INFECTION AND IMMUNITY, Feb. 1984, p. 561-5660019-9567/84/020561-06$02.00/0Copyright © 1984, American Society for Microbiology

Vol. 43, No. 2

Polyamino Acid Enhancement of Bacterial Phagocytosis by HumanPolymorphonuclear Leukocytes, and Peritoneal Macrophages

PHILLIP K. PETERSON,* GENYA GEKKER, ROBERT SHAPIRO, MARK FREIBERG, AND WILLIAM F. KEANEDepartment of Medicine, University Hospitals and Hennepin County Medical Center, University of Minnesota School of

Medicine, Minneapolis, Minnesota 55455

Received 1 September 1983/Accepted 10 November 1983

Cationic polyamino acids are known to enhance a variety of cell-cell interactions by virtue of their abilityto alter electrostatic forces of cell surfaces. In this study, the effect of polyamino acids on phagocytosis of3H-labeled bacteria by human polymorphonuclear leukocytes (PMNs) and peritoneal macrophages wasinvestigated. Negatively charged and neutral polyamino acids did not influence phagocytosis of unopson-ized Staphylococcus epidermidis, whereas protamine, poly-L-arginine, and poly-L-lysine stimulated phago-cytosis in a dose-dependent manner. At 50 ,ug/ml, >30% uptake by PMNs was seen with each of thesecationic polyamino acids. Although cationic polyamino acids promoted PMN and peritoneal macrophagephagocytosis of unopsonized S. epidermidis, Staphylococcus aureus M (encapsulated) and M variant(unencapsulated), and Escherichia coli J5, little effect was seen with the parent E. coli 0111:B4 or aserotype 0222:H16 strain. Pretreatment of bacteria and phagocytes separately demonstrated that thephagocytosis-promoting property of polyamino acids is manifest predominantly on the bacteria. Bacteriapretreated with cationic polyamino acids also elicited a PMN chemiluminescent response, and PMN-associated bacteria were killed, as determined by a fluorochrome microassay. Thus, cationic polyaminoacids promote the phagocytosis and killing of many but not all bacterial strains, and in this respectpolyamino acids function as opsonins.

To perform their bactericidal function, phagocytic cellsmust recognize bacterial cells as foreign. In recent years, anumber of studies have focused upon the important role ofthe opsonic molecules immunoglobulin G and C3b in facili-tating this recognition process. Phagocytic cells have beenshown to possess receptors for these molecules (10, 24), andthe nature of these receptors has been partially characterized(4, 12). Opsonin-independent mechanisms of phagocytosishave also been described whereby specific bacterial surfacecomponents promote phagocytosis (16, 22). Physicochemi-cal surface properties, particularly surface hydrophobicityand charge, have been considered key determinants of theingestion of some of these organisms (16). It has been longappreciated that such nonspecific surface forces are in-volved in the process of phagocytosis (14), and it has beenproposed that even opsonin-mediated phagocytosis may beexplained by changes in certain of these surface properties(21).

Cationic polyamino acids, by virtue of their ability to alterelectrostatic forces of cell surfaces, are known to enhance avariety of cell-cell interactions. In the present study, weinvestigated the effect of highly positively charged poly-amino acids on bacterial phagocytosis by human polymor-phonuclear leukocytes (PMNs) and peritoneal macrophages(PM4+s). This study was based upon the work of previousinvestigators who had demonstrated that cationic polyaminoacids promote the phagocytosis of starch particles by humanPMNs (3), of polystyrene latex beads by rabbit PMNs (2),and of an Escherichia coli isolate by rat PM4+s (15). Theconclusions drawn in each of these studies was that positive-ly charged polyamino acids promote phagocytosis by reduc-ing the electrostatic repulsion naturally existing betweenthese different types of particles and phagocytic cells. Ourgoals in this investigation were to (i) extend these studies tobacterial species with inherent differences in their surface

* Corresponding author.

compositions, (ii) define the site of action of polyamino acidsin the recognition process, and (iii) determine whetherpolyamino acids would trigger PMN responses involved intheir bactericidal activity.

