in triplicate tubes at 37°C for I h.and then the monocytes were lysed during threesuccessive washes with distilled water. This treatmentcompletely lysed the monocytes without damaging thehyphae, as judged by morphological and metaboliccriteria. Triplicate control tubes contained only hy-phae during the 1-h incubation period, but monocyteswere added immediately before lysis with distilledwater. The remaining hyphae were then suspended inyeast-nitrogen base broth supplemented with aspara-gine and glucose, and the suspension was incubatedwith ['4C]uracil (2.0 ,uCi per tube) at 30°C for 2 h,separated onto filters, washed free of unbound radio-isotopes with an automated, multiple-sample harvester(Otto Hiller Co., Madison, Wis.), and then quantitatedfor "4C uptake by liquid scintillation counting. Damageto the hyphae was calculated as the monocyte-mediat-ed percent reduction in isotope uptake: [mean countsper minute in control tubes - (mean counts per minutein experimental tubes/mean counts per minute in con-trol tubes)] x 100 (6).

In some experiments, inhibitors of monocyte func-tion were added during or 20 min before incubations

RESULTS

Metabolic assays for damage to hyphae bymonocytes. Normal human monocytes attachedto and damaged R. oryzae hyphae, as judged bylight microscopy combined with quantitative as-

says of monocyte-induced reduction in [ 14CIura-cil uptake by the hyphae (Fig. 1). Addition of10% autologous serum to the incubation mix-tures did not affect results, regardless of whetherthe sera used were fresh, heated at 56°C for 30min, or positive for antibodies to R. oryvzaedetectable by immunodiffusion. Hyphal damagewas attributable to monocytes: the numbers ofcontaminating neutrophils were judged to beinsufficient to account for hyphal damage, basedupon our previous results (6), and the remaininglymphocytes, depleted of monocytes (by adher-ence to nylon wool or plastic dishes), did notdamage R. oi-vzae hyphae at all. The observedrange of hyphal damage was consistent with theresults of our previous studies of leukocyte-mediated damage to this and other fungi (5, 6).

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294 DIAMOND, HAUDENSCHILD, AND ERICKSON

TABLE 1. Effects of potential inhibitors ofleukocyte oxidative microbicidal mechanisms on the

damage to R. oryzae hyphae by monocytesInhibition No. ofInhibitor (concn) (7,jI expt

Sodium azide (10-4 M) 61.0" 11Sodium cyanide (10-a M) 56.3" 11Halide-free system 42.1" 3

(phosphate-bicarbonate buffer)Catalase (500 to 1,000 U) 53.8" 11Superoxide dismutase (25 p.g) 0 3Histidine (10' M) 32.7" 11Tryptophan (10-' M) 31.4" 41 ,4-Diazobicyclo[2.2.2]octane 43.8"' 8

(10-4 M)Dimethyl sulfoxide 12.3 10

(1.4 x 10-2 M)Mannitol (4.0 x 10-1 M) 11.4 3Polyaspartic acid (10-' M) 0 3Polyglutamic acid (10-5 M) 0 3

' Damage to hyphae was quantitated by monocyte-mediated reductions in ['4C]uracil uptake. Values forhyphal damage in the presence and absence of inhibi-tors (three or more separate experiments, each per-formed in triplicate) were then used to calculate thepercent inhibition of hyphal damage attributable toeach inhibitor.

" P < 0.02; determined by the two-tailed, two-sample Student t test.

As in our prior studies, comparable results wereobtained with radiolabeled compounds otherthan uracil, including amino acids, glucose, andmannose, and the hyphal damage was not re-versible, as judged by repeat incubations ofhyphae with radiolabeled substances.

In contrast to neutrophils or monocytes fromnormal volunteer subjects, neither neutrophilsnor monocytes from patients with hereditarychronic granulomatous disease damaged hyphaesignificantly (Fig. 1). In this disease, phagocyto-sis is unimpaired, but the postphagocytic burstin oxygen consumption by neutrophils andmonocytes fails to occur, so that normalamounts of potentially microbicidal products,including hydrogen peroxide, superoxide anion,and perhaps other oxidative intermediates, arenot produced (1). Thus, these results suggest thecentral importance of oxidative mechanisms inthe R. oryzae hyphal damage caused by mono-cytes. Known inhibitors of potentially microbi-cidal mechanisms were then used to furtherassess the relative roles of oxidative and nonoxi-dative processes in hyphal damage. Inhibitorswere used in concentrations which impairedneither viability nor oxygen consumption bymonocytes, as in our previous studies (5). Like-wise, concentrations of inhibitors which affectedhyphal growth were eliminated, so that somepotential inhibitors (e.g., sodium benzoate)could not be used at all, and not all substances

