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

Journal of Leukocyte Biology 48:258-265(1990)

© 1990 Wiley-Liss, Inc.

Suppression of Late Phase Enhanced VascularPermeability in Rats by Selective Depletion of

Neutrophils With a Monoclonal Antibody

Sakae Sekiya, Takao Yamashita, and Fujiro SendoDepartment of Parasitology, Yamagata University, School of Medicine, Yamagata, Japan

We investigated the role of neutrophils in increased vascular permeability by a selectivereduction of neutrophlls usIng a monoclonal antibody, RP-3. An intraperitoneal Injectionof RP-3 not only selectively depleted peripheral blood neutrophils, but prevented theneutrophil Infiltration to the tissues. Proteose peptone, zymosan, and BCG induced threedifferent types of Inflammatory edema, showing the early phase only, early plus latephase, and the late phase only, respectively. Only the late phase response of zymosanand BCG was Inhibited by a depletion of neutrophils by RP-3, though the early phaseresponse induced by proteose peptone and zymosan was not affected. Reconstitution ofneutrophil-depleted rats by In situ injection of these cells restored the inflammatoryedema induced by BCG, depending upon the number of neutrophils injected.

Key words: edema, zymosan, BCG

INTRODUCTION

Neutrophils infiltrate the tissues in the early phase of a

serial response from inflammation to immunity. They

not only play a major role in defense against bacterial

infection, but may modulate inflammation and immunity

by producing active oxygens [ 1 ,35] , platelet activating

factor [ 1 8] , leukotriene B4 [2] , and interleukin 1 like

molecule [7,31]. The possible role of neutrophils in in-

creased vascular permeability has been discussed for

many years [3, 13, 15,34], but a definite conclusion has

not been obtained. Recently, many in vitro experiments

have shown the interaction between neutrophils and en-

dothelial cells [4,6,8]. Considering these results, we in-tended, in the present study, to clarify the role of neu-

trophils in the formation of inflammatory edema. To this

end, we used a monoclonal antibody that selectively de-

pleted rat peripheral blood neutrophils which we had

recently established [27]. In this paper we demonstrate

that the late phase increased vascular permeability is de-

pendent on the in situ activation of neutrophils.

MATERIALS AND METHODS

Chemicals

The chemicals used were zymosan (Sigma Chemical

Co. St. Louis, MO); Bacillus Calmette-Guerin (BCG)

(Kyowa Chemical Industries LTD. , Tokyo, Japan); pro-

teose peptone (Difco Laboratories, Detroit, MI); diphen-

hydramine (Tanabe Pharmaceutical Co., LTD., Osaka,

Japan); cimetidine (Fujisawa Pharmaceutical Co. , LTD.,

Osaka, Japan); Ficoll-Isopaqe gradient (Pharmacia Fine

Chemicals, Uppsala, Sweden); Eagle’s minimum essen-

tial medium (MEM) (Nissui Pharmaceutical Co. , LTD.,

Tokyo, Japan); ‘25!-bovine serum albumin (BSA) (ICN

Radiochemical, Division of ICN Biochemicals, Inc. Irv-

me California); May-Gruenwald’s solution, Giemsa’s

solution, and Schiff’s solution (E. Merk, Darmstadt,

FRG); and heparin calcium (Shimizu Pharmaceutical

Co., LTD., Shizuoka, Japan).

Animals

Wistar-King-Aptakemann (WKA)/Hok rats were kept

and bred in a pathogenic bacteria-free condition by fil-

tered air flow in the Yamagata University Animal Center

(clean WKA/Hok rats). Specific pathogen free (SPF)

WKA/Hkm rats and SPF BALB/c mice were purchased

from Funabashi farm (Shizuoka, Japan). Six to nine wk-

old male rats were used in the experiments.

Cell Preparation

Rat neutrophils. Ten milliliters of 3% proteose pep-tone were injected into the peritoneal cavities of rats.

