The effects of monoclonal antibodies against iC3b receptors in mice with experimentally induced disseminated candidiasis

The

effects of

monoclonal

antibodies against

iC3b

receptors

in mice with

experimentally induced

disseminated

candidiasis

K. H. LEE,M. S. YOON & W. H. CHUN DepartmentofDermatology andInstitutefor Immunologyand ImmunologicalDiseases,

Yonsei UniversityCollege of Medicine, Seoul,Korea

SUMMARY

CR3(iC3b receptor),composed of CD1 lb/CD18, isa

P2

integrin. A protein that shares antigenic and structural homology with the at-chain of CD1 lb/CD 18 has been isolated fromthe surface of Candida albicans. This molecule is thought to be essential in the pathogenesis of disseminated candidiasis. To evaluate the effects of anti-iC3b receptor antibodies on adhesionbetween human dermalmicrovascular endothelial cells(HDMEC) and C. albicans, and in treatment ofcandidal infection, a binding assay of C. albicans to cultured HDMECwas performed in vitro. An anti-iC3b receptor-specific monoclonal antibody was administered to mice infected with C. albicans. Themice were monitored for mortality and renal involvement by culture and histopathological findings. Flow cytometric analysis demonstrated surface expression of iC3b receptor on C.

albicans. Theadherenceof C. albicansto HDMECwassignificantly decreased by treatmentwith

anti-iC3b receptor antibodies. Anti-iC3b receptor antibodies significantly increased the survival

time and rate while lowering the renal fungal burden. The iC3b receptors are involved in the adherence of C. albicans to vascular endothelial cells and are likely to be involved in the pathogenesisofdisseminatedcandidiasis. Theincreased survivalin mice infected with C.albicans

after treatmentwith anti-iC3b receptorantibodies indicates that thismodality may bebeneficial forfuture developmentofa new therapy forcandidiasis.

INTRODUCTION

Adherence of a pathogen to host tissue is essential in the

pathogenesis ofinfectious disease and presumablytakesplace

inall forms of candidiasis. Studiesconcerningthemodification

orprevention ofinfection in relation toadhesion mechanisms have been performed in animal and human

models."2

The virulence of a species of Candida can be correlated with its

ability to adhereto various cell types, with the mostvirulent C. albicans beingmost adherentto

epithelial

cells. In animal models of invasive disease, the pathogenicities of candidal

strains in vivocorrelate directly with yeast adhesion in vitro.3

Endothelial attachment and subsequentpenetrationare requi-site stages in the induction ofhaematogenously disseminated candidiasis.

Receptors [complement receptor (CR)1, CR2 and CR3]

for C3 degradation fragments (C3b, iC3b, C3dg) are present

Received 14 January 1997; revised 14 May 1997; accepted

14May1997.

Abbreviations: CR, complement receptor; HDMEC, human

dermal microvascular endothelialcell; HUVEC,humanumbilicalvein endothelial cell.

Correspondence: Dr K. H. Lee, Department of Dermatology,

Yonsei University College of Medicine, C.P.O. Box 8044, Seoul,

Korea.

onmanyhuman cellsoperativein theimmuneand

inflamma-toryhostdefenceprocessand may becriticalin the attachment ofinvading micro-organisms.3 C3-bindingstructures are not

specific for mammalian cells ingeneralbut can also befound inmicro-organisms pathogenicfor humans. CR3 is presenton

thesurfaceofmammalianneutrophils, monocytesand

phago-cytes and has been known asCDllb/CD18, Mol,

Mac-i,

or

iC3b receptor. It is composed ofan ao-chain of 165000MW and a 95000 MW

P-chain

common to all leucocytes. There

are iC3b receptors on the surface of C. albicans which share

homology with the a-chain of the

neutrophilic

receptors for

iC3b.-6

Westernblottingandimmunoprecipitation have

con-firmed the similar relative molecular weights of CR3 on the human neutrophil and the iC3b receptors isolated from C.

albicans.6.7

Since indisseminated candidiasis the

fungus

isdeliveredto

target organs through the vascular system, targets for

adher-ence ofblood-borne

fungi

ultimately

include the endothelial cell.Experimentsonadhesion of C. albicanstocultured human umbilical vein endothelial cells

(HUVEC)

demonstrate that

increasing glucoseconcentrations increase adhesion and

mono-clonal antibodies (mAb) recognizing epitopes on neutrophil

CR3 inhibit glucose-enhanced adhesion of C. albicans to

HUVEC.8

However, the exact role of iC3b receptors in

haematogenously disseminated candidiasis in humans hasnot

beenelucidated.

