Pathogenesis of foreign body infection Evidence for a local granulocyte defect

(1)

Pathogenesis of foreign body infection.

Evidence for a local granulocyte defect.

W Zimmerli, … , P D Lew, F A Waldvogel

J Clin Invest.

1984;

73(4)

:1191-1200.

https://doi.org/10.1172/JCI111305

.

Implanted foreign bodies are highly susceptible to pyogenic infections and represent a

major problem in modern medicine. In an effort to understand the pathogenesis of these

infections, we studied the phagocytic function in the vicinity of a foreign body by using a

recently developed guinea pig model of Teflon tissue cages subcutaneously implanted

(Zimmerli, W., F.A. Waldvogel, P. Vaudaux, and U.E. Nydegger, 1982, J. Infect. Dis.,

146:487-497). Polymorphonuclear leukocytes (PMN) purified from tissue cage fluid had

poor bactericidal activity against a catalase-positive microorganism. When compared with

blood or exudate PMN, they exhibited a significant reduction in their ability to generate

superoxide in response to a particulate or a soluble stimulus (72 and 57%, respectively, P

less than 0.001). Not only their total contents in myeloperoxidase, beta-glucuronidase,

lysozyme, and B12 binding protein were significantly reduced (by 62, 21, 47, and 63%,

respectively, P less than 0.01), but also their capability for further secretion of residual B12

binding protein upon stimulation. Ingestion rates of endotoxin-coated opsonized oil particles

were reduced by 25% (P less than 0.05). In an effort to reproduce these abnormalities in

vitro, fresh peritoneal exudate PMN were incubated with Teflon fibers in the presence of

plasma. Interaction of PMN with the fibers led to significant increases in hexose

monophosphate shunt activity and exocytosis of secondary granules (P less than […]

Research Article

Find the latest version:

(2)

Pathogenesis

of

Foreign

Body Infection

Evidence for a Local

Granulocyte

Defect

A

bstract.

Implanted

foreign

bodies

are

highly

susceptible

to

pyogenic infections

and

represent

a

major

problem in modern medicine. In

an

effort to understand

the

pathogenesis

of these

infections,

we

studied the

phagocytic function in the vicinity of

a

foreign body by

using

a

recently developed guinea pig model of Teflon

tissue cages subcutaneously implanted (Zimmerli, W.,

F. A. Waldvogel, P. Vaudaux, and U.

E. Nydegger,

1982,

J. Infect.

Dis., 146:487-497). Polymorphonuclear

leu-kocytes (PMN) purified from tissue cage fluid had poor

bactericidal

activity against

a

catalase-positive

microor-ganism. When compared with blood or exudate PMN,

they

exhibited

a

significant reduction

in

their

ability

to

generate

superoxide

in response

to a

particulate

or a

sol-uble

stimulus (72 and 57%, respectively,

P <

0.001). Not

only

their

total contents in myeloperoxidase,

(-gluc-uronidase,

lysozyme, and B12 binding protein were

sig-nificantly

reduced

(by 62, 21, 47, and 63%, respectively,

P <

0.01), but also their capability for further secretion

ofresidual B12 binding protein upon stimulation. Ingestion

rates

of

endotoxin-coated opsonized oil

particles

were

reduced

by 25% (P

<

0.05).

In an

effort

to

reproduce these abnormalities in

vitro,

fresh

peritoneal exudate

PMN

were

incubated

with Teflon

fibers

in the presence

of plasma. Interaction

of

PMN

with

the

fibers

led

to

significant

increases in hexose

mono-Apreliminaryreportof thiswork was presented at the 22nd Interscience

ConferenceonAntimicrobialAgents and Chemotherapy, Miami Beach,

FL, 4-6October1982.

Dr.Zimmerliwas arecipient ofafellowship from the Swiss National

Research Foundation (grant no. 83.750.0-79). His present address is

Department of Medicine, UniversityHospital, CH-4031 Basel, Swit-zerland. Dr. Lewisarecipientof a MaxCloettaCareer Development

Award.Addressallreprintrequests to Dr. Waldvogel.

Receivedforpublication 20 May 1983 and in revised form 29 De-cember 1983.

Werner Zimmerli, P.Daniel Low,

and Francis A. Waldvogel

InfectiousDiseaseDivision, Department of Medicine, University

Hospital, 1211 Geneva4,Switzerland

phosphate shunt activity and exocytosis of secondary

granules (P

<

0.01). PMN eluted after such interaction

showed

defective bactericidal activity, oxidative

metab-olism,

and granular enzyme content similar to those

ob-served

in tissue cage PMN.

The local injection of fresh blood PMN into tissue

cages at

the time of, or 3 h after, inoculation with 100

microorganisms (Staphylococcus aureus Wood 46)

re-duced the infection rate from 50 of 56 cages to

1 of 21

(P

<

0.001) and 3 of 8 cages (P

<

0.001), respectively.

These results suggest that the in vivo as well as in vitro

interaction of PMN with a nonphagocytosable foreign

body

induces

a

complex PMN defect, which may be partly

responsible for the high susceptibility to infection of

for-eign

bodies.

Introduction

During

the pasttwo

decades,

an

increasing

numberof

prosthetic

devices

have been

implanted

in

orthopedic (1-3), plastic

re-constructive

(4),

cardiovascular

(5-8),

and

general

surgery(9);

foreign materials

have also been used

extensively

for the

treat-ment

of

various

medical

problems (10-12).

Infection isoneof

the

major complications

of such

implants:

it

usually persists

until

removal

of

the

prosthesis (13),

leads to loss of function

(14), and

is

associated

witha

high mortality especially

in

patients

with cardiovascular prostheses (15). Despite ourconsiderable

clinical knowledge

regarding

infections of

prosthetic devices,

little is known about their

pathogenesis (16).

Infections of

prosthetic

devices are characterized by (a) the

high infecting

powerofa lowinoculum, mostly

staphylococci

(1, 17, 18); (b) their protracted evolution

(18,

19); (c) their usually limited spread to the tissues in immediatecontactwith the

implanted foreign

body

(20),

and

(d)

their

persistence

without removalof the prosthesis

(13,

16). Based onthese observations, we have previously postulated that local hostdefense mecha-nismsmight be unable to cope with thelocal invasion of

mi-croorganisms,

and have developedto that effect a

guinea pig

model of foreign body infection, using tissue cages

implanted

subcutaneously (21).

Thismodel

reproduced

all the

previously

mentioned characteristics of the disease and allowed the

col-lectionandanalysis of interstitial fluid and

inflammatory cells,

1191 Pathogenesis of Foreign Body Infection

J.Clin. Invest.

0021-9738/84/04/1191/10 $1.00

(3)

which had been in contact with the implant. We also showed that whereas opsonic activity of sterile tissue cage fluid was

adequate for optimal opsonization

of

Staphylococcus aureus

Wood 46,

the

local tissue cage

polymorphonuclear leukocytes

(PMN)'

were unable to kill the same bacteria in an in vitro

phagocytic

assay (21).

In the present study we have extended these findings by

delineating:

(a) some of the steps responsible for the abnormal

bactericidal

activity of the PMN surrounding the

implanted

tissue

cages; (b) the mechanisms leading to such an

acquired

granulocyte defect; and (c) itspathogenic significance by

pre-venting

infection with local infusion of peripheral PMN.