Twelve hours later, the same volume of proteose peptone

was again administered. Three hours after the last injec-

tion, peritoneal exudate cells (PEC) were obtained by

Received June 12, 1989; accepted January 22, 1990.

Reprint requests: Fujiro Sendo, Department of Parasitology, Yama-gata University School of Medicine, lida-Nishi, Yamagata, 990-23,

Japan.

Vascular permeability =

count of the skin

count of the plasma ( 1 ml)

Microscopic Studies

Vascular Hyperpermeability and Neutrophils 259

peritoneal lavage with MEM (pH 7.4) supplementedwith fetal calf serum (FCS) (1%). Neutrophils were pu-

rifled with Ficoll-Isopaque gradient (specific gravity

I .090) according to the method previously described

[12].

The Monoclonal Antibody

We used a monoclonal antibody (RP-3) that selec-

tively depleted peripheral blood neutrophils of rats by

intraperitoneal injection. The details of the establishment

of RP-3 and its nature were described elsewhere [8,27].

Briefly, the peritoneal neutrophils of WKA/Hok rats

were injected into BALB/c mice, and their spleen cells

were hybridized with P3-X63-Ag8.653 myeloma cells

[14] according to the Oi and Herzenberg method [24].

Then a hybridoma (RP-3) was produced. The ascitic

fluid containing the monoclonal antibody (RP-3) was

centrifuged at 1 ,700g, and the supernatant was used in

all of the experiments. It was stored at -40#{176}Cuntil use.

The antibody titer of each lot of RP-3 was examined witha complement mediated cytotoxicity assay , and was

nearly the same every time. The supernatant of the as-

cites of the parental P3-X63-Ag8.653 myeloma cells (X-

63 ascitic fluid) was intraperitoneally injected as a con-

trol ascitic fluid. The ascitic fluid of the hybridoma

(74D1 1 (1gM)) that was produced by hybridization of

P3-X63-Ag8.6S3 and spleen cells of BALB/c mice

which were infected with Schistosoma japonicum [28]

were also used as another control. The monoclonal an-

tibody 74D1 1 reacted with S. japonicum eggs, but not

with the neutrophils of WKA/Hok rats.

Administration of Monoclonal Antibodies

The ascitic fluid of RP-3, 74Dl 1 , or X-63 was in-

jected into the peritoneal cavities of rats. Six hours later,

various reagents were intradermally injected to evoke

inflammatory edema.

Measurement of Vascular Permeability

The method described by Hellewell and Williams [ 10]

was used. First, the hair of the abdominal skin of the rats

was shaved, and 0. 1 ml of solution of the reagents was

intradermally injected in triplicates. The same experi-

ments were repeated at least twice. After from 3 mm to

4.5 hr, the mixtures of PBS added with ‘25I-bovine se-

rum albumin (10 �i Ci/kg) and Evan’s blue (1%) wereinjected intravenously. After 30 mm, heparmnized blood

(10 U/ml) was obtained by heart puncture. After sacri-

fice, the skins of the rats were stripped off and the in-

jected sites were punched out in 16 mm-diameter pieces

by a cork borer. The readioactivity of the plasma and the

skin was counted by a ‘y-counter. The plasma volume of

the skin was calculated from the following formula:

x 1 ,000 (pA/site).

Ten percent formalin-fixed and paraffin-embedded

specimens were used. Hematoxylin-eosin- and PAS-

stained specimens were observed microscopically.

Measurement of Complement Titer

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

dilution, and were mixed with the test sample. The mix-

ture were incubated at 37#{176}Cfor 90 mm and hemolysis

was determined by absorbance at 541 nm. The amount of

complement required for destruction of 50% of S x 108SRBC in 7.5m1 medium is defined as one CH�0.

Statistical Analysis

The data showed the mean ± SE. The Student’s t test

was employed for statistical analysis.

