Detection and partial characterization of circulating immune complexes in hydatid disease

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0095-1137/83/111021-06$02.00/0

Detection

and Partial

Characterization

of

Circulating

Immune

Complexes in Hydatid Disease

R. D'AMELIO,'t0. PONTESILLI,2L. PALMISANO,2M.PEZZELLA,3 V.VULLO,3 S. DELIA,3 F. DE ROSE,4 F. SORICE,3 ANDF. AIUTI2*

Department ofHygieneandImmunology, Italian Air ForceAerospaceMedicalCentre';andDepartment of ClinicalImmunology, InstituteofInternal Medicine II1,2Instituteof InfectiousDiseasesI,3andDepartment

of Infectious Diseases11,4 University of Rome, Rome, Italy Received 6 October 1983/Accepted 8 August 1983

Thirty serafrom eight patients withdisseminated orlocalized hydatid disease have been examined for thepresenceof

circulating

immunecomplexes(CICs) by the

conglutinin-binding

assayand forimmunoglobulinlevels.Thehighestlevelsof CICswereofthe

immunoglobulin

A(IgA) class,with lower values ofIgG-CIC and IgM-CIC; these results did notcorrelate, exceptforIgG,with thefree immuno-globulin levels. Efforts to

identify

parasitic antigen(s) involved in the CIC formation with different methodshavebeen unsuccessful. In the

follow-up

of each

patient, CIC appeared to be better correlated to clinical conditions than to hemagglutination titers. We have concluded that the presenceofCIC in hydatid disease is probably an expression of B-cell polyclonal activation and that these

complexes are valuable in the clinical monitoringof the disease.

Circulating immune complexes (CICs)

may occurinseveral

pathological conditions

(17). In some

diseases, CICs

could

be responsible

for the

clinical symptomatology,

whereas

in

others, they

probably only

represent an

epiphenomenon

not

directly

related to

specific

symptomatology.

Inthis latter

situation, however, the

identifica-tion

of

CICs

seems to be

important

because they could exert a

modulating effect

on

immunologi-calreactions

(15).

CICs have

been

identified

in many

pathologi-cal

conditions;

in

parasitic

diseases,

particular-ly,

they

seem toplay an

important

role in the

pathogenesis of clinical manifestations. Some

authors,

in

fact,

have

demonstrated

that a B-cell

polyclonal activation is induced by parasitic

antigens (7).

In

hydatid

disease,

B-cell

polyclonal

activa-tion is

notyet

documented,

but a

strong

specific

antibody

response

is normally associated

with

the

disease,

and a

hypothesis

ofCIC

formation

thus can

be

made.

We

studied

30 sera

from

eight patients

with localized or

disseminated

hydatid

disease

bythe

conglutinin-binding

assay to detect and charac-terizeCICs.

MATERIALS AND METHODS

Immunological reagents. Rabbit immunoglobulinG

(IgG)wasprepared byDEAE-cellulose

chromatogra-tAddressreprintrequests to: R.D'Amelio,Department of HygieneandImmunology,ItalianAirForce Aerospace Medi-calCentre, Viale Piero Gobetti 2/A-00185Rome, Italy.

phy (WhatmanDE-52) from normal rabbit serum. Anti-humanhydatid cyst fluid(HHCF)was raisedin

rabbits. Purified anti-HHCFantibodieswereobtained

by affinitychromatography.A50-mg amount ofsheep hydatid cyst fluid was coupled with 3 g of

CNBr-Sepharose 4B (Pharmacia Fine Chemicals) by the method of Cuatrecasas (5). The antibody-containing fractionwaseluted with0.1M glycinehydrochloride

buffer(pH 2.6). Sheep hydatid cyst fluidantigen has been used to obtainonly antibodiesspecifically direct-edagainstparasite antigen.

