Medical Sciences: Corrections Proc. Natl. Acad. Sci. USA 87 (1990) 10133

Correction. In the article "Epididymis is a principal site ofretrovirus expression in the mouse" by Ann AndersonKiessling, Richard Crowell, and Cecil Fox, which appearedin number 13, July 1989, of Proc. Natl. Acad. Sci. USA (86,5109-5113), the authors have requested that the followingcorrection be made. The legend in Fig. 2 has an error in laneidentification and should read as follows: Lanes: 1, testis.

Correction. In the article "Antibiotic proteins of humanpolymorphonuclear leukocytes" by Joelle E. Gabay, RandyW. Scott, David Campanelli, Joe Griffith, Craig Wilde,Marian N. Marra, Mary Seeger, and Carl F. Nathan, whichappeared in number 14, July 1989, of Proc. Natl. Acad. Sci.USA (86, 5610-5614), the authors request that the followingcorrection be noted. On page 5614, in Table 2, middle section(activity against S. faecalis), the LD50 for peak VII reportedas 53 Ag/ml should be 5.3 tug/ml.

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Proc. Nati. Acad. Sci. USAVol. 86, pp. 5610-5614, July 1989Medical Sciences

Antibiotic proteins of human polymorphonuclear leukocytes(bacteria/lysosomes/neutrophils)

JOELLE E. GABAY*t, RANDY W. SCOTTt, DAVID CAMPANELLI*, JOE GRIFFITHt, CRAIG WILDEt,MARIAN N. MARRAt, MARY SEEGER*, AND CARL F. NATHAN**Beatnce and Samuel A. Seaver Laboratory, Division of Hematology-Oncology, Department of Medicine, Cornell University Medical College,New York, NY 10021; and tInvitron Corporation, Redwood City, CA 94063

Communicated by Maclyn McCarty, May 1, 1989 (receivedfor review March 6, 1989)

ABSTRACT Nine polypeptide peaks with antibiotic activ-ity were resolved from human polymorphonuclear leukocyteazurophil granule membranes. All but 1 of the 12 constituentpolypeptides were identified by N-terminal sequence analysis.Near quantitative recovery of protein and activity permitted anassessment of the contribution of each species to the overallrespiratory-burst-independent antimicrobial capacity of thecell. Three uncharacterized polypeptides were discovered,including two broad-spectrum antibiotics. One of these, adefensin that we have designated human neutrophil antimicro-bial peptide 4, was more potent than previously describeddefensins but represented less than 1% of the total protein. Theother, named azurocidin, was abundant and comparable tobactericidal permeability-increasing factor in its contributionto the killing of Escherichia coli.

Polymorphonuclear leukocytes (PMNs) defend against bac-terial and fungal infections by two main mechanisms: pro-duction of reactive oxygen intermediates through the respi-ratory burst and delivery of polypeptide antibiotics fromlysosomal granules into phagocytic vacuoles (1-3). Severalantimicrobial proteins have been isolated from human PMNs.Bactericidal permeability-increasing protein (BPI) (4) [prob-ably identical with cationic antimicrobial protein (5) andbactericidal protein (6)] specifically kills Gram-negative bac-teria. Cathepsin G (7) and three mutually homologous pep-tides termed defensins (8, 9) are active against Gram-positivebacteria, Gram-negative bacteria, and fungi. Lysozyme isactive against some pathogenic Gram-positive organisms(10).