MATERIALS AND METHODSBacterial strains and radioactive labeling. The following

bacterial strains were used: Staphylococcus epidermidisstrains ST and BL, both clinical isolates; Staphylococcusaureus M, an encapsulated strain, and an unencapsulated Mvariant (kindly provided by M. A. Melly, Vanderbilt Schoolof Medicine, Nashville, Tenn.); E. coli ON2 (serotype022:H16, a serum-resistant strain kindly provided by B.Bjorksten, University of Umea, Umea, Sweden); and E. coli0111:B4 and the J5 mutant (gifts of E. J. Ziegler, School ofMedicine, University of California at San Diego). All strainswere maintained on nutrient agar plates at 4°C. Radioactivelabeling for phagocytosis studies was achieved by inoculat-ing several colonies into 20 ml of Mueller-Hinton broth(Difco Laboratories, Detroit, Mich.) containing 0.04 mCi of[2-3H]adenine (specific activity, 5 to 15 Ci/mmol; NewEngland Nuclear Corp., Boston, Mass.). After 18 h ofincubation at 37°C, the bacteria were washed three times inphosphate-buffered saline (pH 7.4) and resuspended in phos-phate-buffered saline to a final concentration of 5 x 107CFU/ml (determined by a spectrophotometric method andconfirmed by pour plate colony counts). For chemilumines-cence (CL) and bacterial killing experiments, bacteria wereprepared in an identical manner but without incorporating aradioisotope in the culture media.

Opsonization with normal serum. Bacteria were preopson-ized with normal human serum (NHS) pooled from threehealthy donors to compare the phagocytosis-promotingproperty of polyamino acids with that of a standard opsonin.Shortly before use, a portion of pooled NHS kept frozen at-70°C was thawed and diluted to a final concentration of10% in Hanks balanced salt solution containing 0.1% gelatin

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562 PETERSON ET AL.

(GHBSS). Bacterial suspensions (0.1 ml; 5 x 107 CFU) wereincubated in 0.9 ml of 10% NHS in plastic tubes (12 by 75mm; Falcon Plastics, Oxnard, Calif.) for 30 min at 37°C; aftercentrifugation, bacteria were resuspended in fresh GHBSS.

Phagocytic cells. PMNs were recovered from heparinizedvenous blood of healthy donors by previously describedmethods (18). Final cell suspensions contained 5 x 106PMNs per ml of GHBSS. Purity of PMN preparations andviability by trypan blue exclusion both exceeded 95%.PM+s were isolated as previously described (23) from

peritoneal effluents of uninfectc J patients treated with main-tenance intermittent peritoneai dialysis for end-stage renaldisease. Final cell suspensions contained 5 x 106 PM(s perml of GHBSS. Cell preparations contaminated with >10%PMNs were not used. Greater than 95% of the cells identi-fied by light microscopy as PM4+s stained positively fornonspecific esterase and were viable by trypan blue exclu-sion.Polyamino acids and treatment of cells. The following

polyamino acids were obtained from Sigma Chemical Co.,St. Louis, Mo.: poly-L-lysine hydrobromide (molecularweight [m.w.], 4,000 or 65,000); poly-L-arginine hydrochlo-ride (m.w., 44,000); poly-D,L-alanine (m.w., 3,000); poly-L-glutamic acid (m.w., 11,000). Protamine sulfate (m.w.,8,000; Eli Lilly & Co., Indianapolis, Ind.) was recoveredfrom spermatozoa of chum or king salmon and contained80.4% arginine. In one experiment, heparin sodium (10mg/ml, stock solution; Abbott Laboratories, North Chicago,Ill.) was used to neutralize the cationic property of prot-amine.

Initial experiments were performed with phagocytic cellssuspended in GHBSS solutions containing polyamino acidsat various concentrations. To help avoid decreased phago-cyte viability due to prolonged exposure to polyamino acids,these phagocyte-polyamino acid mixtures were added imme-diately after being constituted to suspensions of unopsonizedbacteria.

In some experiments, bacteria or phagocytes were pre-treated for 15 min at 37°C with polyamino acids in GHBSS.After centrifugation, these treated bacterial or phagocyticcells were resuspended in fresh GHBSS before use.