1

1997BlackwellScience Ltd

We assessed the role of iC3b receptors on C. albicans in theadhesion of yeast to cultured human dermal microvascular endothelial cells (HDMEC), and the inhibition of this adhesion by mAb against iC3b receptors. In addition, we observed the effects of intravenous injection of anti-iC3b receptor mAb with respect tomortality andlong-terminfection in mice withexperimentally induceddisseminated candidiasis.

MATERIALSANDMETHODS Organisms

One randomly selected pathogenic strain of C. albicans (American Type Culture Collection, Rockville, MD; ATCC 44808) recovered from human blood was used for these experiments. Yeast cells were grown to log-phase in Sabouraud'sbroth, asdeterminedbycolony counts and spec-trophotometric measurement of OD at 420 nm, and were then frozen at -700until use. On theevening before the experiment, aliquots of frozen yeasts were inoculated into RPMI-1640 media (Gibco Laboratories, Grand Island, NY). After attaining log-phase growth, the yeasts were pelleted, washed threetimes with phosphate-buffered saline (PBS) and diluted

spectrophotometrically to anOD of 0 5 at420nm.

Animals

BALB/c mice (Daehan Laboratory Animal Research Centre, Seoul, Korea) weighing around 25 g were used for all experiments.

Monoclonalantibodies

FourmAb directed against epitopes on a subunit of CR3 were Mol (mouse IgM, Coulter Immunology, Hialeah, FL), 44

(mouse IgGl, R&D Systems, Minneapolis, MN), Leu 5 (mouse IgG2a, Becton Dickinson, San Jose, CA), andM1/70. M1/70 (rat IgG2b) was purchased as a hybridoma (ATCC TIB 128) and was purified from supernatant by affinity chromatography on protein G-Sepharose (Hitrap Protein G

column, Pharmacia Biotech, Uppsala, Sweden). The mAb ECCD-2 (anti-E-cadherin, rat IgG2a) was obtained from M. Takeichi (Kyoto University, Kyoto, Japan). ECCD-2 was purified from hybridoma supernatants by protein G-Sepharose affinity chromatography. MEM 48 (mouseIgGI,

R&D Systems, Minneapolis, MN) is a mAb directed against the common ,B-chain ofCR3, leucocyte function associated

antigen-l (LFA-1).

Flowcytometricanalysis

Fifty million colony-forming units (CFU)/ml of C. albicans

werewashed in PBS with 0-5% bovine serum albumin (BSA), counted and aliquotted for staining. Primary antibody at the appropriate dilution was added to each tube. Either an isotype control mAborPBSwithout antibody was used as a first-step control with identical results. The cells were incubated for

30minonice, washed, and second-step antibody, diluted 1/20 in PBSwith 05% BSA, was added. After another 30 min of

incubation, the cells were washed and resuspended in 0-5 ml

PBSwith05% BSA.Propidiumiodide was added immediately before flow cytometric analysis to identify dead cells. Fluorescence was examined by a Becton Dickinson FACStar flow cytometer(Becton Dickinson, MountainView, CA) and

expressed aslogmean channelfluorescence.

