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Department of 1Microbiology and 2 First Department of Internal Medicine, Kumamoto University School of Medicine,. Kumamoto, Kumamoto 860, Japan.

Microbiol.Immunol.,40(6), 415-423, 1996

Bradykinin Proteases Circulation

Generation Triggered by Pseudomonas Facilitates Invasion of the Systemic by Pseudomonas aeruginosa

Yoshifumi Sakata1,2, Takaaki and Hiroshi Maeda*1

Akaike1,

Moritaka

Suga2,

Sumiko

Ijiri1, Masayuki

Ando2,

Department of 1Microbiologyand 2First Department of Internal Medicine, Kumamoto University School of Medicine, Kumamoto,Kumamoto 860, Japan Received

January

25, 1996.

Accepted

March

13, 1996

Abstract: To elucidate the mechanism of bacterial exoprotease in promotion of the intravascular dissemination of Pseudomonas aeruginosa, we examined the possible involvement of bradykinin (whose generation is induced by pseudomonal proteases in septic foci) in the invasion by bacteria, and in access of bacterial toxins to systemic blood circulation. P. aeruginosa 621 (PA 621), which produces very little protease, was injected intraperitoneally into mice together with pseudomonal exoproteases (elastase/alkaline protease). Dissemination of bacteria from the peritoneal septic foci to the blood was assessed by counting viable bacteria in the blood and spleen by use of the colony-forming assay. The results showed that pseudomonal proteases markedly enhanced (10- to 100-fold) intravascular dissemination of bacteria in mice. This enhancement was induced not only by pseudomonal proteases but also by bradykinin. More importantly, the increased spread of PA 621 induced by pseudomonal protease and bradykinin was significantly augmented by the addition of kininase inhibitors, indicating the direct involvement of bradykinin in bacterial dissemination. Similarly, bradykinin caused effective dissemination of pseudomonal toxins such as endotoxin (lipopolysaccharide) and exotoxin A when the toxins were injected into the peritoneal cavity with bradykinin. Furthermore, the lethality of the infection with PA 621 was strongly enhanced by pseudomonal proteases given i.p. simultaneously with PA 621. On the basis of these results, it is strongly suggested that pseudomonal proteases as well as bradykinin generated in infectious foci are involved in facilitation of bacterial dissemination in vivo. Key words:

Pseudomonas

protease,

Bradykinin,

Septicemia

seems possiblethat bacterial proteases facilitatebacterial invasion of the vascular system through potent tissue destruction(9, 17, 20, 25). However,the detailedmechanism of bacterial invasion of the vasculature and entry into the blood circulation remains unclear. We have previously demonstrated that a number of microbial proteases from pathogenic bacteria and fungi activate the bradykinin-generating cascade at one or more steps, i.e., Hageman factor, prekallikrein, and/or high-molecular-weight kininogen (11, 12, 17-19, 21, 24). Thus, bradykinin generation is stimulatedby these bacterial proteases during the infection and acts as a universal mediator in an inflammatory reaction, producing pain, edema formation, and relaxationof vascu-

Pseudomonas aeruginosa is now widely recognized as an important opportunistic pathogen often causing infections complicated by septicemia and sepsis syndrome (3), a serious medical condition in which the host's immune system overreacts to the infection (6). Septicemia is one of the most critical consequences of systemic bacterial infection (27). Most frequently, gram-negative bacilli gain access to the blood from extravascular septic foci via the lymphatics or by the direct invasion of small blood vessels within a local site of infection (16). P. aeruginosa produces a number of extracellular products: exotoxin A, exoenzyme S, and extracellular proteases such as pseudomonal elastase and alkaline protease (5). Accumulating evidence indicates that bacterial extracellular proteases play a critical role in the pathogenesis of various bacterial infections (17). It

Abbreviations: DTPA,

*Address correspondence to Dr. H. Maeda, Department of Microbiology, Kumamoto University School of Medicine, 2-21, Honjo, Kumamoto, Kumamoto 860, Japan. 415

BK, bradykinin;

CFU,

diethylenetriaminepentaacetic

aminetetraacetic

acid;

charide;

ovoM,

ovomacroglobulin;

buffered

saline,

colony

acid;

LD,0, 50% lethal

dose; LPS, PBS,

pH. 7.3; PE, pseudomonal

forming

EDTA,

unit;

ethylenedilipopolysac-

10 mm phosphateelastase.