Methods

Reagents. Cytochrome C type VI, phorbol myristateacetate (PMA),

superoxidedismutase, nitroblue tetrazolium (NBT), Triton X-100,

o-dianisidine-di HCl,

N-formyl-methyonyl-leucyl-phenylalanine

(FMLP), egg-white lysozyme standard,

phenolphthalein-fl-glucuronate,

NADH,

andzymosanwerepurchased from Sigma Chemical Co., St. Louis, MO;

cytochalasin B from Aldrich Chemical Co., Milwaukee,WI; Dextran T-500 andPercoll from Pharmacia Fine Chemicals, Uppsala, Sweden;

endotoxine lipopolysaccharide

B Escherichia coli026: B6 and

micro-coccuslysodeikticusfrom Difco Laboratories, Detroit, MI; phosphate-buffered saline (PBS, Dulbecco's) from Gibco-Bio-CultCo., Glasgow, Scotland;D(+)-glycogenpurum from Fluka AG, Buchs SG, Switzerland; diisodecyl phthalate from Sigfried Zofingen, Switzerland; 57Co-B12,

hexadecan-'4C,

and [1-'4C]glucose from Amersham Corp., Arlington Heights,IL; and"I'indium-oxinefrom Byk-Mallinckrodt, Pretten,The

Netherlands.

Tissue cageimplantation. Rigidperforated polytetrafluoroethylene

(Teflon) tubes (internal and external diameters of 10 and 12 mm, re-spectively; length, 32mm) wereimplanted subcutaneously into guinea pigs, aspreviously described (21). Animalswere used for further

ex-perimentation one orseveral weeks after tissuecageimplantation. Preparation of leukocytes. Acuteperitonealexudates rich in PMN

(>95%)were obtainedin guineapigsby twoi.p. injections of sterile glycogen in0.9% NaCl (0.1%, wt/vol), 175 hand2 h before harvest, respectively(22).Persistent peritonitisas a control for a chronic exudate

generated in the absence ofaforeign bodywasproducedbyrepeated glycogen injectionsover aperiod of2 wk. These latterexudatesshowed the same leukocyte differentialcounts astissuecage fluid,i.e., -40% PMN and60% mononuclear leukocytes(21). Theexudateswerecollected aspreviously described (21). Blood PMN were obtained as a 60-80% pure suspension by the method described by Chenoweth et al. (23). PMN of sterile tissue cage fluidwere aspirated percutaneously from

tissuecages1-2wkaftersurgery and washed with the sameprocedure asblood and exudate PMN (21). Bloodcontaminationin tissue cage

fluidwas0.5-5%atthis postoperative interval.Whennecessary (median number ofPMN permilliliterof tissue cage fluid, 8 X 10'),PMN were

concentratedbycentrifugationand pooled by using cells from various

tissuecages.

1. Abbreviations usedinthispaper:CFU,colony-formingunits;FMLP,

N-formyl-methionyl-leucyl-phenylalanine;

HMS,hexosemonophosphate

shunt; MPO, myeloperoxidase; NBT, nitroblue tetrazolium; PMA,

phorbol myristateacetate.

Whenever mentioned,themixed leukocyte populationwasfurther

purified

on adiscontinuous Percoll gradient, adaptedtoguinea pig

leu-kocyteseparation fromthemethodofHjorthet al.(24). Stock isotonic Percollwaspreparedbydissolving solidNaCIin Percollto afinal con-centration of0.15M.This mixture is referredto as

100%

Percoll(density

s = _{1.136 g/ml). The} _separationswere performed in 17 X 100-mm Falcon tubes (Becton&DickinsonCo.,Oxnard,CA).PMN were sep-arated fromanticoagulatedblood (10 mM EDTA, finalconcentration)

asfollows:3ml ofdextran-sedimented, leukocyte-richplasmawaslayered

onto adouble-densityPercollgradient.Thegradientconsisted of 3ml

of 50% Percoll (diluted withEDTAanticoagulatedguinea pig plasma)

ontopof3 mlof 70% Percoll.Thegradienttubeswerecentrifugedat

350gfor 20min. Themononuclear cells and platelets accumulatedat

the interphasebetween the plasma and the 50% solution, the PMN accumulatedatthe interphase between thetwodifferent

Percoll

solutions,

and theerythrocytes sedimentedto the bottom. The PMNfractionwas

harvested through theuppergradient and washed twicein 15 ml 0.9% NaClat 150gfor 10min. Inordertopurifytissuecageandexudate PMN,anidentical procedurewasused,buton a40% topgradientand

a65% bottomgradient. This allowed thesedimentationof the less dense peritoneal exudate and tissuecage PMNattheinterphasebetween the twodifferent Percoll solutions. After thisprocedure>98% of eachtype ofPMN excluded Trypan Blue.Theyield of eachtype of PMN was

between40 and 60% of theinitialcellcount.

Bactericidalassay. Thebactericidalassayused in theseexperiments

hasbeen previously described byusindetail(21).Weusedtwo micro-organismsasteststrains:acatalase-negative species, i.e.,Streptococcus

faecalis (HC82, wild strain), anda catalase-positive species, i.e.,aS.

aureus

(strain Wood 46). Each of these strainsdependedoncomplement for itsopsonization,asshown bythetotal absence ofPMNkillingafter

opsonization withheat-inactivatedserum

(560C

for30min).The test

incubation contained

0.1

mlof the washed andappropriately diluted bacterial suspension [2X

106

colony-formingunits(CFU)],

0.1

mlof pooledguinea pigserum,and 0.8mlof PBScontaining 1X

106

PMN. Thissuspensionwasincubatedat

370C

for2h inashaking (140 rpm)

waterbath. Counts oftheviable bacteriawereassessedat zerotime, I h,and2 hof incubation, accordingtothe methodpreviouslydescribed (21).Thebacterialkilling by exudate PMN and tissuecagePMN

was

calculated from the results oftheserepetitivecultures. Bacterial

clumping,

whichcanartifactually diminishthebacterial counts in the absence of PMN killing,wasexcludedbymorphologicalcontrols withgramstains

andby control experiments in whichthebacteriawereincubated inthe

absence of PMN. In these testtubes, growth ofS. aureusWood

46

reached

380±43%,

and of S.

faecalis 170±19% (mean±SD),

respectively,

at2 h.

Ingestionrate.Ingestionwasquantitatedby the uptakeratesof

en-dotoxin-coateddiisodecylphthalate particlesaspreviouslydescribed (25) withthe following modifications: diisodecylphthalatewaslabeled with

hexadecane-'4C

(26) andamicromethodwasused (27). Normal pooled guinea pigserum was

used

toopsonizetheparticles.

Superoxidegenerationassay. Superoxideionproductionwas mea-sured asthe superoxidedismutase-sensitivereduction of cytochromec in acontinuousassay asdescribedbyNewburgeret al.(28).Weused

PMA(0.01-2

Ag/ml)

andopsonized zymosan

(1-4

mg/ml)asstimuli (28). The NBTslide test wasperformed according to themethod of Newburgeret al. (29) andscoredasdescribedby Lew et al. (30).For

thisassay

Percoll-purified

PMNwereused.

Enzyme assays. All enzyme assays were performed with

Percoll-purified

PMN.

Total enzymatic activity ofPMN wasdetermined by

adding

0.025-0.1%

(finalconcentration)Triton X-100to the cell

(4)

pensionsinthefollowingassays. Myeloperoxidase (MPO) activitywas

determined inacontinuousassay,byfollowingthechange ofabsorbance

at450 nmwhich accompaniestheoxidationof o-dianisidine-diHC as

describedbyBabiorandCohen (31). Therateof oxidation ofdianisidine

was calculated asfollows:dianisidine oxidation in nanomolesperminute

= 50XAunits (31).Lysozymeactivitywasdetermined by measuring

the rate oflysis ofMicrococcus lysodeikticus at pH6.2 witha turbi-dometricmethod(31). Enzymeactivitywasexpressedintermsof

mi-crogramspermilliliter egg-white lysozymestandard (31).#-Glucuronidase

activitywas assayed bymeasuring the releaseofphenolphthalein from its

f-glucuronate

after 2-h incubation at pH 4.5(31). Activitywas

ex-pressedas nanomolesof phenolphthalein hydrolyzed by 106PMN per

minute (activitynmol/106PMN=0.45XAunits/2 h)(31). B12 binding proteinwas measured byits abilityto take upradioactive cobalamin fromthe medium.B12binding capacity was expressed aspicogramsper

106cellequivalents (31). Lactic dehydrogenasewasassayedby measuring theconsumption ofNADH(31).