Analysis of the role of polypeptide antibiotics in thefunction of PMNs has been held back by three problems. (i)Procedures resulting in isolation ofone polypeptide antibioticoften failed to reveal other antibiotics. (ii) The widely diver-gent conditions used for the bioassay of antibiotics wereoptimal for detection of some species but probably obscuredthe activity of others. (iii) There have been few if any studiescomparing the known antibiotic species in the same assay.Consequently, it has not been possible to assess the relativecontribution of any one of these proteins to the overallrespiratory-burst-independent antimicrobial potential ofPMNs. Nor can it be excluded that previously undiscoveredantimicrobial polypeptides ofhuman PMNs may contribute asignificant proportion of the cells' activity.We dealt with these problems by isolating from human

PMNs the smallest subcellular compartment that containedmost of the respiratory-burst-independent antimicrobial ac-tivity (11). From this compartment-the membrane-associ-ated fraction of the azurophil granule-we used reverse-phase HPLC (rpHPLC) to isolate all 12 of the major poly-peptides and sequenced the N terminus of all but 1, which

may have been blocked. Finally, we compared quantitativelythe individual activities of each rpHPLC peak against mi-crobes of three types (Gram-positive and -negative bacteriaand fungi) under a range of assay conditions previouslyreported to optimize the activity of one or another of thepreviously characterized species.

MATERIALS AND METHODSPMN azurophil granule membranes were isolated and ex-tracted as described (11). Concentrated extracts were ana-lyzed by rpHPLC using a Vydac C4 column (Rainin Instru-ments) and an elution gradient of 0-100% (vol/vol) acetoni-trile in 0.1% trifluoroacetic acid. The resulting fractions weresubjected to SDS/PAGE on a 15% polyacrylamide gel (12).Automated Edman degradation was carried out using anApplied Biosystems model 477A pulse liquid-phase se-quencer. Phenylthiohydantoin amino acid analysis was per-formed on-line using an Applied Biosystems liquid chromato-graph, model 120A. The N-terminal sequences for peaks III,VI, VII, and IX were also verified by direct sequencing ofCoomassie-stained bands electroblotted onto polyvinylidenedifluoride membranes as described (13), except that 0.05%SDS was added to the transfer buffer. Antimicrobial activityagainst Escherichia coli K12 (strain MC4100), Streptococcusfaecalis (ATCC 29212), and Candida albicans (clinical iso-late from Presbyterian Hospital, New York) was tested asdescribed (14). Antimicrobial activity was expressed in totalkilling units [(killing units/ml) x volume of the extract].Killing units are defined as the reciprocal of the dilution ofgranule extract necessary to kill 105 bacteria per ml in 30 minat 37°C or 104 fungi per ml in 60 min at 37°C.

RESULTSIsolation and Identification of Azurophil Membrane Pro-

teins. In an earlier study, nitrogen-bomb cavitates of diiso-propyl fluorophosphate-treated PMNs were fractionated onPercoll density gradients, and antibacterial activity was as-sessed in the fractions enriched in nuclei, cytosol, specificgranules, azurophil granules, or plasma membranes (11).Almost all the activity of the cavitate was recovered in thefractions enriched in azurophil granules and could be recov-ered quantitatively from azurophil membrane material (11).In the present study, we subjected concentrated extracts ofthe azurophil granule membrane to further purification andanalysis.

Abbreviations: BPI, bactericidal permeability-increasing protein;BSA, bovine serum albumin; ECP, eosinophil cationic protein;HNP, human neutrophil antimicrobial peptide; MBP, major basicprotein; PMN, polymorphonuclear leukocyte; rpHPLC, reverse-phase HPLC.tTo whom reprint requests should be addressed at: Box 57, CornellUniversity Medical College, 1300 York Avenue, New York, NY10021.

5610

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 86 (1989) 5611

1.50

'-e 0.75

20 40 60Retention time, min

80

FIG. 1. rpHPLC analysis of azurophil granule membranes. Pro-tein (1-2 mg) from azurophil fraction isolated from 300 ml of bloodwas concentrated 20-fold in a Centricon-10 ultrafiltration unit, made0.1% in trifluoroacetic acid, and subjected to rpHPLC.

