Vol. 45, No. 1INFECTION AND IMMUNITY, JUlY 1984, P. 150-1540019-9567/84/070150-05$02.00/0Copyright © 1984, American Society for Microbiology

Purification and Antibacterial Activity of Antimicrobial Peptides ofRabbit Granulocytest

MICHAEL E. SELSTED, DOROTHY SZKLAREK, AND ROBERT I. LEHRER*Department of Medicine, University of California, Los Angeles, Center for the Health Sciences, Los Angeles, California

90024Received 18 January 1984/Accepted 10 April 1984

Six antimicrobial peptides, corresponding to the family of "Ilysosomal cationic proteins" describedpreviously by Zeya and Spitznagel (H. I. Zeya and J. K. Spitznagel, J. Bacteriol. 91:750-754, 1966; H. I. Zeyaand J. K. Spitznagel, J. Bacteriol. 91:755-762, 1966), were purified from rabbit peritoneal granulocytes bypreparative acrylamide gel electrophoresis and reversed-phase high-pressure liquid chromatography. Each ofthe peptides was of low molecular weight (ca. 4,000) as determined by sodium dodecyl sulfate-polyacrylamidegel electrophoresis. The two most cationic peptides, NP-1 and NP-2, were active against a broad spectrum ofgram-positive and gram-negative bacteria. The remaining four peptides, NP-3A, NP-3B, NP-4, and NP-5, hadmore selective antibacterial activity. None of the peptides was active against Bordetella bronchiseptica, acommon pathogen of domestic rabbits. Antibacterial activity was best expressed at near neutral pH underconditions of low ionic strength.

Zeya and Spitznagel have established that rabbit andguinea pig granulocytes contain a family of low-molecular-weight lysosomal cationic proteins with selective antimicro-bial activity (13-16). These arginine and cysteine-rich mole-cules resided in a distinct class of cytoplasmic granules (17,18) and were considered to be a likely source of antimicro-bial activity in intact granulocytes.Our interest in antimicrobial peptides of leukocytes led us

to develop improved preparative procedures and to purifysix granulocyte peptides from rabbit heterophil granulocytesin quantities sufficient for antibacterial testing.

MATERIALS AND METHODSGranulocyte procurement. Sterile peritoneal exudates

were raised in male or female adult New Zealand Whiterabbits by intraperitoneal instillation of 300 to 500 ml ofnormal saline containing sodium caseinate and Escherichiacoli lipopolysaccharide (Difco Laboratories, Detroit, Mich.)as previously described (4). Exudates, collected, approxi-mately 16 h later, typically contained 1 x 109 to 3 x 109leukocytes, of which >95% were heterophilic granulocytes.In our initial experiments, the heterophils were washedseveral times with phosphate-buffered saline and then ho-mogenized in 0.34 M sucrose. Subsequently, a 27,000 x gpostnuclear sediment was collected for extraction with citricacid as previously described (4). In later experiments, theprocedure was simplified, such that intact, washed granulo-cytes were sonicated directly into 0.1 M citric acid to extracttheir antimicrobial peptides. Peptides produced by eitherprocedure were identical in structure and function.

Peptide purification. The citric acid-soluble material ob-tained by extracting the 27,000 x g postnuclear sediment orthe whole sonicate was dialyzed exhaustively against 0.1%acetic acid in tubing (Spectrum Industries, Los Angeles,Calif.) with a molecular weight cutoff of 3,500, lyophilized,and redissolved at 2 to 6 mg of protein per ml in 1.0% aceticacid containing 3.0 M urea. Samples (0.5 to 1.5 ml) of thismaterial were electrophoresed on acidic, urea-containingpolyacrylamide gels (3 by 160 by 220 mm) (9) for approxi-

* Corresponding author.t Publication number 24 of the Collaborative California Universi-

ties-Mycology Research Unit.

