Suppression of Late Phase Enhanced Vascular ... - CiteSeerX

Journal

of Leukocyte

Biology

48:258-265(1990)

Suppression of Late Phase Enhanced Vascular Permeability in Rats by Selective Depletion of Neutrophils With a Monoclonal Antibody Sakae Department

Sekiya,

Takao

of Parasitology,

Yamashita,

Yamagata

and

University,

School

Fujiro

of Medicine,

Sendo Yamagata,

Japan

We investigated the role of neutrophils in increased vascular permeability by a selective reduction of neutrophlls usIng a monoclonal antibody, RP-3. An intraperitoneal Injection of RP-3 not only selectively depleted peripheral blood neutrophils, but prevented the neutrophil Infiltration to the tissues. Proteose peptone, zymosan, and BCG induced three different types of Inflammatory edema, showing the early phase only, early plus late phase, and the late phase only, respectively. Only the late phase response of zymosan and BCG was Inhibited by a depletion of neutrophils by RP-3, though the early phase response induced by proteose peptone and zymosan was not affected. Reconstitution of neutrophil-depleted rats by In situ injection of these cells restored the inflammatory edema induced by BCG, depending upon the number of neutrophils injected. Key words:

edema,

zymosan,

BCG

INTRODUCTION Neutrophils serial not

infiltrate

response only

the tissues

from

play

a major

role

[ 1 8]

,

leukotriene

to

B4

against

inflammation

[ 1 ,35] [2]

,

phase

immunity.

in defense

infection, but may modulate by producing active oxygens factor

in the early

inflammation

bacterial

and ,

and

of a They

immunity

platelet activating interleukin 1 like

molecule

[7,31]. The possible role of neutrophils in creased vascular permeability has been discussed many years [3, 13, 15,34], but a definite conclusion not been obtained. Recently, many in vitro experiments have shown the interaction between neutrophils and

dothelial tended,

cells

[4,6,8].

Considering

in the

present

study,

these

to clarify

results, the

role

infor has

Pharmaceutical Co. , LTD., gradient (Pharmacia Fine

cimetidine (Fujisawa Japan); Ficoll-Isopaqe

Chemicals,

Uppsala,

Sweden);

(MEM) (Nissui Japan); ‘25!-bovine

tial

me

Division

California);

solution,

and

FRG);

and

Co.,

LTD.,

Eagle’s

minimum

Pharmaceutical serum albumin

medium

Tokyo, Radiochemical,

of ICN

we inof neu-

established [27]. In this paper we demonstrate late phase increased vascular permeability is deon the in situ activation of neutrophils.

Biochemicals,

May-Gruenwald’s

Shizuoka,

Japan).

bred

tered

air flow

(clean WKA/Hkm from old

WKA/Hok rats

Cell

rats

and farm

were

rats

bacteria-free

in the Yamagata

Funabashi male

(WKA)/Hok

in a pathogenic rats). SPF

University

Specific BALB/c

Animal

Japan).

in the

METHODS

tion, used were zymosan (Sigma Chemical MO); Bacillus Calmette-Guerin (BCG) Industries LTD. , Tokyo, Japan); pro-

teose peptone (Difco hydramine (Tanabe © 1990

Wiley-Liss,

Laboratories, Pharmaceutical Inc.

Detroit, Co.,

MI); LTD.,

diphenOsaka,

Received

kept by

fil-

Center

pathogen free (SPF) mice were purchased

(Shizuoka, used

were

condition

Six

to nine

wk-

experiments.

Preparation neutrophils.