416

Y. SAKATA

lar smooth muscle. Recent investigations revealed that bradykinin generated by either pseudomonal elastase or lipopolysaccharide (LPS; endotoxin) plays an important role in the pathogenesis of septic shock (13, 14). In this experiment, we investigated the role of the pseudomonal elastase and alkaline protease in triggering septicemia caused by P aeruginosa, in view of the in vivo action of bradykinin. The present results indicate that pseudomonal extracellular protease facilitates septicemia as well as toxemia through activation of the bradykinin-generating cascade. Materials

and Methods

ET AL

previously (30). Briefly, various concentrations of Pseudomonas proteases such as Pseudomonas elastase and alkalineproteasewas incubatedin the reactionmixture containing2.5 mg/mlof azocasein(AE:Z=30.1) in 40 mm sodium phosphate buffer (pH 7.0) at 37 C for 30 min. Each protease activity was expressed as unit defined as the activity which hydrolyzes 1.0 mg of azocasein/hr. In some experiments, the proteases were preincubated with two types of inhibitors for Pseudomonasproteases,e.g., ovoM and Zinkovinhibitor, at molar ratios of 1:2 to 1:10 (protease vs. inhibitors)at 37 C for 1 hr, followed by protease assay as just described. Bacteria

Animals. Male ddY mice (SPF grade, 6 weeks old, Japan SLC, Shizuoka) were used throughout the study. Pseudomonal elastase and alkaline protease and pseudomonal toxins. Purified P. aeruginosa elastase (39 kDa) and alkalineprotease (48 kDa) were purchased from Nagase Biochemicals, Osaka, Japan. LPS from P aeruginosa serotype 10 (Habs) was purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. P aeruginosa exotoxin A was a kind gift from Dr. Hiroshi No2uchi, Sumitomo Seiyaku Co., Ltd., Osaka, Japan. Oth(7rrea8ents.

Bradykinin

was purchased

from Pep-

tide Institute Inc., Osaka. Captopril and Glu-Trp-ProArg-Pro-Glu-Ile-Pro-OH(SQ20881) were a gift from SankyoCo., Ltd.,Tokyo,and Squibb Institute,Princeton, N.J., U.S.A., respectively. DL-2-Mercaptomethy1-3guanidinoethylthiopropanoicacid (Plummer's inhibitor) was purchasedfrom Calbiochem,La Jolla, Calif.,U.S.A. Diethylenetriaminepentaaceticacid (DTPA) anhydride was obtained from Dojindo Laboratories, Kumamoto. 5℃rCl

3waspurchasedfrom

ICN Radiochemicals,

Costa

Mesa, Calif., U.S.A. Chicken egg white ovomacroglobulin(ovoM) was a gift from Otsuka Pharmaceutical Co., Tokushima. 2-(N-Hydroxycarboxamido)-4methylpentanoyl-L-alanyl-glycine amide (Zincov inhibitor) was kindly supplied by Dr. NorikazuNishino, Kyushu Instituteof Technology,Kitakyushu. The Toxicolor limulus test, a sensitive quantitative assay kit for bacterial endotoxin, was purchased from Seikagaku Kogyo, Tokyo. An enzyme immunoassay kit for bradykinin, MARKIT M, which was developed recently by Prof.Makoto Katori,Kitasato UniversitySchool of Medicine, Kanagawa, and is 100 times more sensitive than a previous immunoassay kit (MARKIT A), was suppliedfrom DainipponPharmaceuticals,Osaka,Japan. All other chemicals were purchased from local commercial sources. Quantification of protease activity. Protease activity of Pseudomonas proteases was measured by using azocasein (Sigma Chemicals) as a substrate as was reported

nosa

and

were

culture.

used.

isolated

from

Two

One an

within

3

days

large

amount

tease).