mately 9 h. Gels were subsequently immersed in a solutionof 0.25% (wt/vol) eosin Y (Sigma Chemical Co., St. Louis,Mo.) in 0.1 N NaOH for 30 s, thus disclosing up to 25transverse magenta-staining bands of protein. Gel stripscontaining the five most cathodal bands were individuallyexcised from eight simultaneously run gels, pooled, andsubjected to electrophoretic elution. The eluted peptideswere dialyzed against 1.0% acetic acid and lyophilized. Eachof the electrophoretically fractionated peptides was subject-ed to further purification by reversed-phase high-pressureliquid chromatography (RP-HPLC) on a Spectraphysics SP8700 system equipped with a Rheodyne 7125 sample injector(2.0-ml loop), a Waters 450 variable wavelength detector, aKipp and Zonen BD 40 recorder, and a large-pore (33.0 nm)Vydac 218 TP C-18 column (1.0 by 25.0 cm). Water-acetoni-trile gradients containing 0.025% trifluoroacetic acid wereused in elution. A C-18 column (0.46 by 25 cm; ,uBondapak,Waters Associates, Milford, Mass.) was used for someanalytical RP-HPLC runs.

Analytical methods. Peptide purity was evaluated by RP-HPLC, acid-urea polyacrylamide gel electrophoresis(PAGE), and sodium dodecyl sulfate (SDS)-PAGE. Estima-tions of peptide molecular weights were made based on themigration of purified samples in SDS-PAGE (9).

Antibacterial assay. Our procedures paralleled those re-cently reported (5). Fourteen bacterial isolates (Table 1),were cultivated for 16 h at 37°C in nutrient broth, mixed, andsubcultured by adding 1 ml to 49 ml of fresh nutrient broth.The subculture was grown at 37°C for 4 h with shaking toprovide mid-logarithmic phase organisms that were washedtwice with buffer by centrifugation (2,000 x g for 10 min) andthen quantitated spectrophotometrically at 620 nm, withpreviously established CFU-optical density relationshipsused as a reference.

Unless other buffers are specified, bacteria were suspend-ed and experiments were performed in 10 mM sodiumphosphate buffer (pH 7.4). In some experiments, bufferswere supplemented with 0.14 M NaCl. The conductivities ofour standard and NaCl-supplemented buffers were 1.36 and12.3 mS, respectively, as determined with a Sybron/Barn-stead PM-70 CB conductivity bridge. In some experiments,10 mM phosphate buffer (pH 5.8; conductivity, 1.36 mS) wasused. Peptide stock solutions were prepared at 500 jxg/ml

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GRANULOCYTE ANTIMICROBIAL PEPTIDES 151

(microbiuret assay) in 0.01% acetic acid and stored at-200C.

Typically, incubation mixtures contained 105 bacterialCFU and 5 ,ug of peptide in 100 p.l of buffer. After incubationfor 20 min at 37°C, 900 p.l of the same buffer was added, andtwo additional serial 10-fold dilutions were made with thatbuffer. Duplicate 100-pd portions from the three serial dilu-tions were spread over appropriate nutrient agar plates andincubated for 24 to 72 h to ensure full colony development.Killing of microorganisms was expressed either as a percent-age of initial colony count or as the log1o NIN, where No isthe initial colony count and N is the colony count afterincubation with peptide. There was no significant alterationin colony count when any of the test organisms wereexposed to peptide-free buffer for 20 min. For experimentsinvolving rapid kinetics (see Fig. 4), incubations werestopped with NaCl-supplemented buffers as previously de-scribed (5).

RESULTS

Nomenclature and purification. When examined by acid-urea PAGE, extracts of rabbit heterophil granulocytes ap-peared to contain five distinct cationic peptides whosecathodal migration greatly exceeded that of lysozyme (mura-midase). These components were designated NP-1, NP-2,NP-3, NP-4, and NP-5, beginning with the most cathodalcomponent. They were recovered from PAGE gels by re-

versibly staining the gels with an alkaline solution of eosin Y,followed by electroelution. When the electroeluted peptideswere further analyzed by RP-HPLC after dialysis, lyophili-zation, and resuspension, NP-3 was found to consist of twodistinct peptides, designated NP-3a and NP-3b. NP-1, NP-2,NP-4, and NP-5 were not further resolved by HPLC andwere deemed to represent individual molecular species.