Isolation and cultureofHDMEC

HDMEC were isolated from human neonatal foreskins by

trypsinization and Percoll gradient centrifugationasdescribed

previously.9"'0

Briefly, foreskins were cut into small pieces,

treated with 0-3% trypsin (Sigma Chemical Co., St Louis,

MD), and 1% ethylenediamine tetraacetic acid (EDTA;Sigma) and individual segments were compressed with a scalpel bladetoexpress microvascular fragments. The microvascular

segments were layered onto a 35% Percoll (Pharmacia AB,

Sweden) gradient in Hank's balanced salt solution (HBSS) and spun at 400g for 15min at room temperature. The fraction with a density less than 1-048 g/mi was applied to

gelatin (Sigma) -precoated tissue-culture dishes and cultured in endothelial basal media (Clonetics Corp., San Diego, CA) withepidermal growth factor 1 ng/ml (Clonetics),

hydrocorti-soneacetate1

gg/ml

(Sigma), dibutyryl cyclicAMP5xI0-5M

(Sigma), penicillin 100U/ml, streptomycin 100

gg/ml,

ampho-tericin B 250 jg/ml (Sigma), and 30% human serum (Irvine Scientific, Santa Ana, CA). The resulting cell cultures were free ofcontaminating fibroblasts asassessedby expression of

CD31 (Fig. 1) and uptake of acetylated low-density lipo-proteins. Experiments were conducted with endothelial cells

atpassages2-6.

Candida albicans-HDMEC adherenceassay

HDMEC were plated in gelatin-coated 96-well flat-bottomed culture plates. An isolate of C. albicans was grown in the

RPMI medium, labelledwith [35S]methionine, washed

exten-sively in PBS and resuspended in HBSS with calcium and magnesium, to which was added 1% BSA

(BSA-HBSS+).

Yeast cellswerethen heldat40 topreventgerminationbefore

incubation with HDMEC (5% germ tubes present for C. albicans). Then 4x104 CFU were added to each well of

confluentHDMEC monolayer,and incubated for 1hrat37°.

Forquantification ofnon-specific bindings, a 100-foldexcess ofunlabelledyeastcellswereaddedtolabelledyeastcells.The

supernatantwas removedfromeach well and themonolayers washed twice with BSA-HBSS+ + to remove non-adherent

yeasts.Thesewerecounted inascintillationcounter(Beckman Instruments, Inc., Palo Ato, CA). Adherent yeast cells and HDMEC monolayers were removed with 1% Triton-X

(Sigma), then solubilized in 0-5M NaOH and counted in a

200

100 101 102 103

FL1

Figure1. Flow-cytometric analysis showing the expression ofCD31 onHDMEC.

©1997 BlackwellScience Ltd,Immunology, 92, 104-1 10

P-scintillation

counter. Thepercentage binding of C. albicans

wascalculatedas follows:

C.p.m. (adherent yeasts-non-specific binding)

100

C.p.m. (totalyeastsadded-non-specific binding)

Inhibition ofadhesion with mAb

C. albicans was grown in RPMI medium and was then

preincubated with 100pl of mAb to ac-subunit ofCR3 for 45min. 100g1 of20

gg/ml

ofmAbwereused. After

preincu-bation withantibodies, the adherence assaywasperformedas

described above in the continuous presenceof antibodies. All

the individual experiments were performed three times

independently. Experimental infection

Mice were divided into three experimental treatment groups for the assessment of anti-iC3b receptor antibody on the

resulting prevention of systemic candidiasis. The threegroups were: Group I, controls, received the vehicle for the mAb

(0-5% human serum albumin) and was inoculated with one dose of 106 CFU of C. albicans grown in RPMI-1640 per mouseintothe lateral tailveins ofagroupof 45normal mice;

Group II,mAb Ml/70, beginning before infection received

mAb M1/70 2mg/kg infused intravenously once per day for 5days (day0today 5). Group III, isotype control, beginning

before infection received mAb ECCD-2 2mg/kginfused

intra-venously once perday for 7 days (day 0today 5);The fourth groupreceived only equivalentamountsofmAbM1/70atthe same time. All animals were monitored for 30 days, and

mortality was recorded. Two groups of 20 mice each were

studied, an only C. albicans-inoculated group and a group

treated anti-M1/70mAb and inoculated with C. albicans as

previously described. On days 0, 2, 4 and 6, five mice per experimental group were selected at random and killed by

cervical dislocation. Thekidneysof each of these animalswere

removed aseptically,transferred into sterilepolyethylene bags,

and homogenized in 5ml of sterile saline. Samples were removed from eachhomogenate, seriallydilutedin0-9%NaCl, platedontoSabouraud dextroseagarplates,and incubatedat