RESULTS

Inhibition of Zymosan Induced Increase inVascular Permeability by the Administrationof RP-3

One milliliter of the ascitic fluid of RP-3, X-63, or

74D1 1 was injected intraperitoneally. Six hours later,

zymosan was injected intradermally. After another 3.5

hr, 125I-BSA was injected intravenously. The vascular

permeability at 4 hr was examined. The vascular perme-

ability induced by zymosan was increased in a dose-

dependent manner in the control rats that had receivedinjections of X-63 , 74D 1 1 , or nothing, but the increase

in vascular permeability was almost completely sup-

pressed in the rats pre-treated with RP-3 within the ex-

perimental condition mentioned above (Fig. 1). Histo-

logical examinations were performed in order toascertain that neutrophils did not infiltrate the connective

tissues, even by stimulation with zymosan in rats of

which peripheral blood neutrophils had been depleted by

RP-3. It was observed that numerous neutrophils had

infiltrated the clump of zymosan in the untreated rats

(Fig. 2A). In the rats pretreated with RP-3, however,

neutrophils hardly infiltrated the zymosan (Fig. 2B). The

number of neutrophils infiltrated in the untreated and

RP-3 treated groups were 70.7±8.5, and 0.7±0.7, re-

spectively, per field (200 times magnification) from the

three randomly selected photographed field from the dif-

ferent untreated and treated rats. This result shows thatintraperitoneal administration of RP-3 not only depleted

a

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260 Sekiya et al.

4).

20

15

10

5

0

50 200

Dose of zymozan ()Jg/O.lmf)

Fig. 1 . Increased vascular permeability induced by zymosanand 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 rep-resent: pretreated with RP-3 ., non-treated ., pre-treated withX-63 A, pre-treated with 74D1 1 A, and the control value injectedwith sterilized saline-. The values show the mean ± SE. Thesame examinatIon was repeated 4 times.

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

Time Kinetics of Inhibition by RP-3 of IncreasedVascular Permeability Induced by Zymosan

The time kinetics of suppression by RP-3 of increased

vascular permeability induced by zymosan was exam-

med. Six hours after the i.p. administration of RP-3,

zymosan (200 p.g/0. I ml) was intradermally injected pe-

riodically before the i.v. administration of ‘25I-BSA. In

control untreated rats, the vascular permeability showed

biphasic. The early phase was a steep curve, and it dis-

appeared within 60 mm. The late phase showed a gentleslope that appeared after 60 mm. The late phase vascular

permeability was almost completely suppressed by the

administration of RP-3, whereas the early phase was not

affected (Fig. 3).

Relationship Between the Number of PeripheralBlood Neutrophils and Vascular PermeabilityInduced by Zymosan

In the previous paper, we demonstrated that the degree

of reduction of peripheral blood neutrophils by RP-3 was

related in a dose dependent manner to the monoclonal

antibody injected [27] . We thus examined the number of

Fig. 2. Histological examination of inflammatory edema in-duced by zymosan in rats treated with RP-3. Zymosan (100 �tg/0.1 ml) was intradermally Injected into the rats non-treated orpre-treated with RP-3 (1 ml) 6 hr before. Three hours later, therats were sacrificed and the tissues were fixed with 10% forma-lin. The specimens were stained with PAS. A: Shows the skintissue of non-treated rats ( x 400). B: Shows that of rats pie-treated with RP-3 ( x 400). In non-treated rats, neutrophils infil-trated the clumps of zymosan and phagocyted them. On thecontrary, neutrophils were hardly found in the tissue of the ratspretreated with RP-3.

peripheral blood neutrophils and vascular permeability

induced by zymosan through the administration of vary-

ing doses of RP-3, and investigated their relation. Two to

0.01 milliliters of RP-3 ascitic fluid were intraperito-

neally (i.p.) injected into rats. Six hours later, zymosanwas injected intradermally. The number of peripheral

blood neutrophils and vascular permeability were exam-med at 3 and 4 hr of zymosan administration, respec-

tively, and the relationship between them was investi-

gated. The vascular permeability was decreased in

proportion to the falling off of the peripheral blood neu-

trophils (the coefficient of correlation was 0.885) (Fig.