PA

nosa

that

was

septic

from

the

of

produces

very

at 33

washed

suspended (i.p.)

in

to

to

kaguma

supernatant

tered

with

volume

by

Danver,

Mass.,

centrated based

on

its

used

0.58

in

Zincov

was inhibitor

The the

which

result

indicated

concentrated

to

in

not that culture

at

81%

size).

be

582.6

of

supernatant

con-

azocasein and

as

alkaline units/rig

respectively.

concentrated

presence

or

culture absence

of

(almost

ratio

of

protease of

90%)

1:10

alkaline the

Inc., the

units/ml

1.25

inhibited a molar

The

one-fifth

of

activity,

Pseudomonas

fil-

(Amicon

showed

the

cen-

was

to

elastase

the

The

by and

pore

against

in

was

621.

activity

found

strongly

elastase but

4 C)

membrane protease

activity

PA

obtained

at

caseinolytic

quantified

pseudomonal

tease:inhibitor)

10

strain strain

concentrated

experiment

of

of

was min

activity

protease

supernatant

that

pseudomonal

this

culture

pseudomon-

kaguma

(0.22-vm

was

units/ƒÊg

Furthermore,

30

The

The

of

kaguma

strain

then

YM

proteolytic

above.

protease

the

a

U.S.A.).

supernatant

described

and

using

bacteria

to obtain

amount

to

for

was

col-

at 4 C)

phosphateThe

P. aeruginosa

filter

supernatant

were

intraperitoneally

aeruginosa

this

a Millipore

resultant

621

Detroit,

min

M

7.3).

mainly

similar

g

PA

below.

P

of

Strain

dissemination.

used

of

(20,000 •~

P. aerugi-

30

0.01

given

described

supernatant

trifugation

a

of

bacteria

(pH

a high

a manner

death

pro-

(Difco,

for

in

was

activity.

in

The

then

supernatant

cultured

hr.

intravascular

contained

its protease

culture

were

as

Culture

broth

(PBS)

strain

al exoproteases

and

16

times

allow

that

exoprotease.

(20,000 •~g

PBS

mice,

The

C for

M saline

in produces

strain

infusion

three

buffered/0.15

septicemia,

strain

isolated

little

centrifugation

had

(elastase/alkaline

in brain-heart

by

and

exoproteases

was

who

resulted

This

aerugi-

strain,

severe

that

onset.

is a clinically

U.S.A.)

lected

with

shock

621

cultured

Mich.,

and

of P

kaguma patient

pneumonia

emboli,

strains

the

immunocompetent

community-acquired septic

different

strain,

(pro-

protease. activity

kaguma

of strain

BACTERIAL

was

attributed

residual

to

fraction

dent

on

the

of

i.p.

with

P.

P.

aliquot

ing

(100 ƒÊg

kininase bacteria,

ture,

and

was

bers

in

es,

after

used

of

a

the that

selective

medium

ide

(NAC

The growth

of

pseudomonal

elastase

on

of P

aeruginosa

Specifically, with

or

into

without

observed In

24

a separate

aeruginosa

2.0 ture with the the

mg/ml

for

with

elastase)

supernatant these spread

of

both

suppressed

P

of

bacteria

elastase almost

Zinkov alkaline

or

of of

at 5.0 and

1 hr

aeruginosa then

into

the

in

P.

the

inhibitor

culture

protease

and

its

only

treated

supernatant

of by

on

partially

621. kagu-

treatment

However,

ovoM (at

maxi-

of

the

to

in

this

were

assay

quantitated

M (19).

but

not

thus, kinin

analysis.

punc-

min

after

and

concen-

centrifugation supernatant

kinins

with to

was

the

the

enzyme

instructions and

kallidin

des-Arg-bradykinin,

the

the

depro-

(final

bradykinin

to

only

inhibitor

immediate

according Both

lavage

ethylenedi-

cardiac

15

resulting

immunoaffinity

in our

Statistical

by

samples

MARKIT

kit;

by and

acid

of

of

Peritoneal

0, 5,

quantitation

assay

administration

obtained

The

blood

a kininase

followed

of

enzyme

1 mM

and

stimulation

Generation circulating

mice.

(EDTA)

the

manufacturer

similar

and

containing

min).