250 for 16 hr. The colonies were then enumerated, and CFU werecalculatedpergramof eachorgan. Allexperimentswere

performed in triplicate. To document the administration of

M1/70mAb sufficientto produce antibodyexcess, samples of serum were analysed for the presence of residual M1/70 antibody. Bloodwascollected fromtheorbital,venousplexus. This wasassessedby indirect immunofluorescence analysis in

which C. albicanswere incubated in serumfor 30minat250,

andthen,afterwashing, incubatedin fluorescein isothiocyan-ate (FITC)-conjugated goat anti-rat immunoglobulin for an additional 30minat250. Antibody bindingto C. albicanswas

quantified byflowcytometry. Formalin-fixedportions of

kid-neys obtained at2, 4 and 6 days were stained with Periodic

acid-Schiff reagent(PAS) to searchfor fungi inkidneys.

Statisticalanalysis

Alldataareexpressedas mean+standarderror.Alldifferences in survival wereanalysed bythe Wilcoxon rank sumtest and Kruskal-Wallistest. Product limit methodwasusedtoanalyse

survival data andrepeated ANOVA wereappliedtocompare

themeanCFUpermeankidney weight.Statisticalsignificance wasdefined as aP-value of <0 05.

RESULTS Expression of iC3breceptoronC.albicans

The iC3b receptor expression was observed by

immunofluo-rescenceflowcytometryfromC.albicanspretreatedwith

anti-iC3b receptor antibody purified from culture supernatant of ATCC MI/70 antibody by affinitychromatography (Fig.2).

Effect of anti-iC3breceptorantibodyonadhesion of C. albicans

toHDMEC

Preliminarystudiesweredonetocomparetherateof adhesion

between HDMEC and C. albicansaccordingtothe number of C. albicans and elapsed time. The binding of 5x105 CFU,

1 x 106CFU and 2x106CFUof C. albicanstoHDMECwere

25-1 +3-8%, 29-9+222% and 283 +1-5%, respectively. The

adherences 30min, 1 hrand 2 hrafterculturewere8 1+19%,

241+37% and 25-4+4-9%. Theadherence increased signifi-cantly after 1 hr of culture. Furtherstudies were carried out

under culture conditions with 106 CFU of C. albicans being

cultured for 1hr.

The adhesion of C. albicans without any prior treatment to HDMEC was 240+336%. Adherences were 25-4+554%,

27 9+1 8%, 26-3+0-3% and 16 5+2-2%whenC. albicanswas treated before inoculation with rat IgG2b,mAb ECCD-2,

anti-CD18,andM1/70 mAb, respectively (Fig. 3).

Effect ofanti-iC3breceptorantibodyonmortalityin micewith experimentallyinduceddisseminatedcandidiasis

Preliminarystudieswereperformedtodeterminethemortality

ratesofBALB/cmiceaccordingtothe numberof C. albicans.

All miceinjected with 5x106CFU of C. albicansculturedin

RPMI-1640 mediaexpired within 1 day.At 1 x106 CFUand

5x105 CFU, the average survival durations were 27+0-6

days and 43+0-6 days, respectively. Inoculation was done with 1 x106 CFU of C. albicans to determine mortality and

effects of the anti-iC3b receptor antibodies. Group I was inoculated withonlyC. albicans. GroupIIwaspretreatedwith

200 2C MAb Mo-I .., I..: 10° 101 102 103 FLi Leu-15 )0 101 102 103 FLI MAbM1/7O 100 101 102 103 FL1

Figure2. Expressionof iC3b receptoronthe surface of C. albicans. Candida albicans was incubated with anti-iC3b receptor mAb anti-Mol, Leu5,44andMI/70andanalysed byfluorescence-activated cell sorter. The expression of iC3b receptors can be observed on the surface of C. albicans.

C 1997Blackwell ScienceLtd, Immunology, 92,104-110

0 14" o-T- "4 100 101 102 103 FLi )o --0 MAb44 .

!"IXPM

"...