Nine peaks were separated by rpHPLC (Fig. 1), with a totalprotein recovery of 77% (Table 1). Fig. 2A shows thepolypeptide profile of each peak after SDS/PAGE underreducing conditions. The polypeptide in peak II, once re-duced, could not be visualized by silver staining, but stainingof gels containing an unreduced sample revealed a singlespecies migrating at 4 kDa (Fig. 2B). Two species, migratingat 29 and 80 kDa, made up peak VI. These have beenseparated by other procedures; only the 29-kDa polypeptidehas antimicrobial activity (unpublished results). The 80-kDaspecies, for which no N-terminal sequence could be deter-mined, will not be discussed further. The polypeptides pre-sent in peaks III, IV, VII, VIII, or IX migrated in more thana single band. The bands within each of these peaks were

closely spaced and had a single N-terminal sequence (Table1); we assume they represent isoforms.The N-terminal sequences of the polypeptides in six of the

rpHPLC peaks matched previously recognized sequences(Table 1). The major peak was peak I (45% of recoveredprotein); this consisted of a mixture of defensins termedhuman neutrophil antimicrobial peptide (HNP) 1, HNP-2,and HNP-3 (15), in a molar ratio of approximately 40:40:20.The peak III polypeptide shared 19 of 20 N-terminal residueswith eosinophil cationic protein (ECP) (16). The peak IVpolypeptide (18% of recovered protein) was identical over itsfirst 16 residues with the N terminus of cathepsin G (17). Thepeak V polypeptide shared its first 17 of 20 amino acids withmajor basic protein (MBP) of eosinophils (18). In somepreparations, a small amount of lysozyme, a soluble constit-uent of the azurophil granule, was also found in peak V (upto 20% of the protein found in this peak). The peak VIIIpolypeptide shared 18 of 20 residues with elastase (19). Thepeak IX polypeptide was identified as BPI on the basis ofidentity of 20 of 20 amino acids (20).Three polypeptides had N-terminal sequences that have

apparently not been reported. The peak II polypeptide ap-peared to be a member of the defensin family. It shared 11 of32 or 33 residues with HNP-1, -2, and -3, including all sixcysteines, and has been designated HNP-4 (unpublisheddata). The 29-kDa polypeptide in peak VI was of particularinterest because its abundance (17% of recovered protein)matched that of cathepsin G and was second only to thedefensins counted as a group. The polypeptides of both peakVI and peak VII displayed N-terminal sequence homology tocathepsin G, elastase, and other members of the serineprotease family (17, 19). Detailed studies of the 29-kDapolypeptide found in peak VI, which has been named azuro-cidin, will be reported separately.

Activity Profiles of Each Species. In prior work, optimalantimicrobial activity of defensins depended on low ionicstrength and the presence of nutrients in the assay buffer (9).Another critical variable for defensins, as well as for cathep-sin G and BPI, was maintenance ofthe assay buffer at neutralto alkaline pH (4, 7, 9). Therefore, before testing each species

Table 1. Protein recovery and N-terminal sequence analysis of polypeptides resolved by rpHPLC: Comparison toknown PMN proteins

Molecular SequencePeak or comparable mass, Recovery,

species kDa ug of protein 1 5 10 15 20Ia 4 200 A C Y C R I P A C I Alb C Y C R I P A C I AIc D C Y C R I P A C I AHNP-1(15) A C Y C R I P A C I AHNP-2 (15) - C Y C R I P A C I AHNP-3 (15) - D C Y C R I P A C I AII* 4 2 V C S C R L V F C R R T E L R V G N C LIII 18 10 X P P Q F T R A Q W F A I Q H I S L N PECP (16) - R P P Q F T R A Q W F A I Q H I S L N PIV 29 76 I I G G R E S R P H S R P Y M ACathepsin G (17) I I G G R E S R P H S R P Y M AV 14 12 T X R Y L L V R S L Q T F S Q A X F T XMBP (18) - - T C R Y L L V R S L Q T F S Q A W F T CVI (azurocidin)* 29 80 I V G G R K A R P R Q F P F L A S I Q NVII* 29 36 I V G G H E A Q P H S R P Y M A S L E MVIII 29 18 I V G G R R A R P H A X P X M V S L Q LElastase (19) - I V G G R R A R P H A W P F M V S L Q LIX 54 12 V N P G V V V R I S Q K G L D Y A S Q QBPI (20) - V N P G V V V R I S Q K G L D Y A S Q Q

Protein recoveries are shown for a typical HPLC run (580 ,ug of concentrated azurophil granule membrane extract wasfractionated and 77% was recovered). rpHPLC-purified polypeptides were concentrated to 50 Il in a SpeedVac (Savant)and identified by N-terminal sequence analysis. The single-letter amino acid code is used. Numbers in parentheses are refs.*Sequences that apparently have not been reported.