(Lys-bradykinin), quite

and

PA

inhibited ovoM.

cul-

circulation

subjected

10

by

i.p.

to

determined counter.

vivo.

trichloroacetic

to

for

Fifteen

sensitive

lavage,

"Cr-

physiological

a gamma

after

elastase,

6%) g

corn-

A was

in

(50 ƒÊg)

adding

immunoassay at

effect

of

(10,000 •~ then

both

The

strain

by

sodium

(200

cavity

samples

of

generated

PBS

peritoneal

teinization

on

radioactive

exotoxin

a highly

acid

of

band

A

200 ƒÊl

with

M)

with

Blood

tration

inhibitors for

aeruginosa

completely

was

of

using

elastase

without

furThe

electrophoresis

bradykinin

(MARKIT

administration

inhibitor

for

systemic

ture,

of P

C in PBS.

tested

cocktail.

was

chelation.

the

in

peritoneal

by

performed

or

with

bradykinin

the

pseudomonal

P

bacteria.

mg/ml

kaguma

was

by

the

in

kinins

i.p.

exoproteases

examined

separated Uppsala,

exotoxin

radioactivity of

by

8.5),

detection.

amount

the

then

conjugate

gel

without the

aminetetraacetic

injected mice

the

Zincov

at 37

of

or

later,

with (pH

a homogeneous

original

(540 ƒÊg)

pseudomonal

was

0.5 •~

supernatant with

i.p.

LIDO

was the

A

Quantitation

for

inves-

or

0.4 •~

LD50)

culture

(ovoM

induced

was

CFU,

rate

treated

elastase, for

inhibitors

lethality

strain

was

by also

supernatant [El,

0.2 •~

the

proteases and

was

administration

strain

protease

10

survival

after

kaguma

Pseudomonas ma

hr

(4 •~ culture

(50 ƒÊg, The

621

injected

bradykinin

were

the

were

counting

was

showed

A

labeled

buffer

"Cr

autoradiographic

with

with

purified

produced

PA

(100

experiment,

of pseudomonal alkaline

621

either

mice.

for

agar

and

lethality

strain

elastase the

the

PA

kaguma

pseudomonal i.p.

effective

NAC

proteases

administration

aeruginosa

the

by

(7).

of

exotoxin

minutes

was

pseudomonal

LD,o)

cetrim-

Tokyo)

showed

in

saline a

aeruginosa.

effect

tigated.

that

appearance

acid

Co.,

with

Mice

agar

that

A

was

Pharmacia,

sulfate-polyacrylamide

labeled

onto

exotoxin

serum.

conjugated

A

radioactive

the exotoxin

was

DTPA-exotoxin

after assay

in

A

A conjugate

with

to

bined

that

soy

colony

nalidixic

agar

dodecyl

dish-

The

LPS

bicarbonate

G-25,

the

min

a commercial of

A

into

30

pseudomonal

(Sephadex

kaguma

administered

exotoxin

mM

DTPA-exotoxin filtration

-labeled

similar

of

10

of

aeruginosa,

was

by

exotoxin in

P

obtained

quantitation

spread

protease

toxins from

(200 ƒÊg),

circulation,

Briefly,

labeled

and

to

tryptic

Chemical

soy

Petri

the gel

rub-

Species

isolated

aeruginosa,

tryptic

in similar

on

the

(Eiken

and

performed.

colonies

P

a typical

as P

The

for

on

and

counted

was

inoculating

agar)

colonies

saline

soy

rinsing

and

ther "Cr

the

anhydride

Sweden).

the

mesh

"Cr.

DTPA

by

was

tryptic

by

sterile

assay

sample

formed by

using

followed

colony-forming

of punc-

blood

experiments,

physiological

blood

identified

agar

assay

with a

for systemic

bacterial

analyzed

for

was

the

treatment.

obtained

samples

were

inhibitor

of

supernatant

of

blood

test)

vs.

10%

inhibitor

bradykinin

LPS

test the

culture Zinkov

(1.0 ƒÊg)

and

of

To

as

injection

in the

without mice,

protease

about

dissemination

(Toxicolor

into

Plum-

cardiac

some

with

kit

into

to

after by

bacteria

surgically

and

referred

taken

In

sterile

which

bacteria

were

viable

grinding

i.p.

or to

thus

the

and

LPS

injection or

(kinin-degrad-

hours

was

with

purified

injected

for

of the

and

in

ovoM

Assay

ratio

1:10,

remains after

i.p.

if the

to

bloodstream.

of

PBS),

SQ20881,

a colony-forming

by

with

of

of

removed

1 ml

were

Three

Laboratories).

homogenization

200 ƒÊl),

henceforth

sample

107 exo-

together

200 ƒÊl

even up

strain

supernatant

kininase

captopril,

cocktail.

by

(Difco

spleen

of

number

quantitated agar

PBS)

a mixture

a blood the

or in

in 200 ƒÊl

(5 •~ little

bacteria

(100

(100 ƒÊg),

inhibitor

the

20%)

increased

inject-

417

SEPTICEMIA

mum,

depen-

were 621

culture

(50 ƒÊg

inhibitors:

inhibitor

other

be

produced

strain

strain

or without

enzyme)

mer's

621

elastase

with

PA

which

concentrated

kaguma

bradykinin mice

the

Mice

strain

PBS), PA

of

aeruginosa

the

to

AND

activity

aeruginosa

pseudomonal

and

thought

dissemination.

