Pharmaco/ogical Research Communications, Vol. 20, No. 11, 1988 983

CROTON OIL PLEURISY INDUCES PULHONARY liYPERR~CI'lt/ITY

ALICIA HERNANDEZ, LUISA DAFFONCHIO, GIUSEPPE BRUNELLI,

ROBERTO PASARGIKLIAN AND CLAUDIO OMINI

Institute of Pharmacological Sciences, University of Milan,

20133 Milan, Italy.

Received in final form 18 July 1988

SUNNAR¥

Inflammatory process of the airways has b@en claimed to be relevant to the

development of bronchial hyperreactivity in different experimental models.

We investigated the consequences of pleural inflammation induced in the

guinea-pigs by croton oil injection into the pleural space. Croton oil

injection was followed by the development of an inflammatory reaction

localized to the pleura as shown by recovery of inflammatory exudate from

the pleural cavity of treated animals. An increased number of white cells

was observed in the pleural fluid of treated animals as compared to control.

Moreover, the croton oli induced inflammation was characterized by

development of pulmonary hyperreactivlty which involved 6oth airway and

vascular smooth muscles. We also studied th is phenomenon in an ~mimal model

of asthma, such as the actively sensitized guinea-piEs. Polymorph6nuclear

leukocyte and particularly ~oslnophil recruitment was increased inthis

experimental condition and a different trend i. the development of the

hyperreactive ;phenomenoh was observed. Our data support the re lat ionship

between inflammatory process w i t h i n the "pleural !space ' and ' i nc reased

reactivity of pulmonary tissues. The posslble involvement of different

classes of white cells in this phenomenon has also been discussed.

0031-6989/88/110983-10/'03.00/0 © 1988 The Italian Pharmacological Society

984 Pharmaco~gicalResearch Commun~ation&Vo~2~ No. 11,1988

INTRODUCTION

Bronchial hyperreactivity, a characteristic feature of asthma, is often

associated with inflammation of the airways (Nadel and Holtzman, 1984).

Inflammation is a defensive response of vascularized tissues to resolve and

repair the damage induced by different stimuli such as pathogen

microorganisms, physico-chemical agents and immunological reactions. The

classical signs of inflammation are characterized by changes in vascular

flow, followed by increase in vascular permeability leading to the

infiltration of the damaged area with white ceils (Kay, 1986). The relative

role of these cells in the genesis of inflammation has been extensively

reviewed (Raphael, 1986), and particularly the role of the different

inflammatory cells in the pathogenesis of bronchial hyperreactivity is still

uncertain (Kay, 1984). Several models of experimental airway inflammation

and bronchial hyperreactivity have been developed (Fabbri et al., 1984;

Marsh et al., 1985). We adapted to the guinea-piE the croton oil induced

pleurisy in rat (Kawamura and Oh-Ishi, 1985) since this classical technique

for the study of antiinflammatory compounds is an easy method to induce

inflammation and collect inflammatory exudate within the pleural space.

Therefore, we assessed if the inflammation induced by croton oil in the

pleural space may also result in the genesis of bronchial hyperreactivity;

in addition we also compared the different cell recruitment in normal and

ovalbumin sensitized guinea-piEs with the development of airway

hyperresponsiveness.

MATERIALS ANDXETHODS

Male guinea-pigs (309-500 g) were used in these experiments. Animals were

first randomly divided into two groups, normal and ovalbumin actively

sensitized guinea-pigs. Active sensitization to ovalbumin (OA, Sigma grade

V) was achieved injecting i00 mg/kg of this antigen intraperitoneally and

subcutaneously 21 days before the experiments. Pleurisy was subsequently

induced in both normal and sensitized gulnea-PiES by intrapleural injection

of 0.15 ml of i% croton oil (Sigma) suspension in saline into the right

pleural %avity under liEht ether anaesthesia as previously described

Pharmacological Research Communications, VoL 20, No. 11, 1988 985

(Kawamura and Oh-lshi, 1985).

Sixteen hours after croton oil injection guinea-pigs were sacrificed by

excess ether anaesthesia. A small hole was cut into the diaphragm and the

pleural fluid was collected into plastic tubes. In control animals, due to

the lack of pleural fluid, the pleural cavity was washed with I ml of

phosphate buffered saline (PBS, pH 7.4) and the wash subsequently collected.

Beth samples were then prccessed for cell content determination. In

particular i0 ul were used for total cell count after dilution with

Trypan-blue (Merck, 0.5% in 0.2% physiological solution) by means of

"improved Neubauer" counting chamber and phase contrast microscopy.