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5612 Medical Sciences: Gabay et al.

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FIG. 2. SDS/PAGE analysis of rpHPLC peaks. Peaks collectedfrom rpHPLC were dried by vacuum centrifugation, washed byresuspension in 0.1% acetic acid, and returned to dryness. (A) Startingmaterial (lane S) and peaks (lanes I-IX) were treated with 2% (wt/vol)SDS and 5% (vol/vol) 2-mercaptoethanol at 100TC prior to SDS/PAGE. Molecular mass marker migration is indicated on the left (inkDa). (B) Reduced (lanes R) or non-reduced (lanes N) peak I and peakII. Molecular mass markers (lane M) were the same as in A. Molecularmass markers (lane m) were myoglobin digest fragments of 17,200,14,600, 8240, 6380, and 2560 Da. Both gels were silver stained.

individually, we tested the effect of these variables on thebioactivity ofthe unfractionated azurophil granule membraneextract. In addition, we included one more variable that wasimportant for the recovery of antimicrobial activity in therpHPLC fractions: the presence of bovine serum albumin(BSA) in the tubes in which the fractions were collected.Experiments (data not shown) indicated a marked increase inrecovery of bioactivity if BSA was added to the tubes priorto, rather than after, further handling of the samples; theoptimal concentration of BSA was 0.02% (final concentra-tion). As indicated in Fig. 3, the microbicidal activity of thegranule extract against E. coli was higher at pH 5.5 than atneutral or alkaline pH. Within the range tested, ionic strengthwas immaterial, but the addition of nutrient broth wasinhibitory. In contrast, with S. faecalis as a test organism,low ionic strength was critical and pH was no longer a keyvariable. Again, nutrient broth was inhibitory.Thus, with the unfractionated extract, each variable tested

affected the bioassays differently depending on the testorganism. This made it necessary to test each fraction againsteach organism under each condition. The detailed results aredepicted in Fig. 4. For purposes of a summary comparison(Table 2), we selected the set of assay conditions yielding themaximum activity of the unfractionated extract against eachtest organism. Because ECP and MBP probably representproteins from contaminating eosinophilic PMNs, rather thanfrom neutrophilic PMNs, the last column ofTable 2 comparesthe activities of the other peaks to the total activity obtainedby summing all peaks except those representing ECP andMBP. For each of the organisms tested, the sum of activitiesof the isolated polypeptides was 80-100% of the activity ofthe unfractionated extract. This allowed us to assign relativepotencies and activities to each antibiotic polypeptide, asdiscussed below.

Activity against E. coli. Two species accounted for almostall the activity against E. coli: BPI and azurocidin, the 29-kDapolypeptide in peak VI. Although the specific activity of BPIwas 10 times higher than that of azurocidin, the greaterabundance of the latter (see Table 1) led us to estimate thatit contributes 75% as much to anti-E. coli activity as doesBPI. Cathepsin G, MBP, and elastase were also active butcontributed only a minor portion of the total activity. Theabundant defensins in peak I had a specific activity 15,000

E co/ia b c d f g

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Activity against:S. foeco/is

a b c d e f 9

50 50 50 10 10 10 105.5 5.5 7.0 5.5 5.5 7.4 7.4

C. olbiconsh i

FIG. 3. Effect of assay conditions onsusceptibility of test organisms to azuro-phil granule extract. The activity of theextract against E. coli, S. faecalis, and C.albicans was tested as indicated. +,Component present; -, component ab- -sent. Assay buffers were sodium citrateand sodium phosphate, at the noted con-

-+ centration and pH. A supplement to- - some of these assay buffers was trypti-io 10 case soy broth (TSB). Results are ex-5.5 5.5 pressed as means ± SEM for three to* * eight experiments, each performed with- - material from a separate fractionation.