Purity of the peptides was confirmed by amino acid se-quence data which will be presented elsewhere. Altogether,rabbit heterophils contained six distinct cationic peptides.The resolution of these peptides by HPLC is shown in Fig. 1.Figure 2 shows the resolution of the HPLC-purified peptideson acid-urea polyacrylamide gels, which show that NP-3aand NP-3b comigrate in this system. The results in Fig. 3,which shows the migration of the six neutrophil peptides on

an SDS-PAGE gel, suggest that the peptides have molecularweights of approximately 4,000.

Antibacterial activity. Antibacterial activity was affectedby peptide concentration, incubation time, pH, and ionicstrength. Figure 4 illustrates the effects of peptide concentra-tion and incubation time on the activity of NP-1 againstPseudomonas aeruginosa PA0579 and Streptococcus aga-lactiae type 1A. Note that the P. aeruginosa was killed withessentially first-order kinetics after exposure to 50 p.g ofNP-1 per ml (Fig. 4a) and that peptide concentration influ-enced the rate and extent of bactericidal activity (Fig. 4b andc). Figure 5 demonstrates that the bactericidal effect ofNP-1 against P. aeruginosa PA0579 was optimal betweenpH 6.5 and 8.0 and was affected by addition of NaCI. Thebactericidal effect was attenuated when the pH was <6.5 or

when the concentration of added NaCl was increased.The activities of all six peptides against selected bacteria

are summarized in Table 1. NP-1 and NP-2 showed thebroadest range of antibacterial activity and were approxi-mately equipotent. Both were active against gram-positiveand gram-negative organisms. Of the species we examined,only Bordetella bronchiseptica appeared to be completelyresistant to all of the peptides.

In contrast to the broad antibacterial spectrum of NP-1and NP-2, NP-4 was active only against type III pneumococ-cus, which was also susceptible to each of the other granulo-

TABLE 1. Antibacterial activity of rabbit granulocyte peptidesa

Organism studied under Bactericidal activityb of the following peptides:indicated conditions Strain or type NP-1 NP-2 NP-3a NP-3b NP-4 NP-5

Staphylococcus aureus 566 >4.1 >4.1 1.3 1.7 0.2 0.4Staphylococcus aureus 502A 2.3 2.4 0.0 0.3 0.1 0.2Staphylococcus aureus J.F. 2.7 2.9 0.4 0.8 0.1 0.1Staphylococcus epidermidis UCLA-622 >4.1 >4.1 1.3 1.7 0.2 0.4Streptococcus pneumoniae Type III 3.8 >3.5 >3.5 >3.5 4.3 1.9Streptococcus agalactiae Type I-A 2.3 2.0 0.6 0.9 0.1 0.5Streptococcus agalactiae Type III 3.9 2.9 0.3 1.2 0.2 0.4Listeria monocytogenes 450 2.1 2.1 0.2 4.3 0.1 0.2Pseudomonas aeruginosa PA0579 >4.2 3.2 3.3 4.2 0.2 0.7Escherichia coli ATCC 29648 1.9 0.8 0.6 1.4 0.1 0.2Klebsiella pneumoniae ATCC 13883 3.0 1.1 2.4 3.4 0.2 1.2Serratia marcescens 1.0 1.0 0.5 0.7 0.2 0.2Haemophilus influenzae Type 3A >1.1 2.0 >1.1 0.7 1.3 1.2Bordetella bronchiseptica UCLA-342 0.1 0.1 0.1 0.1 0.1 0.0

pH 5.8Streptococcus agalactiae Type 1A 0.1 0.1 0.0 0.2 0.0 0.0Listeria monocytogenes 450 0.2 0.1 0.0 0.0 0.0 0.1Pseudomonas aeruginosa PA0579 0.3 0.2 0.1 0.0 0.0 0.1Klebsiella pneumoniae ATCC 13883 0.6 0.5 0.4 0.2 0.3 0.1

High ionic strengthStreptococcus agalactiae Type 1A 0.0 0.1 0.0 0.0 0.0 0.0Listeria monocytogenes 450 0.1 0.2 0.1 0.0 0.0 0.2Pseudomonas aeruginosa PA0579 0.1 0.0 0.1 0.0 0.0 0.0Klebsiella pneumoniae ATCC 13883 0.1 0.0 0.0 0.0 0.0 0.0a Bacteria were exposed for 20 min to 50 pLg of the indicated purified granulocyte peptide per ml under the standard conditions described in the text.

b Bactericidal activity is shown as log1o NJN, where No is the initial concentration ofCFU per milliliter (typically 106), and N is the number of CFU per milliliterafter the 20-min incubation. For additional technical details, consult reference 5.