Phagocytosis mixtures and assay. Uptake of [3H]adenine-labeled bacteria by PMNs and PM+s was determined aspreviously described (23). Briefly, 100 ,lI of phagocytesuspensions (5 x 105 cells) were added to polypropylenevials (Bio-vials; Beckman Instruments, Inc., Fullerton, Cal-if.) containing 100 ,ul (5 x 106 CFU) of unopsonized (sus-pended in GHBSS) or preopsonized (suspended in 10%NHS) bacteria, and after 30 min of incubation at 37°C,phagocytosis was stopped by adding 3 ml of ice-cold phos-phate-buffered saline. Non-phagocyte-associated bacteriawere removed by three cycles of differential centrifugation.Phagocyte-associated radioactivity was determined in a liq-uid scintillation counter and has been shown to reflect thedegree of phagocytosis (25). Phagocytosis was expressed asthe percentage of uptake of total radioactivity by the formu-la: percent uptake = (counts per minute in phagocytepellet/total counts per minute) x 100. All experiments wererepeated at least three times with phagocytes from differentdonors.CL assay. A luminol-enhanced CL assay was used to

measure the generation of reactive oxygen species by PMNs(1, 13). Suspensions of S. epidermidis ST (0.4 ml, 109 CFU)that had been pretreated with polyamino acids, preopson-ized with 10% NHS, or untreated were added to dark-adapted glass vials containing 4.1 ml of GHBSS and 20 ,ul of

luminol (275 ,uM in dimethyl sulfoxide). After backgroundlevels were recorded in a liquid scintillation counter (modelLS 150; Beckman Instruments, Inc.) set in the out-of-coincidence mode, 1 ml of PMNs (5 x 104 cells) was addedsequentially to the appropriate vials, and the CL responsewas observed for 30 min.

Determination of bacterial killing. A modification of afluorochrome microassay (17, 20) was used to determine thePMN-associated killing of bacteria that had been eitherpretreated with polyamino acids or preopsonized with 10%NHS. Phagocytosis mixtures were constituted as describedabove for the phagocytosis assay. After 30 min of incuba-tion, 50-pA samples were removed and deposited on glasscover slips, to which 250 pul of phosphate-buffered saline wasadded. The cover slips were then incubated for 30 min at37°C in 5% CO2. The nonadherent cells were decanted, andacridine orange (1:10,000 in 0.87% NaCI) was added for 30 s.After being rinsed with phosphate-buffered saline, thesecover slips were inverted onto glass slides, blotted, andsealed. Preparations were examined under a x 100 oil immer-sion objective with a UV fluorescence microscope (Leitz,Wetzlar, West Germany). In this assay, live organisms staingreen or orange and dead organisms stain red. Total numbersof bacteria associated with 20 PMNs were counted, andpercent killing was calculated as: [number of dead bacte-ria/(number of dead bacteria + number of live bacteria)] x100.

RESULTSEffect of polyamino acids on phagocytosis of S. epidermidis

by PMNs. The phagocytosis-promoting property of poly-amino acids was investigated initially by incubating unop-sonized S. epidermidis ST with PMNs suspended in variousconcentrations of negatively charged (poly-L-glutamic acid),uncharged (poly-D,L-alanine), or positively charged (poly-L-lysine, poly-L-arginine, protamine) polyamino acids. Mini-mal phagocytosis (<10% uptake) was observed with unop-sonized bacteria alone or in the presence of poly-L-glutamicacid, poly-D,L-alanine, or poly-L-lysine (m.w., 4,000),whereas PMNs phagocytized >30% unopsonized bacteriawhen poly-L-lysine (m.w., 65,000), poly-L-arginine, or prot-amine were in the incubation mixtures at concentrations of250 p.g/ml (Fig. 1). At concentrations of 200 ,ug of poly-L-arginine and poly-L-lysine (m.w., 65,000) per ml, phagocyto-sis of unopsonized staphylococci was similar to that ofbacteria that had been preopsonized with 10% NHS (Fig. 1).When heparin (200 ,ug/ml) was added to PMN mixtures

containing protamine (200 p.g/ml) or when negativelycharged poly-L-glutamic acid (200 p.g/ml) was added to PMNmixtures containing poly-L-lysine (m.w., 65,000; 200 pg/ml),the enhanced phagocytosis seen with each of the positivelycharged amino acids alone was completely abolished (datanot shown). Since heparin combines ionically with prot-amine to form a neutral complex, these data suggest that thephagocytosis-promoting property of cationic polyamino ac-ids is dependent on their positive charge.