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Proc. Natl. Acad. Sci. USA 86 (1989)

Proc. Natl. Acad. Sci. USA 86 (1989) 5613

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FIG. 4. Effect of assay conditions onsusceptibility of test organisms to puri-fied polypeptides. The activity ofthe ninerpHPLC peaks against E. coli, S. faeca-lis, and C. albicans was tested under thesame conditions as in Fig. 3. The activityof each peak against a particular micro-organism in the various assay conditionsis represented as a percentage (shown onthe ordinate) of the activity of the totalgranule extract measured in 50 mM cit-rate (pH 5.5) for E. coli and in 10 mMphosphate (pH 5.5) for S. faecalis and C.albicans. These assay conditions (bars b,d, and i) resulted in the highest activity ofthe unfractionated granule extractagainst these microorganisms, as shownin Fig. 3. Bars without an indicated per-centage correspond to activity below thelimit of detection. Results are means +SEM for three experiments, each per-formed with material from a separatefractionation.

times lower than that of BPI under the conditions used forcomparison, and thus made a negligible contribution to thetotal activity. Defensin HNP-4 found in peak II was 60-foldmore potent than the mixture of HNP-1, -2, and -3.

Activity against S. faecalis. Low ionic strength was re-quired for activity against this organism. Cathepsin G andMBP together accounted for 88% of the total activity againstS. faecalis. Excluding the contribution of MBP, the largestproportion of activity after that of cathepsin G was providedby azurocidin. The contribution of defensins was almostnegligible under these conditions. Under test conditions ofhigher ionic strength, the only species to show some activityagainst S. faecalis was cathepsin G.

Activity against C. albicans. Antifungal activity was alsodependent on low ionic strength (results at high ionic strengthare not shown). Under these conditions, the major species tocontribute to killing of C. albicans were cathepsin G andMBP, with far lower but still appreciable contributions by thepeak I defensins and azurocidin. The specific activity ofMBPexceeded that of the peak I defensins by 70-fold. HNP-4, thedefensin of peak II, was 7-fold more potent than the mixtureof those of peak I.

DISCUSSIONTwelve polypeptides were isolated from the azurophil mem-brane fraction of human PMNs. Eleven of them were iden-tified by N-terminal sequence analysis and their antimicrobialactivities were compared. Four of these polypeptides provedto be potent, broad-spectrum antibiotics. Of these activities,only one-that of cathepsin G-was previously appreciated.Another-that of MBP-probably arose from contaminationof our neutrophil preparation (97%) with eosinophils (3%).Two broad-spectrum antimicrobial polypeptides were dis-covered: a 4-kDa defensin termed HNP-4 and a 29-kDapolypeptide named azurocidin. HNP-4 (contained in peak II)

was more potent than the mixture of HNP-1, -2, and -3contained in peak I but represented less than 1% of the totalprotein. The possibility has not been excluded that HNP-4arose from eosinophils but this seems unlikely as no otherdefensins have yet been identified in eosinophils. In contrast,azurocidin was both an abundant and potent species and maycontribute 75% as much to the killing of E. coli as BPI, whichwas previously thought to account for almost all of thisactivity (21, 22). The localization of azurocidin to the azuro-phil granules of neutrophils has been confirmed by immun-ofluorescence and immuno electron microscopy with amonospecific antibody (D.C., P. Detmers, C.F.N., andJ.E.G., unpublished results).A strength of this analysis was the high recovery in protein