Ten

milliliters

of 3% proteose

were injected into the peritoneal cavities Twelve hours later, the same volume of proteose was again administered. Three hours after the

Chemicals The chemicals Co. St. Louis, (Kyowa Chemical

Irv-

Giemsa’s

Schiff’s solution (E. Merk, Darmstadt, heparin calcium (Shimizu Pharmaceutical

Wistar-King-Aptakemann and

Rat

AND

LTD., (ICN

,

Inc.

solution,

tone

MATERIALS

essen-

Co. (BSA)

Animals en-

trophils in the formation of inflammatory edema. To this end, we used a monoclonal antibody that selectively depleted rat peripheral blood neutrophils which we had recently that the pendent

Japan); Osaka,

peritoneal June

Reprint requests: gata University Japan.

exudate 12, 1989;

cells

accepted

(PEC) January

were 22,

pep-

of rats. peptone last injec-

obtained

by

1990.

Fujiro Sendo, Department of Parasitology, School of Medicine, lida-Nishi, Yamagata,

Yama990-23,

Vascular peritoneal

lavage

MEM

with

fetal calf serum (FCS) with Ficoll-Isopaque

with rifled I .090)

according

to

the

(pH

7.4)

supplemented

gradient method

Vascular

(specific previously

count

count

Monoclonal

Antibody

We used a monoclonal tively depleted peripheral intraperitoneal

were

its nature were described elsewhere [8,27]. peritoneal neutrophils of WKA/Hok rats into BALB/c mice, and their spleen cells

hybridized

[14]

antibody (RP-3) that selecblood neutrophils of rats by The details of the establishment

injection.

of RP-3 and Briefly, the were injected

with

according

P3-X63-Ag8.653

to the

Then

a hybridoma

fluid

containing

centrifuged

Ten

Oi

(RP-3) the

at

titer

was

and

the

ascitic

trol

fluid)

ascitic

(74D1

was

fluid.

1 (1gM))

The that

The

P3-X63-Ag8.6S3

which

and

were

were

also

with

and

in

of by

cells

control.

The

the

cells (Xas a con-

hybridoma

of Complement

mice

japonicum

[28]

monoclonal

an-

eggs,

ture was

jected various

ascitic

fluid

of

into the peritoneal reagents were

inflammatory

RP-3,

but

not

and were

1 , or X-63

was

in-

cavities of rats. Six hours later, intradermally injected to evoke

complement SRBC

required

in 7.5m1

Statistical was

Inhibition Vascular of RP-3

The method used.

described

First,

was shaved, intradermally

Permeability

by Hellewell

the hair

and 0. 1 ml of solution injected in triplicates.

ments were repeated hr, the mixtures

and Williams

of the abdominal

at least

twice.

skin

[ 10]

of the rats

ability

the

skins

of the

borer.

skin

counted

was

the skin

was

The

vascular

of

of S x 108

The

Student’s

in the

of

ascertain

that

rats

were stripped off and the inout in 16 mm-diameter pieces readioactivity of the plasma and the of

74D

rats

(Fig.

2A).

neutrophils number RP-3

of

vascular

permein

that

a dose-

had

received

the

increase

but

almost with

another 3.5 The vascular

increased

rats

was

completely RP-3

sup-

within

the

ex-

mentioned were

above (Fig. 1). Histoperformed in order to did not infiltrate the connective

stimulation

blood observed clump

zymosan

rats

infiltrated

pretreated

in

had been

that numerous zymosan in

of

In the

with

the

rats

were

70.7±8.5,

of

depleted

by

neutrophils untreated

RP-3,

and

had rats

however,

the zymosan (Fig. 2B). infiltrated in the untreated

neutrophils groups

with

neutrophils

hardly

treated

After

The

was

pre-treated

by

RP-3, X-63, or Six hours later,

1 1 , or nothing,

neutrophils

RP-3. It was infiltrated the

to

examined.

in the control ,

perimental condition logical examinations

After

3 mm

t test

Induced Increase in by the Administration

permeability

peripheral

by a ‘y-counter. The plasma volume from the following formula:

calculated

mix-

hemolysis amount

CH0.