MPOslidetestswereperformed accordingtoKaplow(32).

Degranulation assay. Degranulationwasmeasured as thereleaseof B12 bindingprotein into the extracellular fluid. Twodifferent stimuli were used, namely FMLP in the presence ofcytochalasin Band opsonized zymosan.PMN (2.5 X

106/ml)

werepreincubated at

370C

for5min

inthe presence or absence of cytochalasin B (5 ug/ml). At this time, FMLP(10-7 M) oropsonized zymosan (3 mg/ml), respectively, was

added.Theincubationwascontinued foranother 5min (FMLP) or 10 min (zymosan). The reaction was stopped in ice, the PMN were sedi-mented at 1,000rpm/10 min,andB12 binding proteinwas measured asmarkerforsecondary granule exocytosis (31). The absenceof

mea-surablelactic dehydrogenase activityexcluded celldestruction (31).

Re-sultsaregivenas percentoftotal cellular enzyme,determined afterlysis

with Triton-X (0.025%).

PMN turnover assay. In ordertoget an estimate of the time of

exposureofPMNto thetissue cagesurface,westudiedtheirturnover

timeaspreviously described by Cohen et al. (33). Briefly, 5 X

106-1

X10' Percoll-purifiedhomologous blood PMN wereresuspended in 1 mlof PBS. 200-300

ACi

".'indiumoxine

wasadded,andthe PMN were

incubated for 20 minat37°C.After washing twice inNaCI0.9%/plasma

30%, the PMN-associated radioactivity, which usually exceeded 80% of the addedvalue,wasdetermined. These washed cellsweresuspended in 1 mlof PBS and administeredbyintracardiac injectionto aguinea pig withtwoimplanted tissuecages.Leukocytedifferentialcounts were

determined inblood andtissuecagefluidat 2and 30 min, and 2, 4, 20, 24, and27 hafter injection.Totalandcell-associatedradioactivity

were determinedin aliquotsofthe samples witha gamma-radiation

counter. Thecell-associated radioactivity wasusually 70-80% of the added valueduringthe first 30h afterPMNadministration into the

cages. Turnover rate(k)and turnovertime(t,)werecalculated asdescribed by Cohenetal.(33).

In vitro interactionbetweentissue cagematerial(Teflon)andPMN. To testtheinteraction between tissuecagematerial and PMN,weused peritoneal exudatePMN.ThePMN-foreignbody interactionwasstudied

with three types of experiments. First, wemeasured theoxidation of

[1-'4C]glucose

[hexose monophosphate shunt (HMS)] during the

in-cubation with 200mgof Teflon fibers(diameter0.3 mm;Angst&Pfister Co., Zurich, Switzerland). 0.5 ml ofa PMN

_suspension

in PBS

(2

X 107PMN/ml)containing 0.2

ACi

of

[1-'4C]glucose

and0.5mlguinea pig plasmawere incubated with orwithout 200mg Teflon fibersat

37°C invials stoppered bycaps fitted with cups(KontesGlass

Co.,

Vineland, NJ), andcontaining filterpaperwicksimpregnatedwith200

,g of NaOH 8%. The reactionwasstoppedatdifferent incubation time

intervalsafter gentleshakingina waterbathbyinjectionof 0.5mlof

2 NH2SO4 through the stopperintotheincubation medium. Aftera

30-minequilibration period,the cupswere removed andassayedfor

radioactivity. Positive controlswere run ineach assay using 1 ,ugof

PMAasstimulus. These valueswere15-35 timeshigherthan theactivity

ofrestingcells.Negativecontrolswere runwith 0.5 ml 2 NH2SO4in the incubation medium. These valueswere 10-15 times lower than the

activityofrestingcells(34). Second,wedeterminedexocytosis during

contactwith Teflon fibers by measuring B,2 binding protein and

fl-glucuronidase activity(31) released in thesupernatantofaPMN

sus-pension (5 X 106/ml in PBSsupplemented with 5% heated serum).

Third,wemeasuredtheresidualbactericidalfunction,theresidual

su-peroxide production capacity,and the residualenzymatic activity of

PMNafter their incubation with Teflon fibersasfollows. 200mgTeflon fiberswereincubatedduring 1 hwith 107PMN in PBSsupplemented

with10%plasma in polypropylenesyringes of5 ml(Becton & Dickinson

Co.).ControlPMNwereincubated under thesameconditions but with-outTeflonfibers.ToremovePMNfromthe Teflonfibers,thesyringes

wererinsed with 5volNaCl 0.9%and 10 volacid-citrate-dextrose, by gently tappingthesyringebarrelagainstthepalmof the hand(34,35).

This procedure enabledus torecover>50% ofthePMNincubated with Teflon and >70% of the controlPMN. PMNrecovered from different syringeswerepooledandresuspendedin PBSat aconcentration of1

X 107/mland tested fortheir residual bactericidalactivitywith S.aureus Wood 46, for their superoxide productionbystimulation with PMA andopsonizedzymosan,and for their residual MPOactivity(see above).

Experimental infectionsandprotection experiments. S.aureusWood

46,storedinaliquotsofthesameinitialcultureat-70'C,wasusedas infecting microorganism for experimental tissuecageinfections.

Over-nightculturesinMuller-Hintonbrothwerewashedandresuspended in 0.9%NaCl.Tissuecages wereinoculated with0.2 mlofa10-6 dilution,

whichusuallycontained5X

I02103

CFU/ml,asroutinelycheckedby

platingonMuller-Hintonagar. PMNusedforprotectionexperiments

wereharvestedaccordingtothemethoddescribedabove. Foreach

pro-tectionexperiment, I X107 peripheralbloodPMNortissuecage PMN wereinjected inavolumeof 0.2mlof PBS into the tissuecage. The

seruminjected into tissuecagesinsomeexperimentswascollectedand

pooledaspreviouslydescribed(21).Quantitativebacterial cultures with aminimaldetectionlevelof10CFUwereperformedbyaspirationof tissuecagefluid for I wk. Tissuecagefluid showing nogrowth (<10 CFU/ml) 8dafter inoculationwererecordedassterile.

Results

Validation

of

the

experimental

model

1-3wk

after

tissue cage

implantation,

fluidwas

aspirated

under sterile conditions and

analyzed

as to

sterility

andPMNcounts, which

ranged from

7 X

I04'

to 8 X

106/ml

(median

value 8

X

105/ml)

for 39 cages tested.

Injection

of 102 CFUof S.aureus

Wood 46 ledtoinfection in37 of 39 cages,asdocumented

by

macroscopic

examination and

quantitative

bacterial cultures.

Thetwo

only

tissue cages that couldnot beinfectedhad 6X l0 and 3 X

106 PMN/ml, respectively, i.e.,

not the

highest

cell

counts. Weconcluded that infection could be

easily produced

under standard

conditions, irrespective

of the number of PMN

present in the cage fluidatthetime of

inoculation.