could be tested at optimum inhibitory concentra-tions. Sodium azide and sodium cyanide inhibit-ed damage to hyphae by monocytes (Table 1) inconcentrations shown by Klebanoff to primarilyaffect myeloperoxidase activity (13). The omis-sion of halide or the addition of catalase similar-ly inhibited hyphal damage, but the addition ofsuperoxide dismutase did not. Three antagonistsof hypochlorous acid and quenchers of singletoxygen, DABCO, histidine, and tryptophan (9-11, 18, 23, 24), all inhibited damage to Rhizopushyphae. However, the putative scavengers ofhydroxyl radicals, dimethyl sulfoxide and man-nitol (20), had only minimal, insignificant effectson the damage to hyphae, although testing ofthese compounds in higher, potentially moreeffective concentrations was precluded by theireffects on hyphal or leukocytic metabolism. Hy-droxyl radicals, even if not fungicidal them-selves, still might enhance the activity of otherpotentially microbicidal mechanisms. However,it proved impractical to use inhibitors to studythis possibility, as combinations of potentiallyeffective concentrations of these agents alsoadversely affected fungal or monocyte metabo-lism of control samples. In contrast to ourprevious results for quantitative damage of Rhi-zopus hyphae by neutrophils (6), polyanions(polyaspartic or polyglutamic acid) did not inhib-it monocyte-mediated hyphal damage (Table 1).In fact, fractions separated from disruptedmonocytes did not damage R. oryzae hyphae.Damage to hyphae occurred only when granule-rich fractions (but not the cytoplasmic or nuclearfraction) were supplemented with iodide, chlo-ride, or hydrogen peroxide in a concentrationwhich did not damage hyphae by themselves.

Electron microscopy. Samples taken 1 minafter monocytes were added to hyphae revealednormal hyphal and monocyte morphologies,without attachment of monocytes to hyphae.After 20 to 30 min of incubation, monocytes (butnot lymphocytes or neutrophils) were attachedto and spread over hyphal surfaces withoutcompletely engulfing the hyphae, and fungalmorphology remained intact (Fig. 2A). Howev-er, by 1 h of incubation, most of the fungisurrounded by monocytes appeared to havebeen destroyed, with severely disrupted internalmorphology (Fig. 2B), supporting the validity ofthe metabolic measurements of apparent hyphaldamage detailed above.

DISCUSSIONThese experiments established that human

peripheral blood monocytes could damage andapparently kill R. oryzae hyphae by a serum-independent mechanism, even though the hy-phae were too large to be ingested completely.Monocytes were dependent on the activity of

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296 DIAMOND, HAUDENSCHILD, AND ERICKSON

oxidative microbicidal systems in damaging R.oryzae hyphae to an even greater degree thanthat noted for neutrophils in our previous studies(6). Neither neutrophils nor monocytes frompatients with chronic granulomatous diseaseproduce adequate amounts of hydrogen perox-ide and other potentially microbicidal productsof oxidative metabolism (1). In our studies,neither neutrophils nor monocytes from threepatients with chronic granulomatous diseasedamaged hyphae significantly. Monocytes fromone of these patients have been shown to dam-age Candida hyphae normally (5), and mono-cytes from all three of these patients damageAspergillus hyphae within the range observedfor monocytes from normal subjects (R. D. Dia-mond, E. Huber, and C. C. Haudenschild, Clin.Res. 29:383A, 1981). Thus, oxidative microbi-cidal mechanisms seem more critical in mono-cyte-mediated damage to R. oryzae hyphae thanthey do in Candida or Aspergillius hyphal dam-age.

Results with inhibitors of leukocyte functionand normal monocytes were almost identical tothe data from our previous studies of the damageto Rhizopus hyphae by neutrophils (6) and toCandida hyphae by monocytes (5). Inhibition byazide, cyanide, catalase, and halide-free condi-tions supports the importance of the myeloper-oxidase-peroxide-halide system in the damage toRhizopus hyphae by normal monocytes. How-ever, myeloperoxidase deficiency may unmaskalternative oxidative (or perhaps nonoxidative)antifungal mechanisms (5). Moreover, granule-associated myeloperoxidase is lost during thedifferentiation of monocytes into macrophages(15), a fact which emphasizes the potential im-portance of oxidative microbicidal products ca-pable to activity independent of the myeloperox-idase system. These oxidative products mayinclude superoxide anion, hydrogen peroxide (1,7, 12, 15), singlet oxygen, and hydroxyl radicals(1, 9, 10, 24, 25). The singlet oxygen quenchershistidine, tryptophan, and DABCO inhibited thedamage to Rhizopus hyphae, but these com-pounds also antagonize the effects of hypochlo-rous acid, a likely product of the myeloperoxi-dase system (9, 10, 24). Although neitherdimethyl sulfoxide nor mannitol, putative scav-engers of hydroxyl radicals (5, 20), damagedRhizopus hyphae, studies of potentially moreeffective concentrations of these substances ei-ther alone or in combination with other inhibi-tors were prevented by nonspecific effects onleukocyte and fungal metabolism.Leukocyte cationic proteins and other gran-