Effect of cationic polyamino acids on phagocytosis of otherbacterial strains by PMNs and PM4)s. Results of the preced-ing experiment indicated that cationic polyamino acids werecapable of facilitating phagocytosis of an unopsonized S.epidermidis strain by PMNs. We next studied whethercertain of these polyamino acids would similarly affectphagocytosis of other bacterial strains and whether anotherphagocyte population, namely, PM+s, would also recognizeunopsonized bacteria in the presence of polyamino acids.Both protamine and poly-L-lysine increased the uptake of

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POLYAMINO ACID ENHANCEMENT OF PHAGOCYTOSIS 563

10oo

80F70

4' 60

50

* 40

30

20

10n

pomy-L--tysine oly-L-argkiie(m.w. 65.000)

poly-L-glutamic poly-L-Iysieacid (mw. 4,000)

poly-D,L-alanine

1 ftftf~n1:1 xIxilI

rotamma

Tr T

- 105050200 10 50100200 10505000M 105050200 50 50020 10 50lo 0o

Polyamino Acid Concentration (,g/ml)FIG. 1. Effect of polyamino acids on phagocytosis of unopson-

ized S. epidermidis ST by PMNs. PMN uptake of unopsonizedbacteria was determined after 30 min of incubation in the presenceof polyamino acids at the indicated final concentrations. Uptake ofpreopsonized (10% NHS) (0) and unopsonized (O) bacteria in theabsence of polyamino acids was also determined. Values representmeans and ranges of three separate experiments.

unopsonized staphylococci (S. epidermidis strains ST andBL and S. aureus strains M and M variant) by PM4os as wellas by PMNs (Table 1). Phagocytosis of the two S. epidermi-dis strains and of the S. aureus M variant strain was

generally greater when preopsonized (10% NHS) bacteriawere compared with unopsonized bacteria in the presence ofpolyamino acids. In contrast, there was virtually no phago-cytosis of the encapsulated S. aureus M strain when thisorganism was preopsonized with 10% NHS (3% uptake), but.29% uptake was seen when polyamino acids were presentin the incubation mixture (Table 1). Although protamine andpoly-L-lysine increased the phagocytosis of unopsonized E.coli J5, neither the parent E. coli strain O111:B4 nor E. coliON2 were appreciably affected by either of these polyaminoacids (Table 1).

Site of action of polyamino acids. In the above experiments,both unopsonized bacteria and phagocytes were simulta-neously exposed to polyamino acids. To determine whetherpolyamino acids promote phagocytosis by changing the

bacterial cell or phagocytic cell, or both, S. epidermidis STwas pretreated with protamine or poly-L-lysine before beingadded to untreated PMNs and PM+s. Likewise, PMNs andPM+s were pretreated with each polyamino acid beforebeing added to untreated bacteria. When neither the bacteri-al nor the phagocytic cells were pretreated with polyaminoacids, there was minimal phagocytosis (<10% uptake); how-ever, pretreatment of either the bacterial or the phagocyticcell populations resulted in enhanced phagocytosis (Fig. 2).Since pretreatment of the bacterial cells generally produceda greater stimulatory effect on subsequent phagocytosis thandid pretreatment of PMNs or PM4s, the predominant site ofaction of polyamino acids appears to be on the bacterialsurface.CL response of PMNs to polyamino acid-treated bacteria.

Results of the preceding experiment demonstrated that pre-

treatment of S. epidermidis cells with the cationic polyaminoacids protamine or poly-L-lysine promoted recognition(phagocytosis) by PMNs and PM¢s. To determine whetherstaphylococci pretreated with these polyamino acids wouldalso trigger reactive oxygen species production by PMNs,we used a luminol-enhanced CL assay. When compared withthe PMN CL response to untreated control bacteria, a rapidCL response was observed with S. epidermidis that had beenpretreated with poly-L-lysine before being added to PMNs(Fig. 3). A slower CL response was seen with bacteriapretreated with protamine, and the magnitude ofCL was lesswith bacteria pretreated with either polyamino acid than thatobserved with organisms preopsonized with 10% NHS (Fig.3).PMN killing of polyamino acid-treated bacteria. To deter-

mine whether bacteria pretreated with cationic polyaminoacids were killed by phagocytic cells, we used a fluoro-chrome (acridine orange) microassay. S. epidermidis ST, E.coli J5, and S. aureus M were preopsonized with 10% NHSor pretreated with protamine or poly-L-lysine before beingadded to PMNs. After 30 min of incubation, samples were

deposited on glass cover slips. The cells were allowed toadhere to the cover slips for 30 min, and slides were thenexamined with a fluorescence microscope. A majority ofPMN-associated preopsonized and polyamino acid-treatedS. epidermidis and E. coli cells were found to be killed when

