Comparative analysis of serum antibody responses to Pseudomonas aeruginosa exotoxin A by cystic fibrosis and intensive care unit patients

0095-1137/83/090457-06$02.00/0

Copyright ©)1983, American Societyfor Microbiology

Comparative

Analysis of

Serum

Antibody Responses to

Pseudomonas

aeruginosa

Exotoxin A by Cystic Fibrosis and

Intensive Care Unit

Patients

GEORGE CUKOR,l*NEILR.BLACKLOW,1 NANCYA.NOWAK,'CHERYL M.RICH,1 LEWIS E.

BRAVERMAN,2ANDROBERT A. FISCHER1

Divisionsof InfectiousDiseases'andEndocrinology,2 Department of Medicine, University of Massachusetts MedicalSchool, Worcester, Massachusetts 01605

Received25October 1982/Accepted3 May 1983

Pulmonary infection with

Pseudomonas aeruginosa is a major cause of morbid

ity and mortality in cystic fibrosis

(CF) patients. P. aeruginosa toxin is one of

several proposed virulence

factors which

may be responsible for chronic P.

aeruginosa

infections in these patients. With

a highly specific, sensitive, and

quantitative radioimmunoassay

(RIA) and a cell culture assay, the humoral

immune

responses

of CF patients in

termsof total antitoxin, antitoxin

immuno-globulins

A and M, and

neutralizing

antitoxin were compared with those of P.

aeruginosa-infected intensive

careunit patients and controls. The P.

aeruginosa-infected CF patients

were

divided into

severe and moderate disease groups based

on

mortality observed

over an

8-year period.

The

intensive

care unit patients were

divided

by the site of infection and the controls

were healthy children and

uninfected CF patients. Antibodies

to

toxin

werefound in the sera of all subjects

by

radioimmunoassay. Neutralizing antibody

was associated with current

infec-tion. Elevated titers of antitoxin immunoglobulin

A were found only in subjects

with pulmonary

P. aeruginosa

infections.

No significant differences in any

antibody class

were

observed between the

severe and moderate disease groups. In

addition,

no

differences

were

observed in the antitoxin immune

response of

chronically infected CF patients and intensive

care unit patients with acute

pulmonary infections.

Pulmonary infection with

Pseudomonas

aeru-ginosais a

major

cause

of morbidity

and

mortal-ity in cystic fibrosis (CF) patients.

The special

susceptibility of CF patients

to

colonization

with

P. aeruginosa has

long

been under

investigation

but

is still

poorly understood. CF patients

have

been

found

to

have

a

vigorous

serum

antibody

response to P. aeruginosa

which is ineffective in

eradicating

the

bacteria from the

lung but which

may

play

a

role in

restricting

infection

to that

organ

(12, 19).

P.

aeruginosa

possesses a

large

number of

potential virulence factors (21),

and it

has been

suggested that

one

of

these,

exotoxin

A

(P. aeruginosa

toxin)

may

contribute

to

the

severity

of clinical illness

experienced by

CF

patients

(11,

12).

Of the clinical isolates ofP.

aeruginosa,

90%

are

toxigenic

(2).

P.

aeruginosa

toxin

is an

ADP-ribosylating

enzyme which

in-hibits cellular

protein synthesis by

the same

mechanism as the

antigenically

unrelated

diph-theria

toxin

(10).

This

study

was

designed

to assessthe humoral

immune responses

of

CF

patients

to a

highly

purified

P.

aeruginosa toxin

preparation

without

interference

from

other P. aeruginosa

antigens.

We

wished

to test the

hypothesis

that

CF

pa-tients have

a

specific defect in their immune

response to

this

toxin which

might

account

for

their

susceptibility

to

chronic

lung infection with

P. aeruginosa. Low

doses of this

toxin

are

known

to

inhibit the function of

phagocytic

cells

(16).

In

this

report we have

quantitatively

com-pared total

serum

antitoxin,

antitoxin

immuno-globulin

A

(IgA) and IgM, and

toxin-neutralizing

antibody in

groups

of CF

patients

and

intensive

care

unit

(ICU)

patients

with

P.

aeruginosa

infections.