Wetherefore undertookto

analyze

some

_phagocytic

and metabolicfunctions

of thesePMNbefore

experimental infection, taking

PMN from

(5)

<:,60 -f 60

-20

2

Incubationtime (hours)

Figure1. Bactericidal capacity ofPMNobtained from sterile tissue cagefluid 14dafter implantation (v) compared with peritoneal exu-datePMNobtained after stimulation with glycogen 0.1% (o). The

phagocytic-bactericidal assaycontained inafinal volume of 1 ml PBS, 1 X 106 PMN, 2X 106 CFU of S.aureusWood46( ), 2X 106 CFU of S.faecalis (- -),

respectively,

and0.1 mlpooled guineapigserum. Eachpoint represents themeanof six determina-tions.Barsrepresent± 1 SD.

blood and from acute and chronic peritoneal exudates as

con-trols. Viability of tissue cage PMN at the time of their in vitro testing was

confirmed

by a Trypan Blue exclusion of 95-97%

aswell as by their

ability

toreduce NBT (see below). In addition, we determined the turnover rate (k) and time

_(tJ)

of homologous,

"'indium-labeled

blood PMN in the tissue cage. After intra-cardiac

injection,

the homologous, labeled PMN disappeared from the blood with a half-life time of 5.4 h. The turnoverrate

of PMN in the

tissue

cages was 0.17

h-',

and their turnover time, i.e., the average lifetime of a PMN in the tissue cage fluid, was 3.7 h. Thus, any functional or metabolic difference between blood and tissue cagePMN can notbe ascribedto

major

age

differences

of the cell

population

tested.

Evaluation of various

phagocytic and metabolic

activities

of tissue

cage PMN

Bactericidal activity and ingestion rate. Bactericidal activity

of

tissue cage PMN was tested on a

catalase-positive,

i.e., S. aureus

z

a-o

C

-c

"

0

-5

c

Wood

_46,

anda

catalase-negative microorganism,

_i.e.,

_S.faecalis.

Fig.

1 shows that tissuecagePMNwereunabletokill the

catalase-positive

microorganism,

whereas

_they

still

possessed

some

bac-terial

_activity

_against

a

_{catalase-negative}

strain.

_Particularly

striking

werethe results obtained with the

_{catalase-positive}

_strain

S. aureus Wood

_46,

where almost no

killing

could be dem-onstrated eitherat 60 orat 120min

_{despite optimal previous}

opsonization.

These results ledusto

_investigate

several

param-eters

_important

in the bactericidal

_activity

of normal PMN

(36,

37). Small,

but

_{significantly}

_{decreased, ingestion}

rates of

en-dotoxin-coated oil

_particles

wereobserved. We foundan

inges-tionrateof

75±27%

(508±184

cpm,

mean±SD,

n =

5)

for tissue

cagePMN

compared

with

100±14%

(672±92

cpm,

mean±SD,

n =

6)

for

peritoneal

exudate cells

_(P

<

_0.05).

Superoxide production. Fig.

2 shows the

superoxide

pro-ductionrateasafunction of

(a)

the concentration of the soluble

stimulus

PMA,

and

(b)

the

particulate stimulus,

opsonized

zy-mosan. With

_suboptimal

as well as with

_optimal

soluble and

particulate stimulation,

tissue cage PMN

produced

markedly

less

superoxide

than control PMN. The

superoxide

production

rate of chronic

_peritoneal

exudate cells was within the same

rangeas for

blood-derived

PMN. NBT slide testsshowed that

nearly

alltissuecagePMN reduced

NBT,

buttoalower

_degree

than

peritoneal

control PMN

(Table I).

Total

granular

enzyme content.

Inasmuch

as a

nonphago-cytosable

surface may stimulate

exocytosis

under certain

ex-perimental

conditions

(38),

we measured

MPO, lysozyme,

(3-glucuronidase,

and

_{B12 binding}

_protein

activities of tissuecage

PMN and

compared

them with

peritoneal

exudate PMN

_(Table

II).

Total MPO

activity

of tissue cage PMN was reduced to

38%, lysozyme

to

53%,

f3-glucuronidase

to

79%,

and

B12

binding

protein

to37% of control values. MPO slidetestsshowed that

mostof the tissuecagePMN hadalower

granule

contentthan control

PMN,

asestablished

by

ascoringsystem

showing

that 5%

_{(control 0%)}

ofPMN were

_negative,

44%

weakly positive

(control

3%),

and 51%

strongly positive (control 97%).

These results taken

_{together suggested}

that

partial

degranulation

had taken

place during

the

_implanted

foreign body.

5-z 2

0L

10

0

-a

E

4- 3-

2-1

-

0-0 1.0

Opsonizedzy

b

Figure2.(a)Theeffect of PMA concentration onthe

rateof

O2 generation by

tissue cagePMN

(v),

chronicperitonealexudatePMN(o), and bloodPMN

(-).PMAwasaddedatzerotime and O-production

wasmeasured under standard assay conditions(28).

2.0 3.0 4.0 Each pointrepresents themeanoftriplicate determi-(mosan (mg/ml) nations. Barsrepresent±1 SD. (b) Same assay as

de-scribed above but with increasing concentrations of

opsonized

zymosan.

1194 W. Zimmerli, P.D. Lew,andF.A. Waldvogel

0 0.01 0.1 0.5 2.0

[PMA]

I(g/ml) a

(6)

Table I. Reduction ofNBTbyPMN

Purified

from Tissue Cage

Fluid and fromaPeritonealExudate*

PMN reducing NBT

Degreeof Tissue Peritoneal

NBTreduction* cagePMN control PMN

None 7 0

Weak 63 35

Strong 30 65

*_The_slide_test_was_performed_as_{described by Newburger}_et_al._(29),

with PMNin suspensionin the presenceofPMA (I ug/ml).

tIndividualPMN wereexamined under direct microscopy bytwo

examinators inablind fashion and scoredaspreviously

de-scribed (30).

Degranulation.

To test the potential of tissue cage and peri-toneal exudate PMN to mobilize granule components, we in-cubated the cells with two different stimuli. The results are given in Table III. Tissue cage PMN had a decreased ability to de-granulate when compared with peritoneal exudate PMN. The

impaired

degranulation was observed both with a soluble (FMLP in the presence of cytochalasin B), as well as with a particulate (zymosan)

stimulus.

In vitro

interaction

offoreign body

material (Teflon

fibers)

with

PMN

In thatthese descriptive results did not allow us to determine how the PMN

defects

wereacquired, we tried to reproduce them in vitro by adding intact PMN suspended in

10%

guinea pig plasma to a nonphagocytosable surface:

I07

peritoneal PMN wereincubated in the presence and in the absence of 200 mg Teflon fibers for2 h to assess HMS activity, for 1 h to assess endpoint PMN

functions (phagocytic-bactericidal activity,

ox-idative metabolism, and total

residual

MPO

activity),

and for

30 min for

exocytosis. Fig.

3ashows that the interaction between

PMNanda

foreign body

ledtoa modest but

significantly

in-creased metabolic

activity during

the 2 h of incubationas

mea-sured

by

the HMS.

Furthermore, Fig.

3 b shows that the

men-tioned interaction ledtoselective release of the

secondary

granule

marker

B12

binding protein,

asshown

by

the

higher

release of

B12

binding protein

in the Teflon fibers

containing

medium in the absence ofa

significant

release of

_{f3-glucuronidase}

(<5%

of total

(3-glucuronidase

released for the whole

period

tested in both

_conditions).

When these

peritoneal

PMNwereeluted from the Teflon fibers with acid-citrate-dextrose and tested for their residual phagocytic-bactericidal

activity,

oxidative

metabolism,

and MPOcontent, the PMN showeda

significant

decrease in all theseparameterswhen compared with thesamePMN

pop-ulation incubated without Teflon

fibers,

but treated

identically

(Table IV). Thus,

in vitrocontact of exudate PMN with

non-phagocytosable

Teflon surfaces ledtoincreased HMS

activity

and

exocytosis,

and

produced

acell

population

withdecreased functional and metabolic residual activities,aspreviously dem-onstrated in the animal model.