TABLE 1. Effect of polyamino acids on phagocytosis of staphylococcal and E. coli strains by PMNs and PM4s

Mean (range) % uptake by PMNs and PM4s of a:

Unopsonized bacteria in': Preopsonized bacteriacBacterial strain

GHBSS Protamine Poly-L-lysinePMN PMX

PMN PM+ PMN PM+ PMN PMPS. epidermidisST 5 (4-6) 5 (4-6) 36 (32-40) 27 (23-30) 44 (42-46) 22 (19-23) 47 (44-52) 44 (34-51)BL 7 (4-9) 6 (4-8) 34 (33-35) 30 (24-34) 46 (44-48) 26 (24-27) 58 (56-59) 43 (38-44)

S. aureusM 4 (3-6) 2 (1-2) 32 (31-33) 30 (27-33) 45 (40-52) 29 (23-33) 3 (2-5) 3 (2-3)M variant 16 (14-17) 10 (8-11) 29 (23-35) 26 (23-28) 63 (61-65) 27 (22-31) 46 (41-51) 37 (34-40)

E. coliJ5 14 (13-16) 19 (17-23) 39 (37-40) 38 (36-40) 46 (44 47) 33 (32-34) 29 (27-31) 24 (29-28)0111:B4 2 (1-3) 4 (2-5) 6 (3-8) 4 (2-5) 4 (2-6) 3 (2-4) 41 (39-44) 24 (22-26)ON2 3 (3-4) 4 (4-5) 4 (3-5) 4 (3-5) 15 (13-17) 9 (7-11) 54 (54-55) 29 (21-37)

a Numbers represent means (ranges) of three separate experiments.b UnOPSOniZed bacteria were incubated with phagocytic cells in the presence of 200 p.g of protamine, poly-L-lysine (m.w., 65,000), or buffer

(GHBSS) per ml for 30 min, and uptake was measured.C After preopsonization with 10% NHS, bacteria were added to phagocytic cells in GHBSS, and uptake was measured after 30 min.

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564 PETERSON ET AL.

100A O~~~~UntreatedcellsB

[3Pretreated bacteria

80 ElPretreated Phagocytes

6040

20

NONE PROTAMINE POLY-L-LYSINE NONE PROTAMINE POLY-L-LYSINE

Polyamino AcidsFIG. 2. Effect of pretreatment of S. epidermidis ST or phago-

cytes with polyamino acids on subsequent phagocytosis. PMNs (A),PM4¢s (B), and bacteria (A and B) were incubated separately for 15min in GHBSS alone or in GHBSS containing protamine or poly-L-lysine (m.w., 65,000), both at 200 ,ug/ml, before constituting phago-cytosis mixtures that did not contain polyamino acids. Uptake was

determined at 30 min. Values represent means and ranges of threeseparate experiments.

this assay was used (Fig. 4). Essentially none of the PMNswere founj to contain organisms that had been preopsonizedwith 10% NHS, an observation that was consistent with theresults of the phagocytosis experiments with the encapsulat-ed S. aureus M strain. When S. aureus M was pretreatedwith protamine or poly-L-lysine, however, PMNs containedan average of three to four bacteria per PMN, and 44 and62% of cell-associated protamine- or poly-L-lysine-treatedbacteria, respectively, were killed (Fig. 4). Control bacteria,i.e., S. epidermidis ST, E. coli J5, and S. aureus M preop-sonized with 10% NHS or pretreated with protamine or poly-L-lysine and incubated for a total of 60 min in the absence ofPMN, were not killed when the fluorochrome method wasused to assess viability.

DISCUSSIONIn recent years, mounting evidence has suggested that a

variety of phagocyte responses are governed at least in partby alterations in sprface charge. Gallin et al. (7) havereported that chemotactic factors cause a significant reduc-tion in the negative surface charge of neutrophils and that anassociation also exists between decreases iq surface chargeand neutrophil aggregation, adhesiveness, and exocytosis oflysosomal granule contents (6). Weiss et al. (26) have shownthat a charge interaction is involved in the bactericidal actionof one of the constituents of lysosomes, the cationic bacteri-cidal permeability-increasing protein. Additionally, Gins-burg and colleagues have shown that cationic polypeptidesmodulate the bacteriolytic activity of leukocyte extracts (8).