± SE. analysis.

zymosan

manner of X-63

which

sites were punched

by a cork

by

even

of PBS added with ‘25I-bovine serum albumin (10 i Ci/kg) and Evan’s blue (1%) were injected intravenously. After 30 mm, heparmnized blood (10 U/ml) was obtained by heart puncture. After sacrifice,

the mean statistical

at 4 hr was

induced

tissues,

4.5

jected

The

and The

as one

One milliliter of the ascitic fluid 74D1 1 was injected intraperitoneally.

of the reagents was The same experifrom

sample.

of 50%

is defined

of Zymosan Permeability

pressed

was

the test

for destruction

medium

The data showed employed for

in

of Vascular

with

Analysis

dependent injections

edema.

Measurement

mixed

zymosan was injected intradermally. hr, 125I-BSA was injected intravenously.

Antibodies 74Dl

PAS-

Titer

were incubated at 37#{176}C for 90 mm determined by absorbance at 541 nm.

permeability

The

paraffin-embedded

RESULTS of

BALB/c

tibody 74D1 1 reacted with S. japonicum with the neutrophils of WKA/Hok rats. of Monoclonal

Measurement

as-

hybridization

of

specimens

and

Hematoxylin-eosinand observed microscopically.

was

of the

myeloma injected

Schistosoma

as another

Administration

was used

,

supernatant

fluid

spleen

infected used

ascitic

was

assay

produced

used. were

dilution,

supernatant

ascitic

was

formalin-fixed

were

cells

(RP-3)

intraperitoneally

percent

[24].

antibody

nearly the same every time. The cites of the parental P3-X63-Ag8.653

Studies

method

produced.

cytotoxicity

(pA/site).

myeloma

It was stored at -40#{176}Cuntil use. of each lot of RP-3 was examined with

mediated

1 ,000

( 1 ml)

The method described by Mayer [23] was used. First, sheep red blood cells (SRBC) were sensitized with the serum of rabbits immunized with SRBC in the optimal

Herzenberg

monoclonal

1 ,700g,

all of the experiments. The antibody a complement

and

=

of the plasma

specimens

stained

259

of the skin

Microscopic

The

and Neutrophils

x

described

[12].

63

permeability

were pugravity

Neutrophils

(1%).

Hyperpermeability

The and

0.7±0.7,

re-

spectively, per field (200 times magnification) from three randomly selected photographed field from the

the dif-

ferent

that

untreated

intraperitoneal

and

treated

administration

rats.

This

of RP-3

result not

only

shows

depleted

260

et al.

Sekiya

a 20

.0 a

15

a,

E

a

10

0 0

a >

5

0 50

500

200 Dose

of

zymozan

Saline

()Jg/O.lmf)

Fig. 1 . Increased vascular permeability induced by zymosan and its suppression by RP-3. The ascitic fluid (1 ml) of RP-3, X-63, and 74D1 1 was i.p. Injected Into rats, and 6 hr later, 50, 200, and 500 .tg/0.1 ml zymosan was intradermally injected. Vascular permeability was examined at 4 hr. The symbols represent: pretreated with RP-3 ., non-treated ., pre-treated with X-63 A, pre-treated with 74D1 1 A, and the control value injected with sterilized saline-. The values show the mean ± SE. The same examinatIon was repeated 4 times.

the number of peripheral blood neutrophils, vented the neutrophil infiltration to the tissues.

but

pre-

Time Kinetics of Inhibition by RP-3 of Increased Vascular Permeability Induced by Zymosan The vascular med.

time kinetics permeability Six

hours

zymosan

(200

riodically control

of suppression induced by

before The

I ml)

phase

appeared within 60 mm. slope that appeared after permeability

was

almost

administration

of RP-3,

affected

3).