a,

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>

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Time ( hr

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0 500 10001500 Saline

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Fig. 3. Time kinetics of increased vascular permeability in-duced by intradermal injection of zymosan. Approximately 6 hrbefore the zymosan Injection, 1 ml RP-3 was i.p. injected. Zy-mosan (200 �ig/0.1 ml) was intradermally injected at varyingtimes (5 hr-33 mm) before sacrifice. The symbols represent:pre-treated with RP-3., non-treated., saline control - - -. Thelate phase vascular permeability was suppressed by adminis-tration of RP-3. The same experiments were repeated 3 times.

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aa00

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Number of peripheral neutrophils ( /mm3)

Fig. 4. The vascular permeability induced by zymosan wasproportional to the number of peripheral blood neutrophils.Varying doses of RP-3 were i.p. injected into the rats. Six hourslater, zymosan (200 �g/0.1 ml) was Intradermally injected. Thenumbers of peripheral blood neutrophils and vascular perme-ability were examined at 3 hr and 4 hi, respectively.

Inhibition by RP-3 of Increased VascularPermeability Induced by Intradermal Injection ofProteose Peptone and BCG

We examined whether the above mentioned result in

which only the late phase of enhanced vascular perme-

Vascular Hyperpermeability and Neutrophils 261

ability was inhibited by the depletion of neutrophils was

applicable to inflammatory edema induced by reagents

other than zymosan. In the preliminary experiments, we

found that proteose peptone induced only the early phase

edema and that BCG evoked only the late one, respec-

tively. As shown in Figure 5, the early phase edema

induced by proteose peptone was not affected by RP-3,

whereas the late phase edema by BCG was almost com-

pletely inhibited. The results shown in Figures 3 and 5

suggest that the late phase increased vascular permeabil-

ity is dependent on neutrophils and the early phase is not.

The Recovery of Increased Vascular Permeabilityby Reconstitution of Neutrophil Depleted RatsWith In Situ Injection of Neutrophils

The dependency of BCG induced edema on the exis-

tence of neutrophils was further tested by injection of

neutrophils into the BCG-injected site in rats pretreated

with RP-3. The mixture of BCG and peritoneal neutro-

phils was intradermally injected into rats in which neu-

trophils had been depleted by RP-3, and exudate plasma

volume was serially examined. As shown in Figure 6A,

injection of the mixture of neutrophils and BCG resulted

in the recovery of vascular permeability within 33 mm

after the injection. The increased vascular permeability

was, thereafter, gradually decreased. The grade of re-

covery 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.

The Effect of Fresh Rat Serum Injection on theReduced Vascular Permeability Induced by RP-3

In the preliminary experiments we found that RP-3

injection reduced the complement titer (CH�#{216})of the fresh

serum. The results obtained by RP-3 may be ascribed not

to neutrophil depletion but to reduction of complementtiter. In order to test this possibility, we iv. injected fresh

serum of syngenic rats into the rats pretreated with RP-3.

The reduced complement titer was restored nearly to the

level of RP-3 non-treated control rats. However, the re-

duced vascular permeability induced by RP-3 was not

changed (Table 1). And injection of0.2 ml RP-3 depleted

the rat peripheral blood neutrophils, but complement titer

was not changed. The complement titer of rats treated by

0.2 ml of RP-3 was 35.5±0.5CH50, and that of control

group was 36.5±0.5CH��.

DISCUSSION

In the present study, we have demonstrated that selec-

tive depletion of peripheral blood neutrophils with a

monoclonal antibody RP-3 inhibited the late phase re-

(A) (B)

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Time ( hr

B: Shows that of BCG (1 ,000 p.g/slte). Pretreated with RP-3.,non-treated ., and control value injected with sterilized salinealone - - - . The values shown are the mean ± SE.