(Table 1) and antimicrobial activity (Table 2) of the purifiedspecies. Under most assay conditions, the sum of activitiesof the resolved components closely matched (80-100%) thatof the unfractionated extract from which they were derived.This implies that neither synergistic interactions nor mutualinhibition among the component polypeptides need be in-voked. Nonetheless, either phenomenon could occur, inparticular, under suboptimal conditions for antimicrobialactivity. Other antimicrobial polypeptides of the humanPMNazurophil granule may remain to be discovered. We screenedagainst only one microbe from each of three classes. Addi-tional antimicrobial proteins may exhibit activities againstother organisms or, like peak II, exist at levels that makedetection difficult.A limitation of this analysis is that it was restricted to

proteins associated with the membrane of the azurophilgranule. Other subcellular compartments were excluded,based on their lack of bioactivity against E. coli (11). Thus,significant activities elsewhere in the cell against Gram-positive bacteria or fungi may have been overlooked orunderrated. For example, lysozyme, a soluble constituent ofthe azurophil granule, has activity against S. faecalis and C.

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5614 Medical Sciences: Gabay et al.

Table 2. Summary of antimicrobial activities of each peak underthe conditions optimal for activity of the starting material againstthe same organism

Activity, Activity, Activity, % ofLD50, % of % of total less peaks

Peak ,ug/ml ext. total III and VAgainst E. coli

I 225 0.03 0.035 0.04II 3.7 0.03 0.03 0.034III ND ND NDIV 0.38 8 10 11.3V 0.06 8 10VI 0.16 24 30.4 34VII 0.72 2 2.5 2.8VIII 0.15 4.8 6.1 6.8IX 0.015 32 40.6 45.2

Against S. faecalisI 42 0.1 0.14 0.26II 0.6 0.075 0.08 0.16III 9 0.01 0.01IV 0.05 43.8 48 92.8V 0.012 43.8 48VI 1 3 3.4 6.6VII 53 0.006 0.007 0.01VIII 13 0.03 0.034 0.07IX ND ND ND ND

Against C. albicansI 2.8 6.4 5.3 6.9II 0.4 0.6 0.53 0.7III 0.1 4.8 3.9IV 0.11 80 65.8 86.3V 0.04 24 19.7VI 1.6 4.8 3.9 5.2VII 5.6 0.5 0.4 0.5VIII 5.6 0.4 0.33 0.43IX ND ND ND ND

Activity is expressed in three ways: (i) as a percent of that of theunfractionated azurophil granule extract (% of ext.); (ii) as a percentof the total activity recovered in all fractions (% of total); and (iii) asa percent ofthe total activity recovered in all fractions except the twoactivities suspected to arise from eosinophils, peaks III and V (% oftotal less peaks III and V). ND, not detectable.

albicans (23, 24). However, pure human lysozyme (Calbio-chem) was 100-500 times less active than cathepsin G orMBP against S. faecalis and 5-10 times less active than thesetwo species against C. albicans on a weight basis (14) and didnot act synergistically with pure cathepsin G or MBP (un-published data). Quantitatively, the major antibiotic proteinsin the soluble fraction of the azurophil granule are defensins.Half of the total defensin content remained associated withthe granule membrane (data not shown). Thus, our estimatesof the contribution of defensins to overall activity of the cellwere probably not grossly biased by our focus on membrane-associated proteins. Indeed, a survey of the entire azurophilgranule for antimicrobial activity resulted in findings similarto those described here (unpublished data). Finally, resultswere similar when diisopropyl fluorophosphate was omitted(data not shown).

This study points up the major impact of assay conditionson the specific activity of individual antimicrobial polypep-tides. A major question is what set of assay conditions mostfaithfully reproduces the milieu of the phagolysosome. Ex-isting data do not permit an answer; instead, they suggest thatkey factors such as pH are likely to vary with time afterphagocytosis and may also depend on the organism. There isno published information on the ionic strength of phagoly-

sosomal fluid in PMNs. Divalent cations markedly affect theactivity of PMN antimicrobial proteins; the levels of thesecations in PMN phagolysosomes are also unknown.

Purification of the antimicrobial proteins of PMNs, gener-ation of monospecific antibodies against them, and isolationof their cDNAs may lead to a better understanding of theregulation of granule proteins during and after myelopoiesis,insight into the molecular basis of clinical states marked bydefective antimicrobial function, and design of antibiotics.