Similarly,

an

was

protease.

200 ƒÊl

protease.

elastase

activity

bacterial

CFU/mouse;

with

the

alkaline

Analysis ed

Pseudomonas of

PROTEASE

show

antikinin

antibody

B2-receptor

used

agonists

are

assay. All

values

±SE

of three

or four samples.

mice

treated

with

P.

are

expressed

as

The survival

aeruginosa

pseudomonal proteases were evaluated Fisher's exact probability test.

with

means

rates of

or without

statistically

by

418

SAKATA

Fig.

1.

Effect

of

culture

supernatant

protease-producing mouse), P

strain

which

ture;

spleen

use

not

treated

the

colony-forming

of

was

also

proteases

P.

aeruginosa

PA

obtained

i.p. and

and

protease

to

mice.

then

621.

Mice

was The

with

Three

hours The

soy

with

of

agar.

viable

PI,

of

aeruginosa i.p.

200111

of

culture 621

in in

for

the

aeruginosa

PA

the

and

the

the

taken

by

treated

text

for

10

nonCFU/ of

cardiac

with

punc-

inhibitors

of

homogenate

See

the

supernatant

administration

spleen

proteases.

was

strain

to

(5 •~

culture

sample

kaguma

of

621

concentrated

similar

blood

dissemination

a blood

of

pseudomonal

intravascular

10-fold

bacteria,

a manner

the

P.

of the

supernatant

PA

on

with

sample

injection

bacteria

inhibitors

P

injected

or

after

administered numbers

tryptic

strain

were

a 100-

homogenized.

ovoM)

inhibitor.

assay

kaguma

Simultaneously,

given

(Zincov

with

protease-producing

exoprotease.

strain

was

pseudomonal natant

little

kaguma

the

of

produced

aeruginosa

of the

ET AL

culture

of super-

were

quantitated

on

aeruginosa

by

details.

Results

Enhancement of Bacteremia by Pseudomonal Proteases and Involvement of Bradykinin P.

aeruginosa

increase of

in

culture

With

the

200-ƒÊl

was

of

supernatant

of

P

a

dose-dependent

the

after

kaguma

in

the

into

the

blood

and

of

the

supernatant

10-fold

effect

strain PA

1).

dissemi-

and

This

kaguma

aeruginosa

(Fig.

such

blood

controls.

injection

strain

supernatant,

100-fold with of

of cavity

the

injection

compared

semination toneal

showed dissemination

increased

spleen

ture

621

supernatant

the

nation

PA intravascular

of

in

the

cul-

in promoting

621

(from

spleen)

was

dis-

the

peri-

abrogated

by

Fig.

2.

621

bacteremia.

Effect

purified

preincubation of

pseudomonal

the

proteases

values

without

respectively

(Fig.

the

the

substantial

due

event,

these in

facilitated

the to the

the

confirm on

culture

621

the

was in

the

of

the

in Fig.

effectively

bacterial

protease.

of

the

enhancing

effect

i.p. manner

colony

formation

puncture

of

is In

any

protease kaguma

together as

in

P 1.

(50 ƒÊg) (KI)

aeruginosa

after text

cells with

was in

in

P.

PA with

presence

or absence

same

manner

quantitated

blood

of

P

administered the

the

were

the

injection for

(PE)

621

cocktail

sample

as in

by

use

the

Fig.

of

obtained

by

aeruginosa

and

pseudomonal

exhibited

potent

the

cardiac

details.