F(ab')2 fragments from specific IgG and normal rabbit IgG were obtained by pepsin (Worthington

Diagnostics) digestion at a concentration of 1 mg/100

mg ofIgG in acetate buffer (0.1 M, pH 4.5) by the method of Nisonoff et al. (12).

Radiolabeled reagents. Rabbit anti-HHCF

antibod-ies, normal rabbit IgG, rabbit anti-HHCF F(ab')2, normal rabbitF(ab')2, Staphylococcus aureus protein

A(Pharmacia FineChemicals) rabbitanti-humanIgG, andrabbitanti-humanIgMwereradiolabeled with1251

(Radiochemical Centre) by thechloramine T method

(11). The specificactivity of the labeled materialwas about 1mCi/mg.

Bovineconglutinin. A conglutinin-enriched fraction

was obtained by the absorption of heated bovine serum onyeastandsubsequentelutionbythe method

ofLachmann and Hobart(8). Conglutininwasfurther purified by pepsin digestion of contaminants by the method of Maire et al. (9). Conglutinin purification

wasmonitored byconglutination of an EAC43 inter-mediate(8)andimmunochemical analysiswithrabbit

anti-conglutinin and anti-whole bovine serum.

Fur-thermore, the lot used was previously tested to rule outbindingof free humanimmunoglobulin.

Conglutinin-binding assay. The conglutinin-binding

assay wasperformedasdescribedby Casali et al. (3), with anamplificationbyanti-immunoglobulin antibod-1021

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ies(M.Barnet,A.Carpentier, andP.Lambert, Abstr. Intl.Congr. Immunol.4th, Paris, 1980). Brieflyin step one,100,ul ofa1:20dilution inVeronal-buffered saline

(VBS)-Tween20(0.035%)of thesampleswas incubat-ed for 2hat roomtemperature inpolypropylenetubes precoated withpurified bovineconglutinin.Coatingof thetubeswasaccomplished byincubatingconglutinin (5,ul/ml) in carbonate buffer(0.05M, pH 9.6) for3hat

37°C. The tubeswere washed three times with VBS-Tween20just before use. Immunecomplexes which carried fixed C3bi reacted with thesolid-phase conglu-tinin. After incubation, the tubes were washed three times with VBS-Tween 20 to remove all unbound

serumproteins. Instep two,100-pIvolumes of rabbit anti-human IgG, rabbit anti-human IgA, and rabbit anti-humanIgM(Behring Corp.)diluted 1:50 in VBS-Tween 20wereincubated in different tubes for 30 min

at room temperature. After three more washes with

VBS-Tween 20,the amountof rabbitantibodiesfixed tothe complexes wasrevealed by incubating 100 p.l (0.5p.g/mlinVBS-Tween20)of

125I-protein

Afor 3h at room temperature, and finally, after three more

washes,bymeasuring the residualradioactivityinthe

tubes inagammacounter.Resultswereexpressedas

nanogramsof125I-proteinA bound.

Procedure for antigen identification in CICs. For antigen identification, we used amodification of the conglutinin-binding assay (6). Briefly, after step one above, the conglutinin-bound complexes were incu-batedfor3 hat37°C with 100 p.1ofadilutionranging from1 to 10pg/mlinVBS-Tween 20 of radiolabeled IgG or F(ab')2 specifically directed against HHCF. After three more washes with VBS-Tween 20, the

24

23

22

21

20

< 19

*e; 18.

4)

1

M. 1

714

-1

3-1

2-11

-10 Igm-dCI p(0.01 0 00 * *o. 0 009 0 .0.

0|

0 0 0 0 0 0 000 00 0 5.-4 -3 . 2 1 .

tubes were counted in a gamma counter. In these

experiments, the specificity of the binding was

checked by using radiolabeled IgG or F(ab')2 from rabbit serum inparallel with theantigen-specific IgG

or F(ab')2, at the same concentration and specific activity.