Differential leukocyte count was performed on the same pleural fluids

prepared on slide glass and stained with May-GrUnwald-Losung (Merck) and

Giemsa-Losung (Merck) under light microscopy. In a second series of

experiments, after pleural fluid collection, the tracheas and the lungs were

rapidly removed and tracheas (TH), lung parenchymal strips (PS) and

pulmonary arteries (PA) prepared as previously described (Omini et al.,

1985; Gryglewski et al., 1977). Tissues were suspended in i0 ml organ bath

containing oxygenated (02, C02: 95, 5%) Krebs-bicarbonate solution

maintained at 37°C. A load of 1 g for PS, 1.2 g for TH and 0.8 g for PA was

applied to the tissues which were allowed to equilibrate for 60 min before

starting the experiments. The changes in the length of the preparations were

measured via isotonic transducer (mod. 7008) connected to a Gemini 7070 pen

recorder (Basile, Italy). Complete cumulative dose-response curves to

histamine (H) were obtained in each preparation and H effect was expressed

as mm of contraction. Data were analyzed according to Finney's biological

assay (Finney, 1952) and the dose ratio (DR) with 95% confidential limits

calculated. In sensitized animals, active sensitization was verified on a

separate PS challenged with OA (50 /ug/ml); guinea pigs which did not

respond to the antigen with a sustained contraction of the PS were

discharged.

RESULTS

Croton o i l i n j e c t i o n i n t o the p l e u r a l c a v i t y induced an in f lammatory p r o c e s s

986 PharmacologicaI Research Communications, VoL 20, No. 1 I, 1988

as shown by recovery of inflammatory exudate from the pleural cavity of

treated animals. In fact, fluid volume collected 16 h after croton oil

injection was 0.72 ~ 0.17 ml and 1.15 ~ 0.21 ml in normal and sensitized

guinea-pigs respectively whereas no detectable exudate was present in

control animals (Tab. i). Both basal and pleurisy induced cell counts were

TABLE I: Total cell count determined in the pleural fluid obtained from

control and croton oil induced pleurisy in guinea-pigs.

CONDITIONS cell count pleural fluid

(x zo 6) (ml)

NORMAL (N) 1.8 + 0.5 nd

N + PLEURISY 80.7 + 15.0 0.72 + 0.17

SENSITIZED (S) 17.0 + 5.2 nd

S + PLEURISY 226.9 + 60.2 1.15 + 0.21

The figures represent the mean + S.E.M. of at least 5 replications.

Cell count: N vs N + pleurisy, S vs S + pleurisy p < 0.01

N vs S, p< 0.02 N + pleurisy vs S + pieurisy p< 0.05

unpaired Student's "t" test

nd= not detectable

significantly higher in sensitized guinea-pigs as compare d to normal

animals. Moreover, an increase in total leukocyte count was observed in the

pleural fluid of both groups of treated guinea-pigs as compared to the

respective controls (Tab. i). The pleural fluid obtained from both normal

and sensitized croton oil treated guinea-piKs contained mainly

polymorphonuclear leukocytes (PMNs) with a minor number of mononuclear

leukocytes (MNs) as shown in Table 2. In particular, neutrophils were the

most abundant cells present in the exudate whereas no basophils could be

Pharmaco~gicalReseerch Commun~aNon&VoA2~ No. 11,1988

TABLE 2: D i f f e r e n t i a l c e l l count de te rm ined i n the c r o t o n o i l p l e u r a l exudate o b t a i n e d from normal and OA s e n s i t i z e d g u i n e a - p i g s

induced

987

CELLS normal OA sensitized

% total count % total count

N e u t r o p h i l s 65.62 ~ 1 .43 62.62 ~ 1 . 8 6

Eosinophils 0.57 + 0.22 2.07 + 0.44 * w

Basophils nd nd

Lymphocytes 2.75 + 0.85 2.89 + 0.68

Monocytes 31.92 + 1.63 32.38 + 1.92

PMNs 6 6 . 2 0 + 1 . 5 7 6 4 . 6 9 + 1 . 9 0

MNs 3 4 . 6 8 + 1 . 8 6 3 5 . 2 7 + 1 . 8 9

The figures represent the mean ~ S.E.M. of the mean of 9 replications.

* • p <0.02 as compared to normal; unpaired Student's "t" test.

detected. Differential cell count also revealed that the percentage of the

various cell groups examined in the exudate was similar in the two

experimental conditions considered (normal vs sensitized) unless the

eosinophil number. In fact, eosinophil percentage was about 4 fold higher in

the pleural fluid obtained from OA sensitized animals as compared to normal

guinea-piEs (Tab. 2).