ule-associated substances have known antifun-gal properties (14, 16, 19) and may also enhancethe activity of oxidative mechanisms (17). How-ever, polyanions did not inhibit monocyte-medi-

ated damage to Rhizopus hyphae, in contrast toour previous results for neutrophil-mediateddamage to these fungi (6). Partially purifiedfractions of monocytes rich in lysosomal gran-ules similarly had no effect on hyphal viabilityunless hydrogen peroxide and halides were add-ed to supplement granule-associated myeloper-oxidase. Therefore, nonoxidative mechanisms,including granule-associated cationic proteins,did not appear to be involved in the damage ofRhizopus hyphae by monocytes.

Since monocytes can damage the tissue-in-vading forms of Rhizopus, they have the poten-tial for supplementing (2, 3, 22) or substitutingfor (4) the neutrophilic response in host defensesagainst mucormycosis in vivo. Because mono-cyte-mediated Rhizopus damage appears to de-pend so completely upon oxidative mechanisms,antihyphal activity may prove to be particularlysensitive to local conditions in host tissues, suchas anaerobiosis induced by necrosis or metabol-ic abnormalities affecting cellular oxidative me-tabolism.

ACKNOWLEDGMENTS

We thank A. C. Walstrom, Elisabeth S. Huber, StevenLeong, and William Ormerod for technical assistance.This work was supported in part by Public Health Services

grant A115338 from the National Institute of Allergy andInfectious Diseases.

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2. Bauer, H., J. F. Flanagan, and W. H. Sheldon. 1955.Experimental cerebral mucormycosis in rabbits with al-loxan diabetes. Yale J. Biol. Med. 28:29-36.

3. Bauer, H., J. F. Flanagan, and W. H. Sheldon. 1956. Theeffects of metabolic alterations on experimental Rhi.zopusorvzae (mucormycosis) infection. Yale J. Biol. Med.29:23-32.

4. Bauer, H., and W. H. Sheldon. 1957. Leukopenia andgranulocytopenia in experimental mucormycosis (Rhiz-o-pUiS onrzae infection). J. Exp. Med. 106:501-508.

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6. Diamond, R. D., R. Krzesicki, B. Epstein, and W. Jao.1978. Damage to hyphal forms of fungi by human leuko-cytes in vitro: a possible host defense mechanism inaspergillosis and mucormycosis. Am. J. Pathol. 91:313-328.

7. Drath, D. B., and M. L. Karnovsky. 1979. Superoxideproduction by phagocytic leukocytes. J. Exp. Med.141:257-262.

8. Greaves, M. F., G. Janossy, and P. Curtis. 1976. Purifica-tion of human T lymphocytes using nylon fiber columns,p. 217-229. In B. R. Bloom and J. R. David (ed.). In vitromethods in cell-mediated and tumor immunity. AcademicPress, Inc., New York.

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12. Johnston, R. B., Jr., C. A. Godzik, and Z. A. Cohn. 1978.Increased superoxide anion production by immunologi-cally activated and chemically elicited macrophages. J.Exp. Med. 48:115-127.

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14. Lehrer, R. I. 1975. The fungicidal mechanisms of humanmonocytes. I. Evidence for myeloperoxidase-linked andmyeloperoxidase-independent candidacidal mechanisms.J. Clin. Invest. 55:338-346.

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16. Lehrer, R. I., K. M. Ladra, and R. B. Hake. 1975. Non-oxidative fungicidal mechanisms of mammalian granulo-cytes: demonstration of components with candidacidalactivity in human, rabbit, and guinea pig leukocytes.Infect. Immun. 11:1226-1234.

17. Olson, V. L., R. L. Hansing, and D. 0. McClary. 1977.The role of metabolic energy in the lethal action of basicproteins on Candida albicans. Can. J. Microbiol. 23:166-174.

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20. Repine, J. E., J. W. Eaton, M. W. Anders, J. R. Hoidal,and R. B. Fox. 1979. Generation of hydroxyl radical byenzymes, chemicals, and human phagocytes in vitro.Detection with the anti-inflammatory agent, dimethylsulf-oxide. J. Clin. Invest. 64:1642-1651.

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22. Sheldon, W. H., and H. Bauer. 1958. Activation of quies-cent mucormycotic granulomata in rabbits by induction ofacute alloxan diabetes. J. Exp. Med. 108:171-178.

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