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152 SELSTED, SZKLAREK, AND LEHRER

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ELUTION TIME (min)FIG. 1. Chromatogram of purified granulocyte peptides. A mix-

ture containing 20 ,ug of each of the purified peptides was applied toa C-18 column (250 by 4.6 mm; pore size, 33.0 nm; Vydac) anddeveloped with a linear water-acetonitrile gradient buffered with0.1% (vol/vol) trifluoroacetic acid at a flow rate of 1 ml/min. Elutionwas monitored at 220 nm. The dashed line represents the gradient.The individual peptide peaks are identified by peptide (NP) number.A220, Absorbance at 220 nm.

cyte peptides, and Haemophilus influenzae. Although lesscationic than NP-4, NP-5 also had activity against the twoorganisms susceptible to NP-4 and, in addition, was activeagainst Klebsiella pneumoniae. NP-3a and NP-3b had activi-ty intermediate between that of the more highly active NP-1and NP-2 and the less active NP-4 or NP-5. The spectrum ofNP-3b included not only those organisms susceptible to NP-4 or NP-5, but also Staphylococcus epidermidis, Streptococ-cus agalactiae, Listeria monocytogenes, P. aeruginosa, E.coli, and several strains of Staphylococcus aureus. Based onthe results with this test panel, the ranking of these peptidesin descending order of overall antimicrobial effectiveness isNP-1 = NP-2 > NP-3b > NP-3a > NP-5 2 NP-4.The effects of increased acidity (pH 5.8) or ionic strength

(0.14 M NaCl) on microbicidal activity were tested with apanel of 4 organisms which were selected from the 13 used toexamine the activity spectrum of the peptide. The results(Table 1) confirmed the inhibitory effects of low pH and highionic strength that were demonstrated for NP-1 and P.aeruginosa in Fig. 5.

DISCUSSIONGranulocytes subject ingested microorganisms to an on-

slaught of 02-dependent and 02-independent factors thatcontribute importantly to host defenses against infection.

Although interest in the 02-dependent microbicidal mecha-nisms of phagocytes currently dominates research activitiesin this field, 02-independent phagocyte factors also warrantattention.

Metchnikoff reported that bacteria ingested by guinea piggranulocytes became eosinophilic as a consequence (7).Skarnes and Watson (10) and Hirsch (2, 3) have shown thatrabbit granulocytes or their cytoplasmic granules (la) couldbe treated with dilute acids to yield crude soluble extracts(leukin and phagocytin) with antibacterial properties in vitro.In a series of elegant studies, Spitznagel and co-workers (11-

30 18) have shown that guinea pig and rabbit granulocytescontain a family of low-molecular-weight, arginine-rich pep-

-i tides that exhibit antibacterial activity in vitro. Bacteria~ exposed to these peptides adsorbed them, acquiring cationic

properties that rendered them stainable by eosin or analo-z gous stains, presumably explaining the observation of0 Metchnikoff. Transfer of cationic peptides from a specific set- of cytoplasmic granules to phagocytic vacuoles containing< ingested bacteria has also been shown by electron microsco-

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py with ammoniacal silver staining (6).

The studies reported here confirm the general observa-tions of Zeya and Spitznagel concerning the existence of a

family of highly cationic, low-molecular-weight peptides in

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E 1 2 3a3b 4 5 EFIG. 2. Acid-urea polyacrylamide gel of purified granulocyte

peptides. This 12.5% acrylamide gel contained 5 M urea bufferedwith 0.9 M acetic acid and was electrophoresed for 7 h at 150 V andstained with Coomassie brilliant blue R-250. The outside lanes eachcontained 75 ,ug of crude heterophil extract (E), and the remaininglanes each contained 3 ,ug of a purified granulocyte peptide aslabeled by peptide (NP) number.

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INFECT. IMMUN.