(Fig.

administration

vascular was

The

of increased was examof

intradermally

administration

the

rats,

early

i.p. was

the i.v.

untreated

biphasic.

the

after p.g/0.

by RP-3 zymosan

pe-

of ‘25I-BSA. In permeability showed

a steep

curve,

late phase

60 mm. The completely whereas

RP-3,

injected

Fig. 2. Histological examination of inflammatory edema induced by zymosan in rats treated with RP-3. Zymosan (100 tg/ 0.1 ml) was intradermally Injected into the rats non-treated or pre-treated with RP-3 (1 ml) 6 hr before. Three hours later, the rats were sacrificed and the tissues were fixed with 10% formalin. The specimens were stained with PAS. A: Shows the skin tissue of non-treated rats ( x 400). B: Shows that of rats pietreated with RP-3 ( x 400). In non-treated rats, neutrophils infiltrated the clumps of zymosan and phagocyted them. On the contrary, neutrophils were hardly found in the tissue of the rats pretreated with RP-3.

showed late phase suppressed

the early

phase

and

it dis-

a gentle vascular by the was

not

peripheral induced ing doses 0.01

In the previous

paper,

we demonstrated

that

(i.p.) injected

the degree

proportion

antibody

4).

[27]

.

We thus

examined

the

number

and

of injected

and vascular the administration

investigated

RP-3 into

their

permeability of vary-

relation.

Two

to

ascitic fluid were intraperitorats. Six hours later, zymosan

intradermally.

The

number

of

peripheral

blood neutrophils and vascular permeability were exammed at 3 and 4 hr of zymosan administration, respectively, and the relationship between them was investigated. The vascular permeability was decreased in

of reduction of peripheral blood neutrophils by RP-3 was related in a dose dependent manner to the monoclonal injected

of RP-3,

milliliters

neally was

Relationship Between the Number of Peripheral Blood Neutrophils and Vascular Permeability Induced by Zymosan

blood neutrophils by zymosan through

of

trophils

to the (the

falling

coefficient

off

of the

of correlation

peripheral was

blood 0.885)

neu(Fig.

Vascular ability

a,

was

0

other

than

>%

found

that

.0 a, a,

E

a 0

10

0

C,,

and

tively.

As

that BCG shown in

pletely

inhibited.

suggest

that

late

261

of neutrophils

was

edema induced by reagents preliminary experiments, we induced only the early phase

evoked Figure

The

the

and Neutrophils

depletion

only the late one, 5, the early phase

by proteose peptone the late phase edema

ity is dependent

>

by the

to inflammatory zymosan. In the proteose peptone

edema induced whereas

a,

.

inhibited

applicable

25

Hyperpermeability

was not affected by by BCG was almost

results phase

respecedema

shown

in Figures

increased

vascular

and

the early

on neutrophils

RP-3, com-

3 and

5

permeabilphase

is not.

5

The Recovery of Increased Vascular Permeability by Reconstitution of Neutrophil Depleted Rats With In Situ Injection of Neutrophils

-I---

0

0

1

2

3

4

Time

The

Saline

5

Fig. 3. Time kinetics of increased vascular permeability induced by intradermal injection of zymosan. Approximately 6 hr before the zymosan Injection, 1 ml RP-3 was i.p. injected. Zymosan (200 ig/0.1 ml) was intradermally injected at varying times (5 hr-33 mm) before sacrifice. The symbols represent: pre-treated with RP-3., non-treated., saline control - - -. The late phase vascular permeability was suppressed by administration of RP-3. The same experiments were repeated 3 times.

15

neutrophils with RP-3. phils

of BCG

neutrophils into The

was

volume

was serially

after was,

edema tested

on the by

exis-

injection

site in rats and peritoneal

of

pretreated neutro-

intradermally injected into rats in which neubeen depleted by RP-3, and exudate plasma

had

of the

in the

further

the BCG-injected mixture of BCG

trophils injection

induced

was

examined.

mixture

recovery

of vascular

the injection. thereafter,

As shown

of neutrophils

The gradually

in Figure

and

BCG

permeability

within

increased vascular decreased. The

6A,

resulted mm

33

permeability grade of

re-

by injection of the mixture of neutrophils and BCG was dependent on the number of neutrophils added (Fig. 6B). On the other hand, injection of the neutrophils in the absence of BCG did not bring about recovery of hyperpermeability.