V /v 4 M AD. t%-b

Effects

No Ab Irrelevant mAb Anti-CD18 Anti-iC3bR -d -I 0 10 20 %binding 30

Figure 3. The effects ofanti-iC3breceptorantibodiesonthe adhesion

of C. albicans to HDMEC. Candida albicans was pretreated with irrelevant antibody ECCD-2, mAbanti-CD18 against the 1-chain of iC3breceptor,andM1/70against the a-chain of iC3breceptor,added inconcentrations of 20jg/ml at370 for 45 min, and incubated with HDMEC monolayer at 37° for 1hr. Results show the mean percentage+SD ofquadruplicate microtitre wells.

Ml/70mAb 1 hr prior to inoculation with C. albicans and

Group III was pretreated with mAb ECCD-2 1hr prior to

inoculation with C. albicansas control. Immunofluorescence studies revealed strong fluorescence on the surface of C. albicans collected from the serum of mice 6 days after the initialinoculation of M1/70 mAb. Fluorescence wasobserved on the surface of C. albicans by immunofluorescent flow

cytometry from serum collected 2, 4 and 6 days after inoculation.

GroupIVwasinjectedwithonly anti-iC3b antibodiesand all 45 micewerealive 30days afterinjection. However, all 45 mice in bothGroupI andGroupIII expired between 2 and5

100 80 - 60 ._,-40 20 0

Table1. Effectof anti-iC3breceptorantibodyonduration of survival

inmice withexperimentally induced disseminated candidiasis

No.of Survival duration

Inoculationgroup animals (days)

C. albicansonly* 45 2-7+0 7

C. albicans+mAb MI/70t 45 5-3+1 8

C. albicans+control

mAbJ

45 34+009

mAbM1/70only§ 45 >30

*GroupIwasinoculatedwithonly C.albicans.

tGroup II was inoculated with C. albicans and intravenously

injected with anti-iC3b receptor antibody M1/70 at 2 mg/kg of

concentration.

tGroup III was intravenously injected with mAb ECCD-2 at 2mg/kg of concentration and inoculated withC. albicans.

§Group IV was injected with only anti-iC3b receptor antibody

M1/70.

days after inoculation. All mice in Group IIexpired between 2 and 10 days after inoculation. However,the mortality rate inGroupII at 4days after inoculationwas 311%,which was

significantly lower than the mortality rates at this time in

Groups I and III (P=00001, Fig. 4). The average survival durationsforGroupsI, IIandIIIwere2 7 + 0 7days, 5 3+18 days,and 34+09 days, respectively, (Table 1). These differ-encesweresignificant.White blood cell counts andmorphology

of mouseneutrophilswere notdifferent between the irrelevant

mAb-treated group and theM1/70-treatedgroup.

Effect of anti-iC3b receptorantibodyonrenalinvasion of C. albicans in mice withexperimentally induceddisseminated

candidiasis

To evaluate the effects of anti-iC3b receptor antibody in disseminatedcandidiasis,micewereinjectedintravenouslywith 5 x105 CFU ofC. albicanscultured in RPMI-1640. Themice

were killedatrandomand the kidneyswere removed,

homo-genized and cultured on a Sabouraud dextrose agar plate. 10r 0) a) 'D C) J 0 E 0) cl 0) 0 -j 0 2 4 Days 8 10 9 Group \ GroupII 8 . 7 6 5 2 4 Days post-infection 6

Figure 4. Effect of anti-iC3breceptor antibodyon mortality of mice

with experimentally induced disseminated candidiasis. Group Iwas

inoculated with only C. albicans (0). Group IIwasinoculated with C. albicansandintravenouslyinjected with anti-iC3breceptorantibody M1/70 at 2 mg/kg (A). Group III wasintravenously injected with mAb ECCD-2at2mg/kg and inoculated withC.albicans(R).Group IV was injected with only anti-iC3b receptor antibody M1/70 (V). Themortalityandsurvivalduration of the fourgroups werecompared.

Figure5. Effect ofanti-iC3breceptorantibody onrenal invasion of

mice with experimentally induced disseminated candidiasis. GroupI

wasinoculated withonly C. albicans. Group IIwasinoculated with

C. albicansandintravenously injectedwithanti-iC3breceptorantibody M1/70at2mg/kg. Renal specimenswereharvestedevery2days for 6 days to evaluate the invasion of kidneys. A quantitative culture assaywasperformed three times foreachmouse. TheaverageCFU of C. albicans/gofkidneywerecompared.