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GRANULOCYTE ANTIMICROBIAL PEPTIDES 153

,0 --25700

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5 EFIG. 3. SDS-polyacrylamide gel of purified granulocyte pep-

tides. Purified granulocyte peptides (5 ,ug per lane) and crudeheterophil acid extract (E; 80 ,ug per lane) were lyophilized, sus-pended in sample buffer containing SDS and -3-mercaptoethanol,heated to 100°C for 5 min, and applied to this 10 to 30% gradient gelwith appropriate molecular weight standards. After electrophoresisfor 18 h at 150 V, the gel was stained with Coomassie brilliant blueR-250. The molecular weights and migration of the standards (notshown) are indicated on the right of the figure. The standards,purchased from Bethesda Research Laboratories, Gaithersburg,Md., and their subunit molecular weights (in parentheses) wereinsulin (3,000), bovine trypsin inhibitor (6,200), lysozyme (14,300),3-lactoglobulin (18,400), and a-chymotrypsinogen (25,700).

rabbit granulocytes. Because the combined use of prepara-tive gel electrophoresis and RP-HPLC allowed us to preparehomogeneous peptide fractions unavailable to earlier work-ers, our studies cannot be directly compared with thosereported in the literature. Other factors precluding suchdirect comparisons are the choice of different test organismsand different assay conditions. It may be significant, forexample, that the majority of our studies were performed atpH 7.4 in a phosphate-buffered solution, whereas those ofZeya and Spitznagel were typically carried out at pH 5.6 in acitrate-phosphate buffer.As was also noted in our studies of MCP-1 and MCP-2, the

microbicidal cationic peptides of rabbit lung macrophages(5), the antibacterial effects of granulocyte peptides werestrongly pH dependent. The peptides had optimal bactericid-

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ADDITIONAL NaCI (Molar)FIG. 5. lEffect of pH and ionic strength on bactericidal effect of

NP-i for P. aeruginosa PA0579. Bacteria (106 CFU/ml) wereexposed for 20 min to NP-1 (25 p.g/ml) in the standard phosphatebuffer (pH 7.4) supplemented with various amounts of NaCl (dashedline) or in a constant ionic strength buffer (1.36 mS) whose pH wasvaried (solid line). No, Initial concentration of CFU per milliliter; N,number of CFU per milliliter after a 20-min incubation.

al activity between pH 7 and 8 and exhibited relatively littleeffect at pH 5.8. Recent studies have demonstrated that thepH of phagocytic vacuoles within human neutrophils isneutral to mildly alkaline (pH 7.8) during the early post-phagocytic period (1, 8). Comparable data are not yetavailable for rabbit granulocytes. Like MCP-1 and MCP-2(5), the granulocyte peptides also require hypotonic condi-tions (relative to the extracellular milieu) to exert maximalantibacterial effects. There are no available data concerningthe ionic strength of phagocytic vacuoles, nor is it knownwhether mechanisms exist to provide, within such vacuoles,an ionic milieu conducive to the microbicidal activity of thepeptide.We are completing an extensive biochemical characteriza-

tion of the six granulocyte peptides and will report theirchemical features and amino acid sequences in the nearfuture (M. E. Selsted and R. I. Lehrer, manuscript inpreparation). Our evidence to date strongly indicates that thepeptides are products of a single gene family whose membersshow various degrees of sequence homology with MCP-1and MCP-2, peptides purified from rabbit lung macrophages(9). The ability to compare sequences, structures, and func-tions of these peptides should provide a powerful tool foranalyzing the mechanism of their effects.

ACKNOWLEDGMENTS0,1,2.5 01 We thank Jacob Fleischmann, Douglas M. Brown, and Romeo

106t 106I 106 Baldeviso for their expert assistanice.;\\5 \ 5 This research was supported in part by Public Health Service

,1041 41 0 1 grants Al 16252 and Al 18754 from the National Institutes of Health.U.° * o2 1 * 024 X x o4122 LITERATURE CITED

(a) (b) ) 1. Cech. P., and R. I. Lehrer. 1984. Phagolysosomal pH of humanneutrophils. Blood 63:88-95.