In

previous

studies

assays

have

been done for

antibodies

to

toxin in human

sera

by

radio-immunoassay

(RIA) (6, 7, 12),

enzyme

immuno-assay

(11), neutralization

(17),

and

passive

hem-agglutination (18)

and have

established

that

these

antibodies

are

commonly

found

in P.

aeru-ginosa-infected

individuals.

Two

of

thesestudies

have

dealt

with

CF

patients (11,

12),

and both

have

suggested

that more severe CF disease is

associated

with

higher

antitoxin

titers.

Novel

features

of

the assay

employed

in

our

study

include

theuse

of

a

toxin

antigen

of

immunologi-cally defined

purity

andanRIA

design

in which

457

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a

complete

dilution curveisgenerated for each

serumand each serum serves asitsowncontrol

for nonspecific

reactivity. Class-specific

antitox-in

analysis

of CF

patients,

as well as a direct

quantitative

comparison

of the antitoxin

re-sponses

of CF

patients

and P.

aeruginosa-infect-ed non-CF

patients

have not been

previously

reported.

MATERIALSANDMETHODS

Study groups. Serum specimens from CF patients, ICU patients, and healthy children were examined in this study, which was approved by the Committee on Human Studies of the University of Massachusetts Medical Center.

TheCF group consisted of 28 previously studied (1, 8)patients who werebeing followed at the Children's Hospital Medical Center of Boston. At the time of serum collection in 1973, these patients were not acutely ill and wereeitherattending school or work-ing. All were receiving pancreatic enzymes orally as well as multivitamin supplements and prophylactic antibiotics. The current clinical status of these pa-tients, as well as their histories of P. aeruginosa colonization, were determined from their medical rec-ords. The CF patients were divided according to whether they had a P. aeruginosa-positive throat or sputum culture recorded before serum collection in 1973 (13 patients) or not (15 patients). The colonized patients were further subdivided accordingtowhether they were alive in 1981 (seven patients) or not (six patients). This survival criterion was used to divide the patientsinto severe and moderate disease groups.

The secondsetof sera was collected in 1981 from14 critically ill adultpatients who were colonized withP. aeruginosa in the surgical (13 patients) ormedical (1 patient) ICU at theUniversity of Massachusetts Medi-cal Center. Because of the difficulty in locating P. aeruginosa-infected non-CFchildren, it was necessary touseinfected non-CF adultsfor comparison. Sites of symptomatic infection in this group included blood, lung, and urinary tract. In addition, two patients (one with a positive urine culture and the other with posi-tivewound, urine, and sputum cultures) were judged tobe colonized but not clinically ill due to P. aerugino-sa. None of the patients had severely depressed hu-moral immunity. Seven patients ultimately died; Of these, three had bacteremia, three had pulmonary

itifections,and one was colonized with P. aeruginosa.

Innocase was the P.aeruginosa infection regarded as amajor factor contributing to mortality. These

culture-positive ICU patients were divided according to

whether lung infection was present (six patients) or not (eightpatients).

Athird set ofsera was collected from 22 healthy, nonhospitalized children who were in the same age groupastheCF patients studied. These subjects were Massachusetts residents whowerechildren ofmedical personnel, and they servedascontrols. Allsera were storedat -20°C until use.

P.aeruginosatoxin. A highly purified preparation of

P.aeruginosatoxin was obtained fromaculture ofthe toxin-producing strain PA103 by affinity

chromatogra-phyonspecifically-linked NAD agarose. Details of the

purificationof the toxin and its characterization have

been described elsewhere (9). Two control

toxin-deficient strains, PAO-Tl andPA103-29, were kindly provided by D. Ohman (14, 15). Cultivation ofPAO-Tl

wasdone as previously described (9). PA103-29was growninstationary culture with the same media used for the othertwoP. aeruginosa strains.