2, pseudomonal

In

pro-

shown

the

addition,

test

this

ment

inhibitors)

from in

was

the

2),

generated

by

cavity i.p.

injection

inhibitor

vs.

by

cocktail by

elastase

indicating

into

P the

of

the

blood

involveelastase.

aeruginosa

bradykinin

a t-

with

pseudomonal of

blood.

enhanced

determined

alone

strongly

augmeninto

was

0.025

dissemination

peritoneal

increased

aeruginosa

a kininase

(PC

elastase

(Fig.

bradykinin

of

elastase data,

P

dissemination

injection

the

unpaired

of

of

increased

by with

for

elastase

dissemination

Furthermore,

aeruginosa As

of

significantly

pseudomon-

with Fig.

PA

elastase

assay

3 hr

together

strain

pseudomonal

purified

P

See

tation

ninase

bacteremia,

inhibitor

viable

Fig.

inhibitors

pseudomonal

supernatant

pseudomonal

1. The

elastase

aeruginosa

inhibit

translocation

alkaline

P

a kininase

elastase.

Therefore,

protease

that

1,

primarily

above.

the

spleen,

dissemination.

injected same

and shown

as

of

one-seventh

inhibited not

in

suggest

Pseudomonas

al elastase

did

pseudomonal

results

blood

described

presence

bacterial

To

but as

inhibitors

and

experiment

increment

in

mostly

the

the

used

protease

observed

tease

In

elastase,

alkaline

PA

1).

specific

one-tenth in

inhibitors

the

activity

to

inhibitors

protease

Pseudomonas

with

of pseudomonal

PA621 and

spleen (100 ƒÊg)

ki-

BACTERIAL

PROTEASE

AND

419

SEPTICEMIA

A

Fig.

4.

Kinin

generation

pseudomonal

B

blood

elastase

was

measured

at various

time

PBS)

given

was

Fig.

3.

Mice

Acceleration were

absence

of an

aeruginosa

with

authentic

assay of

in

the

was

the

the

bacteria

of and

of P

to

kininase

the

kininase

the

inhibitor

Effect

as

in

cocktail.

The

A,

in

See

the

text

and

with

the

time

3).

This

promoting

simultaneous cocktail,

which

resulted

in

peritoneal In i.p.

of

the

details.

cavity addition,

kinins.

kinins

by

kinin blood

was

contrast,

lavage In

fluid

a separate

ty was

in

found

circulation.

4, an the

However, that

were enzyme

experiment, peritoneal

vivo

by not

mice

within

15

endogenous in

the (•ƒ0.1

of

the

of

given

profile

of in

min

the

after

kininases.

kinin-degrading

cavity

mice

generated

immunoassay strong

after

to

time

kinin

detected

the

amount

of the

the

in

immunoassay

blood

rapidly,

kinins

in

exoprotease

appreciable

possibly

in the

blood

and

kinins

bradykinin

enzyme

decomposed

our

of

Fig. in

by

bradykinin

a specific

showed

injection,

the

peritoneal ng/ml). activi-

mice

PBS, for

(50 ƒÊg

of

a vehicle

was the

mice,

given

i.p.

in

for

and

elastase-treated

kinins 100

Ill the

kinins

in

group.

See

of

pseudomonal i.p.

amount

of

quantitated

pseudomonal

by the counter.

LPS

to

LPS use

mice and of

text

with A

of for

by

'Cr-labeled

in

commercial

radioactivity See

or

together exotoxin

a

toxemia

'Cr-labeled

bradykinin

exotoxin bradykinin

the

blood

assay

kit

A

was

(200 ƒÊg). circulation

for

exotoxin

LPS

was and

A

with

by a

details.

by

inhibitor

action

pseudomonal

elastase.

was

in of

produced

generation

elastase

even

using

shown

pseudomonal

In

and

of

As

of

biological

generation

enhanced

kininase

Enhancement Either

counting

further

the

degradation

prolonged

examined

for

was of

inhibited

administration

was

effect

administration

ill

for

elastase

the

details.

administered The

bradykinin

absence

(BK).

gamma

(Fig.

immunoassay

group

as

in

on

(KI)

or

5.

100

quantitated

of

generated

for-

examined

on

bacteria

Fig.

control

administration

injec-

inhibitors

presence for

colony

was

bradykinin

for

enzyme

pseudomonal

In the

elastase, were

an

i.p. of kinins

of P

3 hr after

cocktail

of

text

using

after

after

amount

or

number

kininase

blood

i.p.

mice

The

(BK). presence

the

at

bacteremia

bacteremia.

mice,

of

1,

inhibitor

B.

the

The

use

Fig.

of of

bradykinin.

aeruginosa

given

effect

potentiation

administration

in

in

A.

by as

bradykinin

621

(100 ƒÊg).

manner The

by

PA

quantitated

same

bacteria.

by

profile

induced

aeruginosa

bradykinin

bradykinin-induced

were

P

organisms

mation tion

of bacteremia

injected

the

by

points

pseudomonal

in

(PE).