In addition, we also used another method. Briefly,

100 p.l each of unlabeled F(ab')2 anti-HHCF and unlabeledF(ab')2from normal rabbit serum,bothata

concentration of 20p.g/mlincarbonate buffer (0.5 M,

pH 9.6), were incubated separately in polypropylene tubes for3 h at37°C and overnightat4°C. After three washeswith VBS-Tween 20, theF(ab')2-coated tubes

were filled with 100 pL. of a 1:20 dilution in VBS-Tween 20 and 100 pL. of

125I-protein

A (0.5 p.g/ml in VBS-Tween 20). After 3 h of incubation at room temperature, thetubeswerewashed three timeswith VBS-Tween 20 and counted in a gamma counter. The purity ofthe F(ab')2 preparation was indirectly con-firmedby the low background obtained in thistest.

Quantitation of immunoglobulins. Immunoglobulin levelsweremeasured by single radial immunodiffusion (10), usingcommercial plates (Kallestad).

Antigen preparation. TheHHCF used as anti-genwascollected from hepatic andpulmonary cysts of sheep infected with Echinococcus granulosus; this material was subsequently filtered, centrifuged, and concentratedby perventilation to a concentration of2

mg/ml ofprotein.

Hemagglutination test. The hemagglutination test wasperformed bythe method of Boyden (2). Titers of

1:400ormorewereconsideredsignificant (4-15).

Patients.Thirtyserafromeight patientswithhepatic

IgG-CIC p<O0.005 0 0 0 00 00 * 0 13 1 2 1 1 10 9 8 6 4 3 2

Controls Hydatidosis Controls Hydatidosis Controls Hydatidosis

FIG. 1. Levelofcirculatingimmunecomplexesin 30serafromeight patientswithhydatiddisease. Asterisk

indicatesmeanplus twice thestandard deviation forcontrolsera.

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orpulmonary hydatid disease were examined. Four patients (no. 5, no. 6, no. 3, and no. 2) presented a single cyst in the liver; three of them underwent surgery (no. 5, no. 3, no. 2), whereas the remaining one was treated with mebendazole (40 mg/kg per day for courses of 2 to 3 months each).

Onepatient (no. 1), who presented with hepatic and pulmonary hydatid disease, was operated on for the removal of the hepatic cyst and then treated with mebendazole; during the first cycle of therapy, he developed aspontaneous rupture of the cyst into a bronchus.

Another patient (no. 8) was a 4-year-old girl with multiplehepatic cysts. She was treated with mebenda-zole;during the second cycle of therapy, amoderate and transient increase of serum glutamic oxalacetic transaminase and glutamic pyruvic transaminase was noted.

The last patient (no. 7) was affected by multiple pulmonary hydatid disease andwastreated with

me-bendazole.

Statistical analysis. Statistical analysis was per-formed by the Student t test. Correlation was

exam-ined bythePearson r test. RESULTS

Levels

of IgM-CIC,

IgG-CIC, and

IgA-CIC in

the 30 sera

examined

and in 30 sera

from normal

control

patients

(matched

for

age

and

sex)

are shown in

Fig.

1. The

highest levels

were

ob-served for

IgA-CIC

(19

serawith values

higher

than

twice

the

standard deviation

above the mean

of the control

sera,

with

a mean

signifi-cantly different from that of

the control sera

[P

0 0 6 10

IgA-CIC Months of disease

IgG-CIC

-MT

FIG. 2. Follow-up of IgM-CIC, IgG-CIC, and IgA-CIC and of hemagglutination titer(HT) in patients 1

through4withhydatiddisease. ngPA, NanogramsofproteinA. 18,

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< 0.001]); in 8 sera,

IgG-CIC

showed values higher than twice the standard deviation above

the

mean of normal control sera

(P

<

0.005);

finally, the

IgM-CIC

mean of the

patient

sera

was only slightly different from that of normal sera(P <

0.01).

Regarding antigen identification

in the

CICs,

we were not

able

to

identify

any

specific hydatid

antigen(s)

by using antibody

or

antibody

frag-ments

directed against

HHCF in all

of the

tests

described above.