In the present study, we used cationic polyamino acids as

probes to test the influence of surface charge on the phago-cytic function of human PMNs and PM4)s. Our results are

consistent with the observations of earlier investigators whoused other sources of phagocytic cells (2, 15) or particlesother than bacteria in their phagocytosis assay systems (2,3). We found that highly positively charged polyamino acidswere capable of altering either the bacterial or the phagocyt-ic cells population in such a way that phagocytosis of

unopsonized bacteria was facilitated. The predominant siteof action appeared to be on the bacterial cell, and as such,cationic polyamino acids function as opsonic molecules.This concept is supported by the recent work of Ginsburg etal. (9), who demonstrated the opsonic properties of cationicpolyamino acids for M protein-positive group A streptococciand Candida albicans.

Bacteria preopsonized with polyamino acids were capableof triggering a PMN CL response and were killed by PMNs,as assessed by a fluorochrome microassay. Although we donot have an explanation for the relatively attenuated CLresponse observed with bacteria pretreated with polyaminoacids, in contrast to the brisk CL response seen with bacteriapreopsonized with serum, it is possible that more efficienttriggering of the respiratory burst is accomplished via recep-tors for opsonic molecules.

Since poly-L-lysine at a m.w. of 4,000 did not enhancephagocytic recognition, in contrast to a higher-m.w. (65,000)form of this polyamino acid, considerations as to surfacecharge density may be important, in keeping with theobservations of de Vries et al. (3), who also found thatprotamine was less effective on a weight/weight basis thanwas poly-L-lysine or poly-D,L-arginine (3), data similar toour experimental results.The cationic polyamino acids poly-L-lysine and protamine

stimulated PMN and PM4 phagocytosis of both S. epidermi-dis strains and both S. aureus strains used in this study.Since the heavily encapsulated S. aureus M strain is knownto be highly resistant to opsonization by NHS (19, 27), theenhancement of phagocytosis seen with polyamino acids isespecially interesting. Kozel (11) hps recently shown thatresistance to phagocytosis of encatsulated Cryptococcus

800

700

600 /

500 - /

a 400Qt3I A/300 t*

200-

A1O'100 1 ..

PN+ GHBSS

MinutesFIG. 3. CL response of PMNs to S. epidermidis ST pretreated

with 10% NHS (0), poly-L-lysine (m.w., 65,000; 200 ,ug/ml) (-),protamine (200 p.g/ml) (A), or buffer alone (GHBSS) (0). PMNs inthe absence of bacteria were also included (PMN + GHBSS).Values are averages of three separate experiments.

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POLYAMINO ACID ENHANCEMENT OF PHAGOCYTOSIS 565

100

00c80

cu 6

0

~0

Z 20

S. epidermis ST Ecoii J5 S.aujeus M

FIG. 4. PMN-associated killing of S. epidermidis ST, E. coli J5,and S. aureus M as assessed by fluorochrome microassay. Bacteriawere pretreated with 10% NHS, protamine (200 ,ug/ml), or poly-L-lysine (m.w., 65,000; 200 ,g/ml) before being added to PMNs. After30 min of incubation, the percentage of the total number of PMN-associated bacteria that were dead was determined. Values repre-

sent means and ranges of three separate experiments.

neoformans is not associated with surface tension (hydrophi-licity) of this organism. Whether cationic polyamino acidsare capable of reducing the negative surface charge of thisencapsulated yeast or of encapsulated bacterial species otherthan S. aureus M, thereby promoting their phagocytosis, iscurrently unknown.The importance of bacterial surface composition in deter-

mining the effect of cationic polyamino acids on phagocyto-sis was underscored by the finding of relatively little influ-ence ofpoly-L-lysine or protanline on phagocytosis of E. coliOlil:B4, in contrast to the enhancement seen With the J5mutant. Since the J5 mutant contains only the core glycolipidin its outer membrane, it may be that an intact lipopolysac-charide confers a greater negative surface charge on theO111:B4 parent strain.