0

a a,

of

covery

C,,

.0

dependency

tence

( hr

.

10

.

E

The Effect of Fresh Rat Serum Reduced Vascular Permeability

a,

a a 0

In

the

preliminary

Injection Induced

experiments

we

on the by RP-3

found

that

RP-3

0

injection >

serum.

reduced the complement titer (CH#{216})of the fresh The results obtained by RP-3 may be ascribed not

to neutrophil titer.

serum

0

0

500 Number

1000

of peripheral

neutrophils

1500

Saline

( /mm3)

The reduced complement level of RP-3 non-treated duced

vascular

changed (Table the rat peripheral was not changed.

ability

group

at 3 hr and 4 hi, respectively.

Inhibition by RP-3 of Increased Vascular Permeability Induced by Intradermal Injection Proteose Peptone and BCG We which

examined only the

whether late phase

the above mentioned of enhanced vascular

of

result in perme-

ml of RP-3

0.2

to reduction

was

induced

1). And injection blood neutrophils, The complement

injected with

fresh RP-3.

by

RP-3

to the the rewas

not

of0.2 ml RP-3 depleted but complement titer titer of rats treated by

35.5±0.5CH50,

was

of complement

we iv. pretreated

titer was restored nearly control rats. However,

permeability

Fig. 4. The vascular permeability induced by zymosan was proportional to the number of peripheral blood neutrophils. Varying doses of RP-3 were i.p. injected into the rats. Six hours later, zymosan (200 g/0.1 ml) was Intradermally injected. The numbers of peripheral blood neutrophils and vascular perme-

were examined

but

depletion

In order to test this possibility, of syngenic rats into the rats

and

that

of control

36.5±0.5CH.

DISCUSSION In the present

tive depletion monoclonal

study, of

antibody

we have

peripheral

RP-3

demonstrated blood

inhibited

that

neutrophils

the

late

selecwith

phase

a

re-

262

Sekiya

et al. (B)

(A) 85

a, U,

U,

0

0

:

80

15

.0

.0

a

a

a,

a,

E

E

a,

a,

a

10

a a

a 0

0

0

0

U)

U,

a

a

>

>

5

5

0

0 0

1

2

3

4

(

Time

Fig. 5. induced

InhibItion

by RP-3

by Intradermal

of Increased

Injection

5

of inflammatory

morphonuclear

permeability

peptone

and BCG.

permeability ate [26,36], selectively,

obtained

edema in rats. The role (PMN) in increased

leukocytes

of polyvascular

using nitrogen mustard [32,34], methotrexand anti-PMN sera [21], which deplete PMN has been previously discussed. The results are

still

In

controversial.

depletion of PMN tioned completely

the

earlier

with the various reagents or partially inhibited

edema induced by thermal [22], and passive cutaneous other hand, others have

injury [32], anaphylaxis shown that

studies,

above meninflammatory

bacterial [17,30]. the late

infection On the phase of

increased vascular permeability induced injury or thermal injury was independent tration tion tory

[I I , 16]. of PMN edema

recently,

by methotrexate and PMN

complementary which

More

suggested

agent that

it was

inhibited infiltration,

suppressed the association

only

by ultraviolet of PMN infilshown that depleboth while

the between

inflammaan antilatter [26], PMN ac-

cumulation and edema formation was complex. Moreover, it was demonstrated that PMN played a role in the inflammatory edema induced by the synergistic effect of complement the conflicting ascribed PMN neither PMN blood

and prostaglandin results mentioned

to the fact

that

the agents

E2 [34]. The reason for above may be partially could

not

2

hr

A: Shows the time kinetics of increased vascular permeability by intradermal injection of 3% proteose peptone (0.1 ml).

sponse

1

3

4

truly

deplete

selectively. Our previous experiences tell us that nitrogen mustard nor anti-PMN sera deplete without affecting the number of other nucleated cells (Sendo, unpublished result). Furthermore, it

has not been clarified that PMN depletion causes reduction of phase (early or late?) vascular hyperpermeability. To our knowledge, this paper is the first demonstration

5

(

Time

vascular

of proteose

0

Saline

Saline

hr

B: Shows that of BCG (1 ,000 p.g/slte). Pretreated with non-treated ., and control value injected with sterilized alone - - - . The values shown are the mean ± SE.