(a)

(b]

..

;7

_4 l_s X. ~_ _ .

Figure6. (a)Histopathological findings ofkidney of mice with

expernmentally

induceddisseminated candidiasis and treated with

anti-iC~b receptor antibody (PAS stainx40). (b) Higher magnification (x 400). Group Iwasinoculated with only C.albicans.

Group IIwasinoculated withC. albicans andintravenouslyinjectedwithanti-iC~breceptorantibody

Ml/70

at2mg/kg. Renal

specimens were harvested every2 days for 6 days toIevaluate the invasion ofkidneys. Specimens were paraffin-embedded and

stained withperiodicSchiffreagent. Thisstainingrevealedmanyfungalsporesandhyphaeintheglomerulusatthe seconddayof infection inGroupI,whereasGroupIIshowedonlyfewfungalsporesandhyphae. G-1-2, Group I-day 2;G-I14,Group I-day 4;

G-I-6, GroupI-day 6;G-1I-2,Group II-day 2; G-11-4, Group

Il-day

4; G-II-6, Group

Il-day

6.

in disseminated

On the days 2, 4 and 6 of infection, Group I had

3 0+0-2x 109CFU, 3-3+1-8x109CFU, 32+O1 x107CFU

of C. albicans, respectively, in 1g ofkidney. By comparison, Group II had [*6+1 3x106 CFU, 28+l1-2x 105 CFU,

1-9+1-2x 105 CFU of C. albicans, respectively, in 1 g of

kidney. Thesedifferencesinrenalinvolvementwerestatistically

significant (P=0-0001, Fig. 5).

Periodic acid-Schiff staining of the extracted renal

speci-mens revealed many fungal spores and hyphae in the renal

medulla on day 2 ofinfection inGroup I. Group II showed

onlya fewfungal spores and hyphae on day 2 of infection.

GroupIshowedheavy infiltrationofinflammatorycellsatthe sixthdayofinfection(Fig. 6).

DISCUSSION

Theimportance of the adherence of Candidain the

pathogen-esis ofdisseminated disease is increasingly recognized. There

have beenreportsthatpathogenicityofcandidal strains in vivo

correlate directly with yeast adhesion in vitro.' However,

studiesonfungus-to-hostcell adhesionmechanisms have been

relatively sparse. It is possible to categorize these adhesion

mechanisms by considering the components ofeach

ligand-receptor.'1 The first category includes protein-protein inter-actions such asthe iC3b receptor on C. albicans. The second

category includes interactions in which a surface Candida

proteinwithlectinactivity recognizessugarresiduesassociated

with the host-cell plasma membrane. The third category

involves acarbohydrate ligand, mannan, onthe Candida cell surface that is recognizedby an unknown host-cell receptor.

Complement fragments adheremore readily to Candida than

toendothelial cells in vitro.4'2CR3onneutrophilsis involved inrecognition of iC3bonthesurface ofmicro-organisms and

subsequent phagocytosis. TheiC3breceptoronthesurfaceof C. albicans hasrecentlybeen isolated andcharacterized.'3The

iC3b receptor in Candida shares antigenic and structural homologywith CR3 onneutrophils.46

As we, and others, have observed the expression of iC3b

receptoris usually below 50%dependingon thetypeof mAb used, condition of culture medium, species or strains of

Candidaused, etc.56'8"12The low expression in ourstudy was

due to the RPMI medium with low glucose concentration.

When theglucoseconcentrationwasincreased, the expression of iC3b receptor was up-regulated (unpublished results).

However, the differenceinthe expression could be attributed

to thetypeofmAbused.