Protective

effect

of

PMN

infusion

into tissue

cages

Becausetissue cages could be almost

uniformly

infected with 100

CFU

of S. aureus, andbecause tissuecage PMNhad been shown subsequentlytobe defective in overall killing,

ingestion

rate, oxidative metabolism, and various enzymatic

activities,

we investigated whether the veryhigh infection rateobtained with aninoculum of 100 CFU of S. aureuscouldbe reduced

by

the local administration of intact PMN. With the infusion of 1 X 107 PMN obtained from peripheral blood, the mean

number ofPMNin the tissuecagefluidwasincreased from 6.1

X 10 (5 X

104-2

X

106)

to2.3 X 106

(1

X

105-7.2

X

106)

per

ml of tissuecagefluid. With 1 X

107

tissuecage PMN the cell

countsincreased from 3 X

105

(5 X

104-5

X

105)

to 1.6 X 106

(9

X

l05-3.3

X

106)

PMN per ml of tissue cage fluid. Fresh

serum wasaddedattime 20 h because opsonic activity of tissue

cagefluidwasdecreasedatthis period of infection(21). Table

V shows the results of different protection regimens. Without

TableII. GranularEnzymatic Activity

of

TissueCagePMN inComparison withPeritonealExudatePMN

Granular enzymatic activity released by Triton X-100

PMNtype MPO Lysozyme fl-Glucuronidase B,2 bindingprotein

nmol/min per 106 PMN _Ag nmol/min per 106PMN pg/J06PMN

Tissuecage 55±7 (10) 0.9±0.13 (6) 1.1±0.07 (6) 20.1±1.1 (6)

Peritoneal 146±8 (5) 1.7±0.14 (6) 1.4±0.01 (6) 54.1±1.1 (6)

SignificanceP* <0.001 <0.001 <0.01 <0.001

MPOactivitywasdetermined inacontinuousassayfollowingthe change ofabsorbanceat450nmthataccompaniestheoxidation of o-dianisi-dine(31).Thelysozymeassaywasbasedonthechangeinlight absorption byasuspensionofMicrococcus

lysodeikticus,

when

lysozyme

actson

it (31).

_{fl-Glucuronidase}

activitywasassayedbymeasuringthe releaseofp-nitrophenolfromp-nitrophenyl

l-D-glucuronide

(31).

B,2

binding

proteinwasmeasuredby itsabilityto take upradioactive cobalamin fromthemedium(31). Mean±SD(n). *Statisticalanalysiswith the unpaired Student'st test.

(7)

TableIII.Extracellular Release ofB,2BindingProteinfrom

Tissue Cage PMNand Peritoneal ExudatePMNIncubated underDifferent Conditions*

B,2binding protein released fromt

Tissue cage Peritoneal Significance§

Stimulus PMN exudated PMN P

PBS 3.5±1.13 3.2±0.25 >0.05 CytochalasinB(5

lg/ml)

+FMLP

(10-7

M) 12.4±1.04 16.2±0.71 <0.01 Opsonized zymosan

(3 mg/ml) 7.5±0.63 10.4±0.21 <0.001

* 2.5 X

106

PMN/ml were preincubated at

370C

for 5 min in the presenceorabsenceof cytochalasin B. Atthistime the mentioned stimulus (FMLPorzymosan)wasadded and the incubation contin-ued for another 5 and 10min,respectively. The reactionwasstopped inice, thePMNwere sedimented at 1,000 rpm/10 min, and theB,2

bindingproteinwasdeterminedasdescribed in Methods(31). Lactic dehydrogenase releasewas<3%.

tPercent of total cellular

B,2

binding protein,

asdetermined

by lysis

with Triton X-100 (0.025% final concentration). Mean±SD from three determinations.

§ Statistical analysis with the unpaired Student's t test.

anyaddition ofPMN and serum,

implanted

tissue cages had

aninfection rate of 91%. The single local

injection of

I07 blood

PMNinto 2 mlof tissue cage fluid at the time of inoculation of 100 CFU of S. aureus did not

afford

anyprotection against

infection.

Incontrast, twoPMN infusions, one at the time of thebacterial inoculation and the other 20 h later were highly protective. The same regimen was significantly protective, when thefirst PMN

infusion

was

performed

3 h

after

the

bacterial

25-

20-

15-

10-5-

,,,,--.

2 15

b Incubationtime(m

challenge, but was ineffective when delayed by 6 h. A control experiment with two injections of

I07

tissuecage PMN provided no significant protection, despite a similar increase ofcell counts (Table V). Thus, implanted tissue cages could be protected from infection by appropriately timed peripheral blood PMN infusions in the first hours after bacterial challenge.

Discussion

Infections

arising

in close contact

with

a

foreign

body or

with

implanted

prosthetic

material are a vexing clinical problem, which to the present time has escaped adequate understanding and

effective

treatment

strategies (16).

Such

infections

are char-acterized

microbiologically

by the high prevalence of

staphy-lococci

(16,

39,40),

histologically

byacellular

infiltrate rich

in PMN,andclinically by the absence

of

spread

beyond

the

foreign

body in most cases,

its protracted

course, and its absence of

cure without removal of the

foreign material (13).

These

em-pirical

observations

have been well illustrated by the classic work of Elek and Conen (41), and by Noble

(42)

who dem-onstrated in a stitch abscess model that the initial bacterial inoculum could be as lowas 100 CFU

of

S. aureus, whereas 107

microorganisms injected subcutaneously

wouldnot

produce

any

lasting infection. Unfortunately,

themodel

developed by

these authors

did

not allow a

quantitative

evaluation of

the

microbiologic,

immunologic, and cellularevents

preceding,

or

associated with, a

foreign

body infection.

We have recently developedananimal model which leads Elek's and Noble's

observation

onestep further

by allowing

the

quantitation

of the

microbiologic,

of the fluid

phase,

and ofthe cellulareventsinduced

by

asterileoraninfected

foreign body

(21). It also reproduces the

main

clinical characteristics of

clinical

prosthetic infection,

including

the

occasional

emergence

of

a

resistant

organism after protracted antibiotic

treatment

(43).

Inasmuchasit is generally

accepted

that

staphylococcal

in-fections

arecontrolled

by

phagocytes,

we

postulated

that one

Figure 3. (a) Results of oxidation of

[1-'4]glucose

to

'4C02

by 10'PMNincubated in 50%plasma/PBS

so-lution with( )orwithout (---) 200 mg Teflon fibers,respectively,and 0.2

,Ci

of

[1-'4C]glucose.

Fig-ureshowscountsper minutetrappedasCO2vs.time. Eachpointis themeanof three determinations. Bars

represent+1 SD.Significances of difference between

"4C02

productionin absence and in presence, respec-.1 _tively,_are_P_<_0.05_at₇_min,_P<0.025at 10 and30

min, andP <0.01 at 15, 60, and 120 min (Student's relatedttest).(b)Extracellular release ofB,2 binding

protein from5 X

106

peritoneal exudatePMN incu-bated with( )orwithout(---)200 mg Teflon fibers. Resultsaregivenaspercent total enzyme

re-leased in themedium after 2, 15, and 30 min of in--

,-..

cubation at37°C.Eachpointrepresents themeanof

30 three determinations. Bars represent± 1 SD. The signif-in) icance of differences was P <

0.01

at 15minand P

<0.02at30 min(Student's relatedttest).