‘neutrophils.

stead

in vitro, was

“

of “PMN”

paper that

inasmuch

as we showed

not changed

by i.p.

injection

the notion that the is dependent on neutrophils rats

remains

late

leukocyte

of RP-3

to be clarified, depletion

[16,17,30]. The present of neutrophil cells

restores

This

result

vascular

(Fig.

than

hyperpermeability,

guinea

This

be simply

result

required

endothelial

confirmed

by BCG. 1 ) Emthe endoappearance

direct

the

intrader-

appearance

This result

assumption showing without

with

assumption

increased

for the

monolayers

pigs

suspensions mixed in neutrophil-depleted

ability [ 19] 2) Intradermal injection neutrophils and BCG in neutrophil-depleted .

between for the

because

of the neutrophil an evident edema

6).

edema other

that reconstitution injection of these

through gaps be a prerequisite

the site of inflammation. ported by the previous cross

in

that

cell emigration is not a requisite for permeability, as described by Hurley Emigration of the neutrophil through may

Whether

the inflammatory edema induced evokes several points to be discussed.

mal injection BCG evoked rats

[27].

since it was previously in rats is more susceptible

study has demonstrated depleted rats by local

igration of neutrophils thelial cells may not of

in the previous

phase inflammatory is applicable to species

shown that hyperpermeability to

on in-

RP-3 killed neither eosinophils nor basophils and the number of peripheral blood eosinophils

or not than

saline

of the late phase inflammatory edema We can use the term “neutrophils”

of dependency ‘

RP-3.,

and

that vascular

Spector

the

[1 1].

endothelium of this

cell

at

may be supthat neutrophils increasing

of the

perme-

mixture rats

resulted

of

Vascular

Hyperpermeability

and Neutrophils

263

(B)

(A) 25

50 C,

C’)

U)

-

20

40

>,

>C

.0

(5 C,

30

15

E

E

C,

C,

0.

0.

CO

10

20

.

0

0 U)

(I)

CO

(5

>

> 5

10

....

.

I

I

0

C

C

I

1

5

10

----1-

cc 60

30

90

120

Saline

Number

( mm

Time

of

added Fig. 6. The recovery of Increased vascular permeability in the rats pre-treated with RP-3 by adding back neutrophils intrader. mally. A: Rats were i.p. Injected with 0.075 mI/bOg RP-3. Approximately 6 hr later, the mixture of 5 x 10 neutrophils and 1,000 BCG, and saline alone were Intradermally Injected. B:

TABLE

1 . The

Effect

Reduced Vascular

of Fresh

Rat Serum

PermeabIlity

Induced

Vascular Exp. group Positive control” RP-3 treatment alonec RP-3 treatment plus Fresh rat serum injection” aTwo

milliliters

of RP-3

permeability (p.1/site)

12.0 5.8 5.3

was i.p.

Injection

on the

by RP-3

Complement (CH0)

titer

±

1.70

36.5

±

0.5

±

0.57

3.8

±

0.2*

±

0.80

23.5

±

6.5*

injected,

and 5 hr later 5 ml of fresh

rat serum was iv. injected. One more hour later, 500 p.g/0. 1 ml zymosan was intradermally injected. Vascular permeability and complement titer were examined at 4 hr. The values show mean ± SE. bPositive control were the rats that were not pretreated with RP-3. cTwo milliliters of RP-3 was i.p. injected 6 hr before zymosan injection. “Two milliliters of RP-3 was i.p. injected 6 hr before zymosan injection and 5 ml of fresh rat serum was iv. injected 1 hr before zymosan injection. *P