Immunization ofmice andtesting ofmouseimmune sera.An adult BALB/c mouse was immunized with 25 ng ofpurified toxin which had been heat-inactivated at 56°Cfor 1 h. Theinjectionwasgivenintraperitoneally with anequal volume ofincomplete Freund adjuvant. The samedose of toxin was used for three biweekly booster inoculations. Forassay of the mouse serum, 25 ,ul of the antigen to be tested was driedontothe surface of polyvinyl microtiter wells by incubation overnightat37°C. Serially diluted mouse serum(50-,u

portions) in phosphate-buffered saline (pH 7.3) with 0.1%NaN3(PBS) was added to duplicate wells of each antigen. After incubation of theplates for 2 hat37°C, the wells were washed five times withPBS, and 50

[lI

(200,000 cpm) of1251-labeledanti-mouseserumrabbit immunoglobulin was added to each well. The plates were reincubated for 4 h at 37°C and washed five times, and the radioactivity bound to each well was determined in a gamma counter. Positive/negative ratioswerecalculatedbydividing themean countsper minute obtained with the immunemouse serumbythat obtained with preimmunizationserumfrom the same animal and tested at the same dilution on the same antigen. A positive resultwasconsideredtobe .2.

Determination of totalimmunoglobulin levels. Total serumIgG, IgA, and IgMwere determinedby radial immunodiffusion(HylandDiagnostics, Deerfield, Ill.). Determinationofserumantitoxin titerbyRIA.Wells

ofa polyvinylmicrotiterplate werecoatedovernight

atroomtemperature with 100,ul(4.4 ng) ofpurifiedP. aeruginosa toxin diluted in PBS. Control wellswere coated with PBScontaining 1% bovineserumalbumin. All wellswerewashedtwice withPBS,filled with PBS containing 1% bovine serum albumin, and incubated overnight at4°C, and the plateswere againwashed. Sera frompatientswerediluted in PBScontaining1% bovineserumalbumin and0.05%Tween-20,and 25pul of eachserumdilutionwasthenaddedtoeach oftwo toxin-coated wells and two control wells. After incu-bation at 37°C for 2 h, the plates were washed five times with PBScontaining0.05%Tween -20, and 50 pul(200,000 cpm) offreshly prepared

125I-labeled

detec-tion antibody wasadded toeach well. After another incubationat37°C for 2 h and five washings with PBS containing 0.05% Tween -20, the radioactivity bound to each well was determined in a gamma counter. Affinity-purified detection antibodies specific for total human immunoglobulins and human IgM and IgA were purchased from Kirkegaard and Perry Labora-tories, Gaithersburg, Md. and were iodinated by a previously describedchloramine-T procedure (3). Po-sitive/negative ratios were calculated by dividing the mean counts per minute bound to two toxin-coated wells by thatbound by the same serum dilution to two wellscoatedwith bovine serum albumin. The recipro-cal of the highest dilution of serum yielding a

posi-tive/negative ratio of.2.0 was considered to be its

titer.

Neutralization assay. Neutralizing antibody was measured bydetermining the rate of protein synthesis of cell cultures exposed to toxin which had been preincubated with dilutions of sera from the patients.

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ANTIBODIES TO P. AERUGINOSA TOXIN 459

L-929 mousefibroblasts were grown to confluence (2 x 106 cells per culture) in minimal essential medium (MEM) with 10%o fetal calf serum in 24-well culture dishes. The cultures were washed with Hanks bal-anced salt solution, and 300 ,ul of a toxin-serum mixture was added to duplicate wells. The mixture was prepared by incubating (1 h, 37°C) 500-1± (4 ng) portions of toxin diluted in MEM with 0.1% fetal calf serumwith 250p.l of various dilutions of patients' sera prepared inPBS without NaN3. Next, 200,u1of MEM with 0.1% fetal calf serum was added to each well, and the plates were incubated for 4 h. Cultures were washed oncewith Hanks balanced salt solution, and 1 mlof serum-free MEMcontaining 0.1 ,uCi of a3H-L