A

comparison with clinical conditions

(Fig.

2 and 3)

demonstrated

a

quite

different

behavior

between

CICs

and

hemagglutination titers.

Pearson's r test showed no

correlation

be-tween

IgA and

IgA-CIC

(r=

0.2418)

or

between

ngPA

20

1

5-5

HT

IgM and IgM-CIC (r = 0.088), whereas there was a significant correlation between IgG and IgG-CIC levels (r = 0.5288; 0.01 >P> 0.001).

DISCUSSION

In our

patients

we found different classes of

immunoglobulins included

in

CICs,

with a prev-alence of IgA > IgG >

IgM;

this polyclonal response probably reflects the physiological switch from IgM to IgG and IgA, as may be expected in chronic diseases.

Regarding the antigenic component ofCICs, we were unable to

identify parasitic antigens;

thiscould probably be explained on the basis of

one or more of thefollowing hypotheses: (i) the

gPA No.6

CR

Hepatic HT

mebendazole

)O \ "25600

203

/ 3200

,\

\I

1

0

.i:.::ii.

A

10I00

ngPA1 No. 8

9

Disseminated

'25600

20.

,3200 5.

5-15 0

IgA-C-C IgM-CIC....---__

IgG-CIC_--HT

1 4 7

Months of disease

0

HT

25600

3200

FIG. 3. Follow-up of IgM-CIC, IgG-CIC, and IgA-CIC and of hemagglutination titer (HT) in patients 5 through8 withhydatiddisease. ngPA, Nanogramsofprotein A.

No.7 g Pulmonary

mebendazole

0 4 8

mebendazole mebendazole

1 "l~~~~~~~~.

1

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absence of specific parasitic antigen(s)

in the

CICs,

at

least

as

recognized by

our antiserum (The

complex antigenic

mosaicism

of

the Echi-nococcus may

partially

account for these

diffi-culties.);

(ii)

the

steric

inhibition of the

antigen

within the complex due to the possible large size

of

the

CICs; (iii)

the molar ratio of the

CIC

components

(in fact,

the method we

employed

shows

a

good

performance only in conditions of

equivalence

or

slight antigen

or antibody

ex-cess); (iv)

the

possibility

thatCIC formation is a consequence of a B-cell polyclonal

activation,

with

consequent

antibody

response

against

het-ero-

and

autoantigens,

but not

against

Echino-coccus

antigens.

Even

if

we

failed

to demonstrate the presence

of

a

specific hydatid antigen(s)

in

CICs,

the

follow-up

of

our

patients strengthens

our

hy-pothesis

about the value of CICs in

monitoring

the

disease.

In

fact, CICs

seemed to present a

more strict

parallelism

with clinical course when

compared

with the

hemagglutination

titer. In

patient

no.

5, for

example,

we

observed

astable

decrease

of CIC

levels

after

surgery

during

a

48-month

follow-up,

whereasthe

hemagglutination

titer showed

an

irregular behavior;

in

patient

no.

4, who was

affected

by

a

disseminated

hydatido-sis and

was

successfully treated with

mebenda-zole, CIC levels

gradually

decreased, while

hemagglutination titers exhibited

an

unexplained

oscillation;

in

patient

no.

1,

the

increase of CIC

values between the

first and second

serum sam-ples was

probably related

to an

episode of

cyst rupture

into

a

bronchus, associated with

mas-sive

liberation

of

antigenic material from

the cyst and

subsequent

passage

of

the material

into

the

bloodstream;

and

finally, in patient

no. 8, a

girl with disseminated

hydatidosis

successfully

treated

with mebendazole,

an

increase of CIC

levels in month

7

of

follow-up

was

observed

associated with

a

rise of

serum

glutamic

oxalace-tic

transaminase and

glutamic

pyruvic

transami-nase,

probably

due to

toxic

hepatitis,

a

condition

in

which

other authors have demonstrated the appearance of

circulating

specific

antibodies

to altered

hepatocytes

(16).