(data

not

shown). Moreover,as described below, various materials such as bacterial toxin and LPS as well as bacterial cells seem to be deliveredquicklyto the blood circulation after simultaneous i.p. administration of bradykinin. Rapid clearance appears to occur in the case of kinins generated de novo in the peritoneal cavity of the mice. Thus, failureto detect kinins in peritoneallavagefluid of mice injected with the pseudomonal elastase is most likely due to the rapid degradation and clearance of kinins from the peritoneal cavity. All these results indicate that Pseudomonas exoproteases significantlyenhancebacterialdisseminationfrom local bacterialfoci to the systemic circulation,at least in part through bradykinin-generatingactivity. In fact, the lethal effect of PA 621 was markedly augmentedwhen this strain was administeredi.p. to the mice together with either the culture supernatantof the

420

Y. SAKATA

Table 1. Lethality natant

Fig. 6. A schematic sible involvement

induced

of P aeruginosa

representation

by P aeruginosa

kaguma

of the mechanism

of the bradykinin-generating

kaguma strain or the purified Pseudomonas (Table 1).

PA 621 in mice enhanced

by culture

super-

strain

of bacteremia

cascade

ET AL

activated

elastase

Enhancement of Pseudomonal Toxemia by Bradykinin To detail the action of bradykinin in Pseudomonas bacteremia, the effect of bradykinin on the movement of

and bacterial by bacterial

toxemia proteases

mediated

by bacterial

in the spread

proteases.

of bacterial

Note the pos-

cells and toxins.

pseudomonal endotoxin and exotoxin, such as LPS and Pseudomonas exotoxin A, from the peritoneal cavity into the blood circulationwas investigated.The delivery of LPS and 'Cr-labeled exotoxin A was significantly enhanced when these toxins were given to the mice with the bradykinin (Fig. 5); the blood concentration

BACTERIAL of LPS was

increased

4-fold,

and that

showed an almost 2-fold increase. These data suggest that bradykinin in the septic foci, which

could

of exotoxin

generated

be stimulated

al proteases, enhanced not only bacteremia toxemia of P aeruginosa.

PROTEASE A

de novo

by bacteribut also the

Discussion

It is now well known that the virulent P. aeruginosa produces a number of extracellular toxic factors, e.g., exotoxinA, exoenzymeS, elastase,and alkalineprotease (5). Much attention has been paid to the important role of pseudomonal proteases (elastase and alkaline protease) in the pathogenesisof P aeruginosa infection (2, 9, 10, 14, 15, 17, 22-25). In particular, we have been studying the pathogenic potential of pseudomonal proteases and focusing on their potent stimulatory activity for bradykinin generation (17, 24). We previously reported that extracellular proteases from various microbes includingP aeruginosa activate the bradykinin-generatingcascade at several steps in vitro (12, 17, 21) and cause increased vascular permeabilityin vivo(10, 11, 17, 24). Both pseudomonal elastase and alkalineprotease proteolyticallyactivate Hageman factor and also act directly on high-molecularweight kininogens, which is followed by generation of bradykinin (17, 24). Thus, generation of kinins triggered by bacterialproteasesin the septic foci may be one of the most critical reactions in the host infected with pathogenicbacteria. The results obtained in this experiment make it clear that extracellular proteases of P. aeruginosa and bradykinin, which is produced endogenously via activation of the kinin-generatingcascade by pseudomonal protease or exogenously administered to mice, facilitated the bacteremia as well as toxemia caused by P aeruginosa. In addition, kinin-potentiating kininase inhibitors augmentedsuch effects, putatively involving activation of the kinin-generatingcascade by virtue of their inhibiting kinin degradation and enhancing local kinin concentration. This is the first definite demonstrationindicatingthe possibleinvolvementof the kiningenerating cascade in the mechanism of P aeruginosa invasionof the systemiccirculationsystem,which causes septicemia. Although the mechanism of enhanced intravascular dissemination of bacterial cells and toxic products as causedby bradykininis still incompletelyunderstood, it may be that the invasionof P aeruginosa and passageof bacterial toxins toward the intravascular lumen are amplified by bradykinin through its potent action in causing the intracellularjunctions of endotheliumin the