amino acid mixture (New England Nuclear Corp., Boston, Mass.) wasadded to each well. After further incubation of the plates for 1 h, the cultures were washedfive times with cold (4°C) Hanks balanced salt solution. The cells were dissolved in 3 N NaOH, and the 5% trichloroacetic acid-precipitable radioactivity wasdetermined for each culture. The mean counts per minute obtained with six control cultures which re-ceivedonly MEM with0.1% fetal calf serum but not toxin was taken as100%1protein synthesis. The recip-rocal of thehighest dilution of serum which produced aninhibition ofprotein synthesisof<50%owas consid-ered to be its neutralization titer. Cultures which received only toxin without any patient sera showed 80 to90%oinhibitionofprotein synthesis. The relative averagedeviation from themeanfor all data reported

was <10o.

Statistical analysis.Confidence limitswere

calculat-edby the formula describedby Campbell(5). Probabil-ity values were determined by the use of one-way analysis ofvariance followed by the Newman-Keuls test for multiple comparisons with unequal sample

sizes(20).

RESULTS

Specificity

of theassay for detection ofantibody

to P. aeruginosa toxin. We

intended

in

this

study

to

examine the

antibody

response

specific

to P.

aeruginosa

toxin without interference from

oth-er

pseudomonal

antigens.

An

affinity

chroma-tography-purified

toxin

preparation

known

to

be

enzymatically,

biologically,

and

immunological-ly active and shown

to

have

a

gel electrophoretic

pattern

consistent with

purified

P.

aeruginosa

toxin (9)

was

used

as

antigen.

To

further

show

specificity,

wetested serum

from

amouse

immunized

with the

purified

toxin

preparation

for

its

reactivity

in RIA with five

antigens

(Table 1).

The results

(Table 1)

show

that the serumfrom the

toxin-immunized

mouse

reacted

only

with the P.

aeruginosa

toxin and

PA103

antigens.

The lack of

reactivity

with the

PAO-T1

and

PA103-29

antigens

shows that

im-munization with

the

purified

toxin

preparation

did

not

elicit detectable

antibodies either to

antigens shared

with the

toxin-deficient strains

or to

media

components.

PA103-29

is a 99%

toxin-deficient

strain,

with allother

characteris-tics

identical

tothe

parental

PA103 strain

(14).

A

stationary

method

of culture

was

used

for

TABLE 1. Reactivity of antitoxin hyperimmune mouse serum with various antigens

Antigen Serum RIA titer

Purified P. aeruginosa toxin .102,400

PA103(toxigenic)a .102,400

PAO-T1 (nontoxigenic)a <10

PA103-29 (toxindeficient)a <10

Bovine serum albumin <10

aSpent medium and cells from culture.

PA103-29 in an attempt to further diminish toxin synthesis without affecting any other antigens. PAO-T1, which was grown in shaker culture, is

a nontoxigenic strain, but it differs from

PA103

in other

respects as well (13).

Study

groups and total immunoglobulin levels.

Six

groups of subjects were examined in this

study. Descriptions

of these groups are given in

Table 2, which also shows the mean

class-specific

total serum immunoglobulin levels for

each

group. Total IgG,

IgM,

and IgA contents of

all sera used in this study were determined by

radial immunodiffusion.

No significant

differ-ences among the four pediatric groups were

found with regard

to total IgG, IgA, or

IgM.

The

mean

values for

IgG and IgA for each of the six

study

groups

fell within

2 standard deviations of

reported normal mean age-specific

immunoglob-ulin levels (4). The IgM levels obtained

were

slightly elevated for all study

groups.

Antibody response to P. aeruginosa toxin.

(i)

Total serum antitoxin.

The

total

toxin-specific

serum

antibody

titers

were

determined

by

RIA

(Fig.

la).

The

two groups

with

severe

lung

infections,

comprising

ICU

patients

with

lung

infection and

P.

aeruginosa-colonized CF

pa-tients who died within

8 years

of

serum

collec-tion, had the

highest

levels

of antitoxin. The CF

group

who

died had

significantly

higher

antitox-in

titers than the

uncolonized CF

(P<0.01),

control

(P

<

0.05), and ICU without

lung

infec-tion (P

<

0.05)

groups.