In conclusion, results of the present study add support tothe hypothesis that changes in the physicochemical proper-

ties of the microbial or phagocytic cell surface can influencethe recognition process. Although the relative importance ofalterations in electrostatic forces versus interfacial tensionremains controversial, it is nonetheless clear that suchsurface properties can profoundly affect phagocytic cellfunction. The opsonic activity of molecules such as immuno-globulin G and C3b may also be in part explained byconsiderations such as net basic surface charge, as hasrecently been suggested in studies reported by Fischetti (5)of opsonic antibodies directed against the anti-phagocytic Mprotein of the group A streptococcal cell wall. A unifyinghypothesis may yet emerge linking both opsonin-dependentand opsonin-independent mechanisms of phagocytosis tounderlying changes in physicochemical surface properties.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grantAI-08821-10 from the National Institute of Allergy and InfectiousDiseases and by a host defense research grant from Baxter-TravenolLaboratories.

We thank Kim Podany and Bridget Stellmacher for their assist-ance in the preparation of this manuscript.

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15. Nagura, H., J. Asai,Y. Katsumata, and K. Kojima. 1973. Role ofelectric surface charge of cell membrane in phagocytosis. ActaPathol. Jpn. 23:279-290.

16. Ohman, L., J. Hed, and 0. Stendahl. 1982. Interaction betweenhuman polymorphonuclear leukocytes and two different strainsof type 1 fimbriae-bearing Escherichia coli. J. Infect. Dis.146:751-757.

17. Pantazis, C. G., and W. T. Kniker. 1979. Assessmnent of bloodleukocyte microbial killing by using a new fluorochrome mi-croassay. J. Reticuloendothel. Soc. 26:155-170.

18. Peterson, P. K., J. Verhoef, f). Schmeling, and P. G. Quie. 1977.Kinetics of phagocytosis and bacterial killing by human poly-morphonuclear leukocytes and monocytes. J. Infect. Dis.136:502-509.

19. Peterson, P. K., B. J. Wilkinson, Y. Kim, D. Schmeling, and

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P. G. Quie. 1978. Influence of encapsulation on staphylococcalopsonization and phagocytosis by human polymorphonuclearleukocytes. Infect. Immun. 19:943-949.

20. Smith, D. L., and F. Rommel. 1977. A rapid micro method forthe simultaneous determination of phagocytic-microbicidal ac-

tivity of human peripheral blood leukocytes in vitro. J. Im-munol. Methods 17:241-247.

21. van Oss, C. J. 1978. Phagocytosis as a surface phenomenon.Annu. Rev. Microbiol. 32:19-39.

22. Verbrugh, H. A., J. R. Hoidal, B. T. Nguyen, J. Verhoef, P. G.Quie, and P. K. Peterson. 1982. Human alveolar macrophagecytophilic immunoglobulin G-mediated phagocytosis of protein-A positive staphylococci. J; Clin. Invest. 69:63-74.

23. Verbrugh, H. A., W. F. Keane, J. R. Hoidal, M. R. Freiberg,G. R. Elliott, and P. K. Peterson. 1983. Peritoneal macrophagesand opsonins: antibacterial defense in patients undergoingchronic peritoneal dialysis. J. Infect. Dis. 147:1018-1029.

24. Verhoef, J., P. K. Peterson, and P. G. Quie. 1977. Humanpolymorphonuclear leukocyte receptors for staphylococcal op-sonins. Immunology 33:231-239.

25. Verhoef, J., P. K. Peterson, and P. G. Quie. 1977. Kinetics ofstaphylococcal opsonization, attachment, ingestion and killingby human polymorphonuclear leukocytes: a quantitative assayusing 3H-thymidine labeled bacteria. J. Immunol. Methods14:303-311.

26. Weiss, J., M. Victor, and P. Elsbach. 1983. Role of charge andhydrophobic interactions in the action of the bactericidal/per-meability-increasing protein of neutrophils on gram-negativebacteria. J. Clin. Invest. 71:540-549.

27. Wilkinson, B. J., S. P. Sisson, Y. Kim, and P. K. Peterson. 1979.Localization of the third component of complement on the cellwall of encapsulated Staphylococcus aureus M: implications forthe mechanism of resistance to phagocytosis. Infect. Immun.26:1159-1163.

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