The relationship between hyperglycaemia and candidal infection has been well documented.14 However, the exact mechanism by which sensitivity to candidal infection is

increased isunknown.There havebeenreportsthat expression ofiC3breceptoronCandida andadhesiontoculturedHUVEC correlate with glucose concentrations.56 Moreover, adhesion to cultured HUVEC is suppressed by 50-80% with mAb directedagainst theac-chain of the iC3breceptor.8

As there are marked differences between the endothelial cells ofvarious

vessels,9"10"15

andasC. albicans infectsthe skin andmucousmembranes easily,weperformedadhesion experi-mentsusing culturedHDMEC isolated from skin. Ourresults show that adherence of C. albicans correlates with reaction

time and number of organisms. But there was a marked

decrease when anti-iC3b receptor mAb were added. These

findingsaresimilar toprevious studies usingHUVEC.8

Intercellular adhesionmolecule-l (ICAM-1),whichactsas

a ligand for neutrophil CR3, has no role in the adhesion of iC3breceptoron C. albicans.8"6 Recently, the host ligandfor

the iC3b receptor on C. albicans has been characterized as

iC3b."' C3 is synthesized from endothelial cells'8 and it is

plausibletoassumethatC3 isdegraded byproteolyticenzymes

in the serum orcell with the resultant iC3battaching to the endothelial surface and acting as a ligand for the iC3b

reetr7,18,19

receptor.7""

In the present study, binding of C. albicans to HDMEC

was partially inhibited by mAb M1/70. This would be

explainedbythefindingof 50000, 100 000, 130 000 and 165 000 MWproteins inextractsofC. albicansreacting with anti-iC3b

receptor antibodies in immunoblotting and

immunoprecipit-ation studies.6'7 Hence, thecombination of thisvariability of the iC3b receptorepitopesand specificity of each mAbmight

have resulted in the partial inhibition as seeninour study. We confirmed that the iC3b receptor on C. albicans is essential for adhesiontoculturedHDMEC. Thusweassumed that prevention of the yeast-to-host cell adhesion could block thedissemination of Candidato surrounding tissue. haemato-genously disseminated candidiasis was induced in mice by intravenous injection with C. albicans. The mortality rate of the group injected with anti-iC3b receptor mAbwas signifi-cantly lower than that of the normal control. The 50% survival

rateincreasedsignificantly. Theamountofyeastand the extent

of invasion of renal tissue was significantly less. The affinity

constantforthe iC3breceptoris 2-1+0 5x106L/M for human neutrophils20 and virtually identical to that of C. albicans.5 However, Candida possess 2x106 iC3b receptors per yeast

which is nearly 100 times higher than neutrophils.5 The increased survivalrateinmicetreated withanti-iC3breceptor

antibodies indicatesthat thismodality mightbe beneficialfor future development of a new therapy for disseminated

candidiasis.

TheiC3breceptor on Candida ishomologous in srtucture to human CR3, however, it is not the same molecule. There isno doubt that antibodies directly against the Candida iC3b

receptorwouldbelikelytobindbetterto Candida andtohave betterinhibitory effects. However, at this time, wecould not

obtain the purifiedcandidal iC3b receptor anditsmAb. It is desirable that similar experiments using the above antibody

becarriedoutasanextstep toevaluatethe clinical usefulness

ofanti-iC3breceptorantibodyinthetreatmentof haematogen-ouscandidiasis.

The anti-iC3b receptormAb may have deleterious effects

on host neutrophils and aggravate the course ofdisease, but

ourin vivo results indicate that theyhave atherapeutic effect

in haematogenously disseminated candidiasis without signifi-cant alterations in host neutrophils. Attachment to the iC3b

receptor on C. albicans blocks adhesion to endothelium and subsequent systemic invasion. Adherence to the endothelium iscorrelatedwithvirulence. However, it istooearlytodiscuss

theinteractions betweenintravenously injected C. albicans and

the anti-iC3b receptor mAb based solely on the current experiment. We plan to use radioisotope-labelled anti-iC3b antibodiesforfuturestudies andtoevaluatethesafety profiles

ofanti-iC3b mAb inhumans.

ACKNOWLEDGMENTS

Theauthorsthank Dr Masatoshi Takeichi (Kyoto University, Kyoto,

Japan) for providing ECCD-2 mAb. We are also grateful for the

experttechnicalassistanceof Nam Soo Jang. This paper was supported

in part by the Non-Directed Research Fund, Korea Research

Foundation., Korea.

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