The

group

of colonized

CF

patients

who

were

alive 8

years

after

serum

collection had

a more

varied

response,

with

no

statistically

significant

difference when

com-pared

with

the response

of

the

above three

groups.

(ii)

Antitoxin

IgA.

Only

the

three

groups

with

P.

aeruginosa

lung

infections

showeda

marked

IgA response to

toxin

(Fig. lb).

The

toxin-specific

IgA

titers

for

these threegroupswere

all

significantly

higher

than the titers for the

con-trol, ICU without

lung

infection,

and

uncolo-nized CF

groups. It

is

noteworthy

that the

levels

of titer for

the

ICU

group

without

lung

infection

remained close

to

control levels. Differences

among these three

IgA-responding

groupswere

not

significant.

(iii)

Antitoxin

IgM.

The

infected

CF

patients

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TABLE 2. Study population

Groupa Age(yr) Totalserumimmunoglobulin (mg/dl)(mean ± SE)

(mean ± SE) IgG IgA

IgM

CONT 12.0 ± 1.4 1179 ±50 124± 12 151 ± 10

ICU-PA-LUNG 59.5 + 8.6 1398 ± 198 286± 56 159± 23

ICU-PA-NOT LUNG 51.9 ± 6.3 1239 ±249 302± 56 130 ± 28

CF-PA-DEAD 13.7 ± 1.6 1220 ± 166 184± 35 164± 38

CF-PA-ALIVE 10.7 ± 0.8 1074 ± 99 114± 20 127± 25

CF-NOT PA 11.3 ± 1.1 1213 ±94 125 ± 14 137 ± 16

aDescription of groups: CONT, 22 healthy nonhospitalized children (8 males);

ICU-PA-LUNG,

6

ICU

patients with P. aeruginosa lunginfections (5 males);ICU-PA-NOT LUNG, 8 P. aeruginosa-colonized ICU patients withoutlung infection(8 males);CF-PA-DEAD, 6 P. aeruginosa-colonized CF patients who died within 8 yearsofserumcollection(nomales); CF-PA-ALIVE, 7 P.aeruginosa-colonizedCFpatientswhowerealive 8 yearsafterserumcollection(5males);CF-NOTPA,15CFpatientsnotcolonized with P. aeruginosa(sixmales).

exhibited widely variable IgM responsestotoxin

(Fig.

lc),

and

no

significant

differences among

the groups werefound.

(iv)Toxin-neutralizingserumantibody.

Toxin-neutralizing antibody

was measured in a

tissue

culture assay

(Fig. ld).

This type

of

antibody

1000 -C

x0o1001

10,000-1,000 D

**1,6

100

CONT ICU-PA- ICU-PA- CF-PA- CF-PA-

CF-LUNG notLUNG DEAD ALIVE notPA

1 2 3 4 5 6

Study Group

FIG. 1. AntibodyresponsetoP. aeruginosatoxin. Bars representgeometric mean titersfor each group

and lines represent the 95%confidenceintervalof the

mean. See Table2 fordefinition ofthe studygroups.

(A) Total antitoxin; (B) IgA; (C) IgM;(D)neutralizing antitoxin.Symbols: *,P<0.05 by theNewman-Keuls

testwhencompared with the indicatedgroup(s);**,P <0.01,asabove.

was

detectable

only

in the four infected groups.

No

significant

differences in

antibody

titers

amongthese groups were

noted, although

only

the CF group who

died showed

a

consistently

high

neutralizing

antibody

response,

significant-ly

higher

than the two uninfected groups

(P

<

0.01).

DISCUSSION

Several

findings

have

emerged

from our

test-ing

of

serumsamples from CF and ICU

patients

and control

subjects

for

specific antibody

to P.

aeruginosa

toxin. First,

every

subject examined

had serum antibody to the toxin which was

detectable by RIA.Inuninfected

subjects

this is

presumably

due to environmental exposure to

the

ubiquitous

P.

aeruginosa bacterium.