Note. After this manuscript was submitted, we became aware of aprior report of isolation of HNP-4; its antimicrobial activity was nottested (25).

We thank J. Snable for technical assistance and P. Detmers, R.Nachman, H. Shuman, and S. Wright for critically reading themanuscript. This work was supported by Grant BC-586 from theAmerican Cancer Society and Grants Al 23807 and CA 43610 fromthe National Institutes of Health. J.E.G. is a Cornell Scholar inBiomedical Sciences.

1. Klebanoff, S. J. (1988) in Inflammation, eds. Gallin, J. I.,Goldstein, I. M. & Snyderman, R. (Raven, New York), pp.391 444.

2. Elsbach, P. & Weiss, J. (1988) in Inflammation, eds. Gallin,J. I., Goldstein, I. M. & Snyderman, R. (Raven, New York),pp. 445-470.

3. Lehrer, R. I., Ganz, T. & Selsted, M. E. (1988) in Hematology/Oncology Clinics ofNorth America, ed. Curnutte, J. T. (Saun-ders, Philadelphia), pp. 159-163.

4. Weiss, J., Elsbach, P., Olsson, I. & Odeberg, H. (1978) J. Biol.Chem. 253, 2664-2672.

5. Shafer, W. M., Martin, L. E. & Spitznagel, J. K. (1984) Infect.Immun. 45, 29-35.

6. Hovde, C. J. & Gray, B. H. (1986) Infect. Immun. 54, 142-148.7. Odeberg, H. & Olsson, I. (1975) J. Clin. Invest. 56, 1118-1124.8. Drazin, R. E. & Lehrer, R. I. (1977) Infect. Immun. 17, 382-

388.9. Ganz, T., Selsted, M. E., Szklarek, D., Harwig, S. S. L.,

Daher, K., Bainton, D. F. & Lehrer, R. I. (1985) J. Clin.Invest. 76, 1427-1435.

10. Spitznagel, J. K. (1984) in Contemporary Topics in Immuno-biology, ed. Snyderman, R. (Plenum, New York), pp. 283-343.

11. Gabay, J. E., Heiple, J. M., Cohn, Z. A. & Nathan, C. F.(1986) J. Exp. Med. 164, 1407-1421.

12. Laemmli, U. K. (1970) Nature (London) 227, 680-685.13. Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10038.14. McGrogan, M., Simonsen, C., Scott, R., Griffith, J., Ellis, N.,

Kennedy, J., Campanelli, D., Nathan, C. & Gabay, J. (1988) J.Exp. Med. 168, 2295-2307.

15. Selsted, M. E., Harwig, S. S. L., Ganz, T., Schilling, J. W. &Lehrer, R. I. (1985) J. Clin. Invest. 76, 1436-1439.

16. Gleich, G. J., Loegering, D. A., Bell, M. P., Checkel, J. L.,Ackerman, S. J. & McKean, D. J. (1986) Proc. Natl. Acad. Sci.USA 83, 3146-3150.

17. Salvesen, G., Farley, D., Shuman, J., Przybyla, A., Reilly, C.& Travis, J. (1987) Biochemistry 26, 2289-2293.

18. Wasmoen, T. L., Bell, M. P., Loegering, D. A., Gleich, G. J.,Prendergast, F. G. & McKean, D. J. (1988) J. Biol. Chem. 263,12559-12563.

19. Sinha, S., Watorek, W., Karr, S., Giles, J., Bode, W. & Travis,J. (1987) Proc. Natl. Acad. Sci. USA 84, 2228-2232.

20. Ooi, C. E., Weiss, J., Elsbach, P., Frangione, B. & Mannion,B. (1987) J. Biol. Chem. 262, 14891-14894.

21. Weiss, J., Victor, M., Stendahl, 0. & Elsbach, P. (1982) J. Clin.Invest. 69, 959-970.

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Proc. Natl. Acad. Sci. USA 86 (1989)