AND SEPTICEMIA

421

vasculature to open up (1). Enhanced vascular permeability inducedby bradykininwill result in buildupof the tissue hydrostatic pressure, which will then facilitate bacterial translocation from local septic tissue to the inside of the vascular lumens. Furthermore, it is possible that exacerbationof bacteremiaand toxemiamightbe caused by increased lymphatic flow that is accessible to the circulation system. Specifically,bacterial cells and toxins could enter the blood circulation system via the increased lymphatic flow. In any event, a novel pharmacologicalaction of kinin, i.e., enhancement of intrusion of bacterial cells and products, is clarified, and this effect may have implications for the pathogenesis of septicemiacaused not only by P aeruginosa but also by other species of pathogenic bacteria. A number of reports have demonstratedthe strong tissue-destructivepotency of pseudomonal elastase in various animal models of P aeruginosa infection, such as pulmonary and corneal infections (20, 23) and dermal infection of burned sites (10). This potent action of pseudomonalproteasesis based on the proteolyticdegradation of matrixproteins,e.g., elastin,denaturedcollagen (gelatin), and fibronectin (25). In addition, our recent investigationindicatesthat pseudomonalelastase strongly activatedmatrixmetalloproteasessuch as progelatinase and procollagenase,which were purifiedto homogeneity from human neutrophils, through limited proteolysis of the zymogens by the bacterial protease (unpublished observation).A similar observationin a model of corneal infection with P aeruginosa in rats was obtained by Matsumotoet al (20). Further,it has also been reported that pseudomonalelastase destroys a wide range of substances of the host used for defense, such as immunoglobulinA (8), complement (28), and a plasma serine protease inhibitor (26), and that it suppresses neutrophil function (15). Therefore, in addition to the direct destruction by pseudomonalproteasesof host tissues and defense mechanisms, activation of the bradykinin-generatingcascade by the Pseudomonas exoproteases might accelerate the triggeringof septicemiaof pathogenic bacteria showing strong tropism for blood vessels. It was recently reported that pseudomonal proteases are the most important factor promoting P aeruginosa septicemiain a mouse model of gastrointestinalinfection (4). Moreover, Holder et al (9) and Snell et al (29) found that P aeruginosa virulence is maximally expressedwhen both Pseudomonas exotoxinA and exoproteases are secreted by the pathogenic strain of P aeruginosa. This synergistic effect of proteases and exotoxin may be explained by the effect of bradykinin, generatedby stimulationwith the proteases at the infectious foci, on delivery of pseudomonal toxins to the

422

Y. SAKATA

systemic circulation, as shown in Fig. 5. Yamamotoand coworkersrecently showed the important role of kininsin the pathogenesisof septic shock analyzed in a guinea pig model by using purifiedPseudomonas elastase (14). Their experiment suggested that kinin, of which generation is stimulated by the pseudomonal elastase in the systemic circulation, is the responsible mediator in the induction of hypotension and shock in P aeruginosa infection. Therefore,kinins induced by Pseudomonas exoproteases may play the most critical role not only in bacterial invasion of the blood circulation,triggering bacteremia,but also in septicemia, as in shock and multiple organ failure. In conclusion, as summarized in Fig. 6, it is strongly suggested that pseudomonal proteases as well as bradykinin generated in infectious foci are crucial principles in the pathogenesisof Pseudomonassepticemia,in that they facilitatebacterialdisseminationand exacerbate septicemia. Septicemia and sepsis syndrome are often encountered as serious complications of opportunistic infections caused by gram-negative bacilli (such as P aeruginosa) with multiple drug resistance. Therefore, attempts to treat septicemia by focusing on the host's derived factors such as kinins and the endogenous protease cascade now become essential.

ET AL

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We thank Ms. Judith Gandy for editorial review, and Ms. Rie Yoshimoto for typing the manuscript. Thanks are also due to Prof. Makoto Katori for his generosity in providing the enzyme immunoassay kit MARKIT M. This work was supported by a Grant-in-Aid for Scientific Research from the Monbusho (Ministry of Education, Science and Culture), Japan, and by a grant from Yakult Honsha Co., Tokyo.

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