Occur-rence of serum

antitoxin

detectable by RIA or

enzyme immunoassay in healthy

subjects

has

been

reported by

other

investigators

who did not

base their

calculation

of positive results on the

values

obtained

with a negative control serum

(6, 11, 18). In the present study the serological

test was

designed

sothat each serum served as

its owncontrol, inasmuch as the reactivity of the

serum with

purified

toxin and control antigen

wascompared. Infected ICU and CF patients as

a group had higher levels of antitoxin than

controls. Uninfected CF patients had control

levels of total serum antitoxin.

Second, toxin-neutralizing antibody appears

to be specifically associated with current P.

aeruiginosa

infection because this type of

anti-bodywasnotdetected in the uninfected groups.

It may be postulated that neutralizing antibody

is either of much shorter duration than RIA-detectable antibody or that neutralizing antibody

is notformed by mere environmental exposure

to P. aeruginosa.

Third,

antitoxin serum IgA was found in

sub-stantialamountsonly inpatients with lung

infec-tions and not in uninfected subjects or in ICU

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ANTIBODIES TO P. AERUGINOSA 461

patients with extrapulmonary

infections.

Pre-sentation of the toxin antigen

on

the

pulmonary

mucosal

surface

appears to be essential for the

production

of large amounts of antitoxin IgA,

regardless of the acute or chronic nature of the

infection.

Fourth,

no

significant

quantitative differences

in

any

antitoxin immunoglobulin

class were

ob-served between P. aeruginosa-infected CF

pa-tients with

severe

and moderate disease.

Severe

and moderate CF disease

were

defined

retro-spectively based

on

the

survival of the patient

although

at

the

time of

serum collection all

subjects

were

considered

to

be clinically

well.

The

patients with

severe

CF disease

generally

tended

to

have

high antitoxin titers, but

the

patients with moderate CF disease

had

some-what

more

variable

responses. However, we

were unable to make any judgments as to the

condition

of

an

individual

patient

on

the

basis of

his antitoxin titers because

many

of

the

children

with moderate

disease had high levels

of

antitox-in.

Our

findings

differ from

a

previous

report

(12),

which correlated high levels of

serum

anti-toxin with increased

severity

of CF disease,

although the clinical grouping of patients

was

done

in

a

different

way

from

ours. In the

previ-ous report a

modified Shwachman

scoring

sys-tem was

used which did

not

include the

presence

or

absence

of

P. aeruginosa

colonization

as an

evaluation

component

for the determination of

CF

severity.

Indeed,

if

only CF patients with

positive

P.

aeruginosa

cultures

were

consid-ered, the

findings

in

both

studies would be

similar. Infection with

P. aeruginosa

is

recog-nized

as

having

a

major

effect

on

the

severity of

CF

(13). Among

our

13

infected CF patients,

only

7

survived

for

8

years,

whereas all 15

uninfected

CF

patients

were

alive

at

the end of

the

same

period.

Finally, it is particularly noteworthy that

no

significant

differences in

any

of

the

antitoxin

antibody

response

levels

were

noted between

P.

aeruginosa-infected

CF

patients and the ICU

patients with

P. aeruginosa

lung infections.

It

is

therefore

unlikely that

a

toxin-specific

humoral

immune defect is

present

in CF

patients. The

possibility

of

a

toxin-specific

secretory

IgA

de-fect in the

lung

still

remains,

and the

examina-tion ofantibody in sputum is the object offurther

work. The functions

of

phagocytic

cells are

sensitive

to

inhibition

by

low

levels

of

P.

aeru-ginosa

toxin

(17),

and the presence

of

unneutra-lized

toxin

in the

lung

would result in the

inacti-vation

of

a

major bacteria

clearing mechanism.

ACKNOWLEDGMENTS

Thisstudywassupportedbygrant G131B fromtheCystic

Fibrosis Foundation.

We aregratefultoMaryEllenClifford for technical

assist-ance,Mary Williams for aid in obtaining clinicalinformation, andLeslie Lipworth for statistical advice.

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