0 1 2 3 4 5 Saline

Time ( hr

Fig. 5. InhibItion by RP-3 of Increased vascular permeabilityinduced by Intradermal Injection of proteose peptone and BCG.A: Shows the time kinetics of increased vascular permeabilityby intradermal injection of 3% proteose peptone (0.1 ml).

0 1 2 3 4 5 Saline

262 Sekiya et al.

5

sponse of inflammatory edema in rats. The role of poly-

morphonuclear leukocytes (PMN) in increased vascular

permeability using nitrogen mustard [32,34], methotrex-

ate [26,36], and anti-PMN sera [21], which deplete PMN

selectively, has been previously discussed. The results

obtained are still controversial. In the earlier studies,

depletion of PMN with the various reagents above men-

tioned completely or partially inhibited inflammatory

edema induced by thermal injury [32], bacterial infection

[22], and passive cutaneous anaphylaxis [17,30]. On the

other hand, others have shown that the late phase of

increased vascular permeability induced by ultraviolet

injury or thermal injury was independent of PMN infil-

tration [I I , 16]. More recently, it was shown that deple-

tion of PMN by methotrexate inhibited both inflamma-

tory edema and PMN infiltration, while an anti-

complementary agent suppressed only the latter [26],

which suggested that the association between PMN ac-

cumulation and edema formation was complex. More-

over, it was demonstrated that PMN played a role in the

inflammatory edema induced by the synergistic effect of

complement and prostaglandin E2 [34]. The reason for

the conflicting results mentioned above may be partially

ascribed to the fact that the agents could not truly deplete

PMN selectively. Our previous experiences tell us that

neither nitrogen mustard nor anti-PMN sera deplete

PMN without affecting the number of other nucleated

blood cells (Sendo, unpublished result). Furthermore, it

has not been clarified that PMN depletion causes reduc-

tion of phase (early or late?) vascular hyperpermeability.

To our knowledge, this paper is the first demonstration

5

of dependency of the late phase inflammatory edema on

‘ ‘neutrophils. “ We can use the term “neutrophils” in-

stead of “PMN” inasmuch as we showed in the previous

paper that RP-3 killed neither eosinophils nor basophils

in vitro, and the number of peripheral blood eosinophils

was not changed by i.p. injection of RP-3 [27]. Whether

or not the notion that the late phase inflammatory edema

is dependent on neutrophils is applicable to species other

than rats remains to be clarified, since it was previously

shown that hyperpermeability in rats is more susceptible

to leukocyte depletion than that in guinea pigs

[16,17,30].

The present study has demonstrated that reconstitution

of neutrophil depleted rats by local injection of these

cells restores the inflammatory edema induced by BCG.

This result evokes several points to be discussed. 1) Em-igration of neutrophils through gaps between the endo-

thelial cells may not be a prerequisite for the appearance

of vascular hyperpermeability, because direct intrader-

mal injection of the neutrophil suspensions mixed with

BCG evoked an evident edema in neutrophil-depleted

rats (Fig. 6). This result confirmed the assumption that

cell emigration is not a requisite for increased vascular

permeability, as described by Hurley and Spector [1 1].Emigration of the neutrophil through the endothelium

may be simply required for the appearance of this cell at

the site of inflammation. This assumption may be sup-

ported by the previous result showing that neutrophils

cross endothelial monolayers without increasing perme-

ability [ 19] . 2) Intradermal injection of the mixture ofneutrophils and BCG in neutrophil-depleted rats resulted

(A) (B)

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Vascular Hyperpermeability and Neutrophils 263

60 90 120 Saline

Time ( mm

Fig. 6. The recovery of Increased vascular permeability in therats pre-treated with RP-3 by adding back neutrophils intrader.mally. A: Rats were i.p. Injected with 0.075 mI/bOg RP-3. Ap-proximately 6 hr later, the mixture of 5 x 10� neutrophils and1,000 � BCG, and saline alone were Intradermally Injected. B:

TABLE 1 . The Effect of Fresh Rat Serum Injection on theReduced Vascular PermeabIlity Induced by RP-3

Exp. group

Vascular permeability

(p.1/site)

Complement titer

(CH�0)

Positive control” 12.0 ± 1.70 36.5 ± 0.5

RP-3 treatment alonec 5.8 ± 0.57 3.8 ± 0.2*

RP-3 treatment plus 5.3 ± 0.80 23.5 ± 6.5*

Fresh rat seruminjection”

aTwo milliliters of RP-3 was i.p. 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 com-plement 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 injec-

tion.“Two milliliters of RP-3 was i.p. injected 6 hr before zymosan injec-

tion and 5 ml of fresh rat serum was iv. injected 1 hr before zymosan

injection.

*P<0.0l.

in increased vascular permeability within a short time

(Fig. 6A), compared with that which was observed when

BCG alone was injected into the skin of untreated rats

(Fig. SB). This result suggests that the late appearance of

. I

I

C C I I � I

1 5 1 0 Saline BCG Neutrophil

Number of neutrophils

added back (X166)

The rats pretreated with RP-3 as shown in A were Intradermallyinjected with the mixture of the varying number of neutrophilsand 1,000 � BCG; neutrophils (1 x 10w) alone; and BCG (1,000

ILg) alone; and saline. The exudate plasma volume was exam-

med at 33 mm.

neutrophil dependent edema may be explained by the lag

time of neutrophil emigration through the gaps of endo-

thelial cells ( 1-3 hr). 3) Induction of inflammatory

edema by in situ stimulation of neutrophils suggests that

vascular injury induced by activated neutrophils may be

responsible for neutrophil dependent hyperpermeability.

Injuries of endothelial cells by products of neutrophils in

vitro which were recently reported [6,9,25,29,35] may

support the validity of this assumption. Which factor(s)

reported in vitro is responsible for in vivo vascular per-

meability remains to be clarified by using inhibitors of

each of the factors.

Before we reach the final conclusion on the role of

neutrophils for vascular hyperpermeability from our ex-perimental results, we need to explain about a possible

effect on vascular hyperpermeability of the in vivo com-

plement depletion induced by administration of RP-3.

The answer is as follows: 1 ) Reconstruction of neutrophil

depleted rats with in situ injection of neutrophils recov-

ered the vascular hyperpermeability, whereas this treat-

ment did not affect the complement titer of serum.

2) Administration of 200 pA RP-3 which resulted in a

significant inhibition of vascular hyperpermeability did

not reduce the value of CH50 of the serum. 3) Intrave-

264 Seklya et al.

nous injection of fresh serum from syngeneic rats intoWKA rats whose serum complement titer was reduced

by RP-3 treatment restored the value of CH50 of the

serum. However, this treatment did not restore the sup-

pressed vascular hyperpermeability induced by RP-3 in-

jected as shown in the result. These results indicate that

complement depletion induced by RP-3 injection is not

responsible for the decreased vascular hyperpermeability

observed in our experiments.

Finally, we have demonstrated that dependence on the

neutrophils of inflammatory edema is restricted to the

late phase one. The dependence of the late phase vascu-

lar permeability on PMN was also described earlier

[ 1S ,32]. However, it was not shown whether the deple-

tion of neutrophils affects the early response of hyper-

permeability. Here, we have extended previous results

and added a novel result which shows no effect on the

early phase edema of neutrophil depletion.

It has been postulated that inflammatory edema, de-

pendent on neutrophils, may be pathogenic in certain

diseases such as adult respiratory distress syndrome

[20,33]. The experiments using rats, of which neutro-phils are selectively depleted by RP-3, may be helpful

for analysis of the pathogenesis of these diseases. As a

next step the use of other neutrophil stimuli such as CSa,

platelet activating factor, and NAP- 1, a novel chemo-

tactic peptide [5], may be required to ensure that our

conclusion is applicable to vascular hyperpermeability in

general.

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