1196 W. Zimmerli, P. D. Lew, and F. A. Waldvogel

(8)

TableIV.Effect of Preincubation ofPMNwith

Nonphagocytosable

Teflon Fibers: Residual

Functional

and

Metabolic Activities

Incubation ofI _X107 peritoneal

exudate PMNat370CforIh

With 200 mg

Signifi-Assay Without Teflon Teflon cance* P

Phagocytic-bactericidal

test, % 67.1±3.6(6)t 12.1±9.9 (6) <0.001

Superoxide production, nmol

Oi/min

per106

PMN

Phorbol myristate acetate 10.3±1.3 (3) 5.4±1.3 (6) <0.001 Preopsonized zymosan 2.6±0.2 (3) 1.3±0.8 (3) <0.05

Myeloperoxidaseactivity, nmolo-dianisidine oxidized/minper106

PMN 73.4±6.8 (3) 49.4±6.8 (5) <0.001

The

phagocytic-bactericidal

activitywasdeterminedasdescribed in

Zimmerlietal.(21). The resultsarereportedaspercent killing of2 X 106 CFU S.aureus after 45-min incubation with 2 x 106PMN/ ml. Thesuperoxide productionwasmeasured with the method

de-scribed by Newburgeretal.(29).Theresultsarereportedasnmol

O/minper

106

PMNafterchallenge with the mentioned stimulus. MPO activitywasdeterminedinacontinuousassayfollowingthe

change of absorbanceat450nmthat accompanies the oxidation of

o-dianisidine(31). Resultsarereported asnmol o-dianisidine

oxi-dized/minper

106

PMN.

*Statistical analysiswith theunpaired Student'sttest

f Mean±SD

(n).

of the factors

leading

to

foreign body

infection could bealocally

acquired

phagocytic

defect.

In our previous study, we

dem-onstrated

that the

fluid-phase

bathing steriletissue cages pro-moted adequate

opsonization,

but that the PMN isolated from

sterile

tissue cage fluidwere unableto killS. aureusWood 46

adequately.

Inthe present

study,

wehave extendedtheseresults by

showing

that

this

phagocytic-bactericidal

defect(a) involves multiple steps in the

phagocytic

events, including

decreased

ability to ingest

particles, decreased

granule content, and de-creased

capacity

to mount a respiratory burst; (b) that it can

be

reproduced

in vitro by

incubation

of

peritoneal

PMNwith

nonphagocytosable

Teflon

fibers; and

(c)

thatit is of

pathogenic

importance, in that local infusion ofPMNprotects against tissue cage

infection.

Several ofour results deserve some comments. First, we

considered

that

peripheral

blood

PMNwere notfully

adequate

controls for ourtissue cage

studies,

because

peripheral

blood and exudate PMNhave been recentlyshown to differ in some

of

their

characteristics

(44).

By

repeated glycogen

injectionswe

were able to

produce chronic

peritoneal

exudates inthe absence

ofa

foreign

body, as

determined

by

cytological

criteria. Care

was taken to harvest these exudates at a time

interval

similar

tothe first

aspiration of

tissue cage

fluid,

andto

purify

the cells under the same

conditions.

We

therefore believe

that the

chronic

peritoneal

PMN

provided

an adequate

control for

the tissue cage PMN. In

addition,

we

tried

to rule out that

tissue

cage PMN were

defective

merely because

of aging

or

incipient cellular

death. Indeed, our turnover

studies showed

short

half-lives

for tissue cage PMN. NBT

slide

tests and Trypan Blue

exclusion

tests

confirmed

the

viability of

the cells

studied.

Moreover, the NBT

histochemical

technique

allowed us to

demonstrate

that

thePMN defect involved the majority of the cells in the cage and

did

notinvolve two

different

subpopulations.

Second,

the very poor

bactericidal activity of

tissue cage PMN

demonstrated

against S. aureus, but much less

dramatically

against

catalase-negative

S.

faecalis,

and the markedly

decreased

oxidative

metabolism of tissue

cage PMN upon

stimulation

with

opsonized

zymosan are

reminiscent

of early

observations

made on PMN from patients with chronic

granulomatous

disease

(36,

37). However, Lew et al. (30) have recently shown that, if PMN from a patient with a variant of chronic

granulomatous

disease generate even small

amounts

of

superoxide,

the PMN

can mount a

significant bactericidal

activity against

catalase-positive organisms.

Thus, the described defect, although striking, cannot be the sole

explanation

for the poor

bactericidal

activity of tissue cage PMN against S. aureus. We

postulate

that this

defect

is duetothe

combined synergistic

effect of partial defi-ciencies at various steps of the

phagocytic

process, and also includes

decreased

ingestion rates and

decreased

granule contents

aswell as the ability to

degranulate

further. MPO and

lysozyme

were markedly

decreased

and these enzymes have been shown

to

potentiate

the

bactericidal activity of phagocytes

(45). Because B12 binding protein, a

secondary

granule marker, was the most decreased

function,

our findings

confirm

the

observation

of Wright and Gallin (46) showing a selective release of

secondary

granules during

adherence

to foreign surfaces.

What is the cause of the defects found in tissue cage PMN? Several

possibilities

had to be

investigated,

the most obvious

onesbeinga

detrimental

factor present in tissue cage fluid and toxicto

incoming

PMN, anda

PMN-PMN interaction

between

residential

and incoming

phagocytic

cells. Neither the

prein-cubation

of

peritoneal

PMN with 50% tissue cage fluid nor the

coincubation

oftissue cagePMNwith

peritoneal

PMN

decreased

the

phagocytic

bactericidal activity of

these cells

(unpublished

observations).

We

therefore postulated

that the tissue cage PMN

population

was damaged by a

temporally

limited contact with the foreign bodytowhich they had been

attracted.

This damage

could

be

dueto frustrated

phagocytosis

along the

nonphagocytosable

sur-face, as

proposed

inanother

experimental

situation by Klock

andStossel (34), Klock and Bainton (35), and Wright and Gallin

(46).

These authors showed that adherence ofPMN to nylon fibers is

associated

with variable degrees of cell

spreading (47)

and

activation

of oxygen

metabolism

and

exocytosis

of

granules

(46) leadingto membrane damage (34) andto

defective

bac-tericidal activity (35).In order to detect whethersimilar defects could be

elicited

inoursystem, we

incubated

peritoneal

PMN

(9)

Table V. Local Treatment withInfusions ofPMNinto Tissue Cages

Local injection of PMN at time (h) of:* Significance

Localinjectionof 0.2 ml

0 3 6 20 serum at20h* Infectionrate Pt P2t

n %

I07blood PMN

- - - - _50/56=₉₁

+ - - - - 5/5 = 100 NS

+ - - - + 3/4 =75 NS

+ - - + + 1/21 = 5 <0.001

- + - + + 3/8 =38 <0.001

- - + + + 6/6 =100 NS

107 tissuecage PMN

+ - - + + 2/4 =50§ NS <0.05

The peripheral bloodPMNand thetissuecage PMNused in theseexperimentswerepurifiedasdescribed inMethods. Theserum wascollected andpooledaspreviouslydescribed(21).For eachprotection

experiment,

1X 107

peripheral

blood PMNwere

injected

in 0.2 ml of PBS.

Inthe controlexperiment, I _X ₁₀₇_tissue_{cage PMN} _{from several tissue}_cages_of_one_{guinea pig}_were

_injected

_{in 0.2}_ml_{of PBS.} _*_{Time after}

inoculation of 100 CFU of S. aureusWood 46intotissuecages. ZerotimemeansthatPMNwere

injected

2 minafterthebacterial inoculation.

tStatistical analysis: Fisher'sexact test.PI = treatment vs. notreatment;P2=treatmentwith tissuecage PMNvs. bloodPMNtreatment; NS =>0.05. §Inthisparticularexperiment,thesimultaneous infectionrateof fourunprotectedanimalswas50%. Statistical

comparison

was

donewith pooled results of control animals.

with Teflon fibers in plasma and foundamodest but significant

increase

in the HMS

activity of

PMN incubated with Teflon

fibers in

comparison

with the controls. These results indicatea

stimulation of

metabolic

activity by

adherence to

nonphago-cytosable material

(48).