These

observations

suggest

that, in

our

pa-tients, CICs belong

to different populations, some

involving hydatid antigens

and

specific

responses, and others probably consisting of auto- or heteroantigens as documented by the high titer of anti-rabbit F(ab')2 activity [studies arein progress to further characterize this

anti-F(ab')2

activity].

The discordance between our data and previ-ous data relative to the low

positivity

of CIC levels in

hydatid

disease(13) or to the significant increase of CIC levels after treatment with me-bendazole (1) could probably be explained on thebasis of the different methods

employed

to

detect CICs.

Finally, the

positive significant correlation

observed between the levels of free IgG

and

IgG-CIC is probably not due to a possible interfer-ence

of

free

immunoglobulin

levels in the

con-glutinin-binding

assay (U. DiMario

and

K.

Guy,

personal

communication), but likely

to the

clon-al

expansion

of IgG, with possible involvement

of

IgG auto-

and

heteroantibodies

in the CICs. In

conclusion,

the

study

of

our

patients with

hydatid disease

showed that CICs can be useful

for

monitoring clinical conditions since they

reflect both

specific

and

nonspecific

symptoma-tologies

more

reliably

than does

hemagglutina-tion

titer.

Furthermore, even

if in

this condition

the

typical features of immune complex diseases

are

lacking,

the presence

of

CICs

is probably

relevant

from

a pathogenic

point of

view

be-cause they

could

exert a

modulating effect

through

the

interaction with

receptors

for

IgG-Fc

fragments

and

C3

on the

surface of

circulat-ing cells.

LITERATURE CITED

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2. Boyden, S.V.1951. Theadsorption of proteinon erythro-cytes,treated with tannicacid, and subsequent hemagglu-tination withantiproteinsera. J. Exp.Med. 93:107-120. 3. Casali, P., A.Bossus,N. A.Carpentier, and P. H.

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4. Castagnari, L., and A. Tolu. 1964. L'emagglutinazione indiretta nelladiagnosi biologica dell'idatidosi. Policlinico Sez. Med. 71:395-399.

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7. Greenwood,B.M.1974.Possible role ofBcellmitogen in hypergammaglobulinaemia in malaria and trypanosomia-sis. Lancet i:435-438.

8. Lachmann, P.J., andK.J. Hobart. 1978. Complement technology.In D. M.Weir(ed.),Handbook of experimen-talimmunology-1978,vol.1.Blackwell Scientific Publi-cations, Ltd., Oxford.

9. Maire, M. A., M. Barnet, and P. H. Lambert. 1981. Purification of bovineconglutinin using pepsin digestion. Mol.Immunol.18:85-90.

10. Mancini, G., A.0.Carbonara, and J. F. Heremans. 1965. Immunochemicalquantitation of antigens by singleradial immunodiffusion. Immunochemistry2:185-189. 11. McConahey,P.H., and F.J. Dixon. 1966. Amethod for

trace iodination of proteins for immunological studies. Int.Arch.Allergy Appl.Immunol. 29:185-189. 12. Nisonoff, A., F. C. Wissler, L. N. Lipman, and D. L.

Woernley. 1960. Separationof univalent fragmentsfrom the bivalent rabbit antibody molecule by reduction of disulfide bonds.Arch. Biochem.Biophys.89:230-244. 13. Richard-Lenoble, D., M. D. Smith, M. Loisy, and P. J.

Verroust. 1978. Human hydatidosis: evaluation of three

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16. Vergani, D., G. Mieli-Vergani, A. Alberti, J.Neuberger, A. L.Eddleston,M. Davis, and R. Williams. 1980. Anti-bodiestothe surface of halothane-alteredhepatocytesin patients with servere halothane-associated hepatitis. N. Engl. J.Med. 303:66-71.

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