5 10 10 20 20 60 la.Cohn, Z. A., and J. G. Hirsch. 1960. The isolation and proper-ties of the specific cytoplasmic granules of rabbit polymorpho-

INCUBATI10ON TIME (MSIN) nuclear leukocytes. J. Exp. Med. 112:983-1004.FIG. 4. Effects of NP-1 on viability of P. aeruginosa PA0579 (a 2. Hirsch, J. G. 1956. Phagocytin: a bactericidal substance from

and b) and Streptococcus agalactiae type IA (c). The concentrations polymorphonuclear leucocytes. J. Exp. Med. 103:589-611.of NP-1 were 50 ,ug/ml (a) or as indicated in (b and c). Samples 3. Hirsch, J. G. 1960. Antimicrobial factors in tissues and phago-shown in (a) were processed for rapid kinetics as described in cytic cells. Bacteriol. Rev. 24:133-140.reference 5. 4. Lehrer, R. I., K. M. Ladra, and R. B. Hake. 1975. Nonoxidative

400~~~~~sm

E 1 2 3a3b 4

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fungicidal mechanisms of mammalian granulocytes: demonstra-tion of components with candidacidal activity in human, rabbit,and guinea pig leukocytes. Infect. Immun. 11:1226-1234.

5. Lehrer, R. I., M. E. Selsted, D. Szklarek, and J. Fleischmann.1983. Antibacterial activity of microbicidal cationic proteins 1and 2, natural peptide antibiotics of rabbit lung macrophages.Infect. Immun. 42:10-14.

6. Macrae, E. K., and J. K. Spitznagel. 1975. Ultrastructurallocalization of cationic proteins in cytoplasmic granules ofchicken and rabbit polymorphonuclear leukocytes. J. Cell Sci.17:79-94.

7. Metchnikoff, E. 1905. Immunity in infective diseases, p. 198. InF. G. Binnie (translator). Cambridge University Press, London.

8. Segal, A. W., M. Geisow, R. Garcia, A. Harper, and R. Miller.1981. The respiratory burst of phagocytic cells is associatedwith a riie in vacuolar pH. Nature (London) 290:406-409.

9. Selsted, M. E., D. M. Brown, R. J. DeLange, and R. I. Lehrer.1983. Primary structures of MCP-1 and MCP-2, natural peptideantibiotics of rabbit lung macrophages. J. Biol. Chem.258:14485-14489.

10. Skarnes, R. C., and D. W. Watson. 1956. Characterization ofleukin: an antibacterial factor from leucocytes active againstgram-positive pathogens. J. Exp. Med. 104:829-845.

11. Spitznagel, J. K. 1961. The effect of mammalian and other

INFECT. IMMUN.

cationic polypeptides on the cytochemical character of bacterialcells. J. Exp. Med. 114:1063-1078.

12. Spitznagel, J. K., and H.-Y. Chi. 1963. Cationic proteins andantibacterial properties of infected tissues and leukocytes. Am.J. Pathol. 43:697-711.

13. Zeya, H. I., and J. K. Spitznagel. 1963. Antibacterial andenzymic basic proteins from leukocyte lysosomes: separationand identification. Science 142:1085-1087.

14. Zeya, H. I., and J. K. Spitznagel. 1966. Cationic proteins ofpolymorphonuclear leukocyte lysosomes. I. Resolution of anti-bacterial and enzymatic activities. J. Bacteriol. 91:750-754.

15. Zeya, H. I., and J. K. Spitznagel. 1966. Cationic proteins ofpolymorphonuclear leukocyte lysosomes. II. Composition,properties, and mechanism of antibacterial action. J. Bacteriol.91:755-762.

16. Zeya, H. I., and J. K. Spitznagel. 1968. Arginine-rich proteins ofpolymorphonuclear leukocyte lysosomes. Antimicrobial speci-ficity and biochemical heterogeneity. J. Exp. Med. 127:927-941.

17. Zeya, H. I., and J. K. Spitznagel. 1969. Cationic protein-bearinggranules of polymorphonuclear leukocytes: separation fromenzyme-rich granules. Science 163:1069-1071.

18. Zeya, H. I., and J. K. Spitznagel. 1971. Characterization ofcationic protein-bearing granules of polymorphonuclear leuko-cytes. Lab. Invest. 24:229-236.

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