In a second type of

experiments,

we

detected

significant

increasesinthe releaseofa

secondary

granule marker in presence

of

Teflon fibers.

In athird series

of

exper-iments,

wetested the

phagocytic-bactericidal

and

metabolic

ac-tivities of

PMNafter their incubation with Teflon

fibers,

which

had been

carefully

washedto exclude the presence of

phago-cytosable material. The results showed

that,

in the absence of any

phagocytosis,

the variousfunctional and metabolic

param-etersof fresh PMN were

markedly reduced,

an

additional

ar-gument in

favor

of

PMN-Teflon interaction

and PMN

stimu-lation.

These results taken together suggest that this

interaction

leadstopartial exhaustion ofPMN

function. Interestingly,

in-cubation periods

of only 1 hledto a

population of

cells with remarkably similar defects to thosefound in

vivo,

i.e.,

inthe tissue cagePMN.

PMNinfusionswere used in order todemonstratethatthe observed

phagocytic

defectwas

operational

in the

development

of

foreign

body

infection,

andthatits restoration would abrogate this high risk. Such techniques have been used previously by others in another experimentalmodel topreventpseudomonas pneumonia in neutropenic dogs (49). As the infusions led to PMN counts

frequently

found

under standard

conditions

in sterile

tissue

cage

fluids,

andastheinfusion of tissue cage fluid

PMNprovidedno

significant protection,

weconclude that the protection

afforded

byfresh blood PMN must be due to a

qual-itative rather than quantqual-itative improvement of thelocal phago-cytic activity. However, the protection obtained with fresh PMN

infusions

was

highly

dependentontheir timing, and delays be-yond 6 h led invariably to infection. Although our

findings

provide an explanation for the high susceptibility of foreign material toinfection, other factors must beoperational in its persistence, oneofthese being late loss of opsonic activity of the infected tissue cagefluid (21).

In conclusion,ourdata show that in a subcutaneous foreign body animal model, local PMN have low ingestion and bac-tericidal activities associated with low levels of granularenzymes

anddecreased abilitytomount arespiratory burst.Wepostulate, basedonourdata, that this defect is secondary to the prolonged stimulation of thePMNby the foreign, nonphagocytosable

sur-face. Although this defect probably explains partially the

high

susceptibility of prosthetic material to infection, we do notwant

to imply that this is the sole pathogenic mechanism. Recent studies by twoother groups have shown that foreign surfaces

irreversibly attach some microorganisms which then form a

glycocalyx (50-53). We postulate that the combination of a PMN defect and increased bacterial adherence act in concert tofavor infection on prosthetic material.

Acknowledgments

We thank Mrs. E. Hugglerfor technical assistanceandMrs. N. Deola

for secretarial help.

Thiswork wassupported byagrantfromtheSwiss NationalResearch

Foundation(grant no.3.869.0.81).

(10)

References

1.Charnley,J. 1972.Postoperative infection after total hip replace-mentwithspecial referencetoaircontaminationin theoperatingroom.

Clin. Orthop. Relat.Res. 87:167-187.

2. Sheppeard, H., D. K. Cleak, D. J. Ward, andB. T.O'Connor. 1980. A review ofearly mortality and morbidity in elderly patients

followingCharnley total hip replacement.Arch. Orthop. Trauma.Surg.

97:243-248.

3. Melton, L. J., R. N.Stauffer,E. Y. S. Chao, and D. M. Illstrup. 1982.Rates of total hip arthroplasty. N.Engl. J. Med. 307:1242-1245. 4.Courtiss,E.H., R. M.Goldwyn,and G. W.Anastasi. 1979.The

fate ofbreast implants with infectionsaround them. Plast. Reconstr.

Surg. 63:812-816.

5.Szilagyi, D.E., R. F.Smith,J. P. Elliott,and M. P. Vrandecic.

1972. Infection in arterial reconstruction with synthetic grafts. Ann.

Surg. 176:321-333.

6. Goldstone, J., and W. S. Moore. 1974. Infection in vascular prostheses. Am. J. Surg. 128:255-232.

7. Bernhard, V. M. 1975. Management of infected vascular prostheses.

Surg. Clin. N. Am.55:1411-1417.

8. Bhat,D.J.,V. A.Tellis,W.Kohlberg,B.Driscoll,and F. J.Veith.

1980. Managementof sepsisinvolving expandedpolytetrafluoroethylene grafts for hemodialysisaccess. Surgery. 87:445-450.

9.Georgiade, N.G.,E. H. King,W. A.Harris,J. H.Tenery, and B. A. Schlech. 1975. Effect of three proteinaceous foreign materials of

infectedandsubinfected wound models. Surgery. 77:569-576. 10.Rubin,J., W. A.Rogers, H. M. Taylor, D. Everett, B. F. Prowant, L. V. Fruto, and K. D. Nolph. 1980. Peritonitis during continuous

ambulatory peritoneal dialysis.Ann.Intern. Med.92:7-13.

11.Ladefoged,K., F. Efsen, J.Krogh-Christoffersen,and S.Jarnum.

1981. Long-term parenteral nutrition.II.Catheter-related complications.

Scand. J. Gastroenterol. 16:913-919.

12.Christensen, G. D., A. L. Bisno,J.T. Parisi, B. McLaughlin,

M.G. Hester, and R. W. Lutzer. 1982. Nosocomial septicemia due to multiply antibiotic-resistant staphylococcus epidermidis. Ann. Intern.

Med. 96:1-10.

13.Schimke, R.T., P. H. Black, V. H. Mark, and M. N. Swartz. 1961. Indolent staphylococcus albus or aureus bacteremia after

ven-triculoatriostomy.N.Engl. J.Med. 264:264-270.

14.Buchholz, H. W., R. A. Elson, E. Engelbrecht, H.Lodenkamper,

J.Rottger,and A. Siegel. 1981.Managementof deep infection oftotal

hip replacement. J. Bone Jt. Surg. Br. Vol. 63-B:342-353.

15.Gardner,P., J. R. Saffle, and S. C. Schoenbaum. 1977. Man-agement of prosthetic valve endocarditis. In Infections of Prosthetic

HeartValves and VascularGrafts.R. J.Duma,editor. UniversityPark Press, Baltimore. 123-142.

16.Dougherty,S.H., and R. L.Simmons. 1982. Infectionsinbionic

man: thepathobiology of infections inprostheticdevices. Curr. Prob. Surg. 19:219-319.

17. Lidwell, 0. M.,E. J. L.Lowbury,W.Whyte,R.Blowers, S.J. Stanley, andD.Lowe.1982.Effectofultracleanairinoperatingrooms ondeep sepsisin thejoint aftertotalhip ofknee replacement: a ran-domisedstudy. Br. Med. J.285:10-14.

18. Hunter,G., and D. Dandy. 1977. The naturalhistory ofthe

patient withaninfectedtotalhip replacement.J. Bone Jt.Surg.Br. Vol. 59-B:293-297.

19. Fitzgerald, R. H.,Jr., D. R. Nolan, D. M.Ilstrup, E. R. Van

Scoy, J. A. Washington II, and M. B. Coventry. 1977. Deepwound

sepsis folowingtotal hip arthroplasty. J. Bone Jt. Surg. Am. Vol. 59-A:847-855.

20.Burri,C. 1978. Die infizierte Osteosynthese. Helv. Chir. Acta.

45:545-552.

21. Zimmerli, W., F. A.Waldvogel,P.Vaudaux, and U. E. Nydegger. 1982. Pathogenesis of foreign bodyinfection: descriptionand

charac-teristicsofananimal model.J. Infect. Dis. 146:487-497.

22. Hisatsune, K., K. Kobayashi, S. Nozaki, and I. Muramatsu. 1978. Phagocytosis-stimulating activity of tuftsin analogs. Microbiol.

Immunol. 22:581-584.

23. Chenoweth,D. E., T. A. Lane,J. G. Rowe, and T. E. Hugli.

1980.Quantitative comparisonsofneutrophil chemotaxis in four animal

species. Clin. Immunol.Immunopathol. 15:525-535.

24.Hjorth, R.,A. K.Jonsson,and P. Vretblad. 1981. Arapidmethod

for purificationof humangranulocytes using Percoll: a comparison with dextransedimentation.J Immunol. Methods. 43:95-101.

25. Stossel, T. P., R.J.Field,J. D.Gitlin,C. A.Alper,and F. S. Rosen. 1975. Theopsonic fragmentof the thirdcomponentof human complement(C3). J. Exp.Med. 141:1329-1347.

26. Davies, W. A., and T. P. Stossel. 1981. External membrane proteinsofrabbit lungmacrophages. Arch. Biochem. Biophys.

206:190-197.

27. Forsgren, A., D.Schmeling,and0.Zettervall. 1977. Quantitative phagocytosis by human polymorphonuclear leucocytes: use of radio-labelled emulsions to measure the rate of phagocytosis. Immunology.

32:491-497.

28. Newburger, P. E., M. E. Chovaniec, and H. J. Cohen. 1980.

Activityand activation ofthe granulocyte superoxide-generating system.

Blood. 55:85-92.

29.Newburger, P. E., J. S. Pagano, J. S. Greenberger, A. Karpas,

andH. J.Cohen. 1980. Dissociationofopsonized particle phagocytosis andrespiratoryburstactivity in an Epstein-Barr virus-infected myeloid cell line.J. Cell. Biol. 85:549-557.

30. Lew, P. D., F. S. Southwick, T. P. Stossel, J. C. Whitin, E.

Simons,and H. J. Cohen. 1981. Avariant ofchronic granulomatous

disease: deficient oxidative metabolism dueto alow-affinity NADPH oxidase.N.Engl.J. Med. 305:1329-1333.

31.Babior,B.M., and H. J.Cohen. 1981.Measurementofneutrophil

function:phagocytosis, degranulation, the respiratory burst and bacterial killing. In Leukocyte function. M. J. Cline, editor. Churchill-Livingstone

Co.,New York. 1st edition. 1-38.

32.Kaplow,L.S. 1965. Simplified myeloperoxidasestain using

ben-zidine dihydrochloride. Blood.26:215-219.

33. Cohen,A.B.,M. Rossi,D.Geczy,and L.Knight. 1982.

Neu-trophilturnoverinnormalrabbitlungs.J. Clin. Invest.69:794-798. 34. Klock,J. C.,and T. P. Stossel. 1977. Detection, pathogenesis

andprevention ofdamage to human granulocytes caused by interaction

withnylonwoolfiber.J. Clin.Invest. 60:1183-1190.

35. Klock, J.C.,and D. F.Bainton. 1976. Degranulationand ab-normal bactericidal function of granulocytes procured by reversible

adhesiontonylon wool. Blood.48:149-161.

36.Mandell, G. L.,and E. W. Hook. 1969.Leukocyte bactericidal activity in chronic granulomatous disease: correlation of bacterial hy-drogen peroxide productionandsusceptibilitytointracellular killing.J. Bacteriol. 100:531-532.

37. Kaplan, E. L., T. Laxdal, and P. G. Quie. 1968. Studies of polymorphonuclearleukocytesfrompatientswithchronic granulomatous disease of childhood: bactericidalcapacity for streptococci. Pediatrics.

41:591-599.

38.Goldstein,I.M., and G.Weissmann. 1979.Nonphagocytic stim-ulation ofhumanpolymorphonuclearleukocytes: rolesoftheplasma

membrane.Semin. Hematol. 16:175-187.

39.Ericson,C.,L.Lindgren,andL.Lindberg. 1973. Cloxacillinin

(11)

the prophylaxis ofpostoperativeinfections ofthe hip. J. Bone Jt. Surg. Am. Vol.55-A:808-813.

40. Fitzgerald, R. H., Jr., L. F. A. Peterson, J. A.Washington II, R. E. VanScoy, and M. B. Coventry. 1973. Bacterial colonization of wounds and sepsis in total hip arthroplasty. J. Bone Jt. Surg. Am. Vol. 55-A:1242-1250.

41. Elek, S. D., and P. E. Conen. 1957. The virulence of staphy-lococcuspyrogenesforman: astudy of the problems ofwound infection. Br. J. Exp. Pathol. 38:573-586.

42. Noble, W. C. 1965. The production ofsubcutaneous staphy-lococcalskinlesions in mice. Br. J. Exp.Pathol. 46:254-262.

43. Tshefu, K., W. Zimmerli, and F. A. Waldvogel. 1983. Short termrifampin administration intheprevention/eradication of foreign

bodyinfection.Rev. Infect.Dis. 5(Suppl.):S474-S480.

44. Van Epps, D. E., and M. L.Garcia. 1980. Enhancement of

neutrophilfunctionas a resultofprior exposure tochemotactic factor.

J.Clin.Invest. 66:167-175.

45. Root, R. K., and M. S. Cohen. 1981. The microbicidal

mech-anismsofhumanneutrophilsandeosinophils.Rev.Infect.Dis. 3:565-598.

46. Wright, D. G., and J. I.Gallin. 1979. Secretory responses of human neutrophils: Exocytosis of specific(secondary) granules by human neutrophils during adherence in vitro and during exudation in vivo. J. Immunol. 123:285-294.

47.Wright, D. G., and J. C. Klock. 1979. Functionalchanges in

neutrophils collected by filtration leukapheresis and their relationship

tocellular events that occurduringadherenceofneutrophilstonylon

fibers.Exp. Hematol. 7(Suppl.):11-23.

48.Tedesco, F., S. Trani,M. R. Soranzo, andP. Patriarca. 1975. Stimulation ofglucose oxidation in human polymorphonuclear

leu-cocytes by C3-sepharose and solubleC567. FEBS(Fed.Eur.Biochem.

Soc)Lett. 51:232-236.

49. Dale, D. C., H. Y. Reynolds, J. E. Pennington, R. J. Elin, T. W.Pitts, and R. G. Graw, Jr. 1974. Granulocyte transfusion therapy

ofexperimentalpseudomonaspneumonia. J. Clin. Invest. 54:664-671. 50. Costeron,J. W. 1982.Effect of antibioticsonadherentbacteria.

InActionof Antibiotics in Patients.L. D.Sabath,editor. Hans Huber, Bern. 160-176.

51. Costerton, J. W., G. G. Geesey, and K. J.Cheng. 1978. How

bacteria stick. Sci.Am. 238:86-96.

52. Peters, G., R.Locci,andG.Pulverer. 1981.Microbial colonization ofprosthetic devices. II. Scanningelectronmicroscopy of naturally in-fected intravenouscatheters. Zentralbil.Bakteriol. (B) 173:293-299.

53. Peters, G., R. Locci, and G. Pulverer. 1982.Adherence and

growthof coagulase-negative staphylococcionsurfaces of intravenous

catheters. J.Infect.Dis. 146:479-482.