In another series of experiments We verified the possible changes in the in

vitro contractile effect of H in the different anatomical structures of the

lung. These data show that the pleural inflammatory process induced by

croton oil injection was accompanied by development of pulmonary

hyperreactivity. In fact H dose-response curves performed in TH and PS taken

9 8 8 Pharmaco~gicalResearch Commun~ation~Vo4 2~No. 11,1988

from normal croton oil treated guinea-pigs were significantly (p < 0.01)

potentiated as compared to control tissues. In addition, maximal H responses

did not significantly differ between the two groups. The two dose-response

curves performed in TH (Fig. IA) taken from normal and pleuritic guinea-pigs

were parallel and the DR calculated was 6.15 (95% confidential limits

2.56-14.78). However, a different trend in the development of the curves was

observed in PS with a non parallel shift to the left of H dose-response

curves in pleuritic strips as compare~ to control (Fig. 2A). The DR

calculated for these experiments was 3.17 (95% confidential limits

1.40-7.17). The hyperreactivity was not restricted to the airway smooth

muscles since in a vascular tissue such as the PA, the croton oil treatment

affected the H induced contraction determining a shift to the left of H

dose-response curves of about 2 times (DR 1.99, 95% confidential limits

0.97-4.09; p <0.05).

Active sensitization per se markedly potentiated the H induced contraction

in TH but did not modify the reactivity of PS (Fig. 1, 2); in fact the DR

calculated in TH betweeen normal and sensitized tissues was 4.68 (95%

FIGURE i: Effect of croton oil induced pleurisy on histamine dose-response

curves in normal (panel A) and sensitized (panel B) guinea-pig tracheas.

TRACH[A

160- IANORMAL 10 N centre! 150- SENSITIZEO ~ plesrlsy

140" i~- f20- 12,56.14,~=T~ IfO- 0R=6.15 0R:!62(~ . - . 100-

, 90- 80-

00- i 50' E 40.

30, 20" 10-

(M)HISTANIN[

Figures represent the mean ~ S.E.M. of at least 5 replications. DR= dose rabio. In parenthesis 95% confidential limits.

Pharmacological Research Communications, Vol. 20, No. 11, 1988 9 8 9

confidential limits 2.50-8.73; p <O.Ol), whereas that in PS was 2.56 (95%

confidential limits 0.73-9.09; ns). The hyper~eactive phenomenon was

observed in sensitized animals after croton oil injection only in PS. In

fact, H dose-response curves performed in this tissue were significantly (p(

0.01) shifted to the left of those in control sensitized tissues in a

parallel manner (Fig. 2B), with no significant changes in H maximal

contraction. The DR calculated in these series of experiments was 3.38 (95%

confidential limits 1.45~7.89). On the other hand no potentiation of H

contractile activity was evident in TH and particularly the dose-response

curves of H performed in sensitized pleuritic TH were slightly shifted to

the right as compared to control sensitized TH (Fi~. IB), with a DR of 1.82

(95% confidential limits i.i0-3.04; p <0.05).

In some experiments, normal animals were sacrificed 4 h after croton oil

injection. At this time neither pleural exudate was present nor development

of hyperreactlvity was observed. In fact, H dose-response curves performed

FIGURE 2: Effect of croton oil induced pleurisy on histamine dose-response

curves in normal (panel A) and sensitized (panel B) guinea-pig parenchymal

strips.

160" 150- 140- 136- 120- 110- 100-

, , 00"

~ liO- ~ 7P ~ 6P

• 40- N" ==.

2A NORMAL

PAR(NCHYMA

(I.40-7.17)

yO,

;-7 ;I 3'x

20 ~ coetr;| pleurisy

SENSITIZEn

[M) HISTAMINE

Figures represent the mean ~ S.E.M. of at least 5 replications.

DR = dose ratio. In parenthesis 95~ confidential limits.

990 Pharmaco~gicalResearch Communication~VoL2~ No. l l , 198B

in PS taken from 4 h treated guinea-pigs were not statistically different

from control (DR 2.35, 95% confidential limits 0.70-7.94; ns).

DISCUSSION

Croton oil or carrageenin induced pleurisy in rat is a common used method

for acute inflammation studies (,Kawamura and Oh-Ishi, 1985; Harada et al.,

1982); we adapted the methodology to guinea-pig in order to verify if this

localized inflammation within the p!eural space may generate a hyperreactive

phenomenon of the airways.

The croton oil induced inflammation resulted in the pulmonary

hyperreactivity which seemed to involve the entire lung structure. In fact,

potentiation of H activity was evident both in the airway domain and in the

vasculature, indicating that the same mediators might affect both airway and

vascular smooth muscles. This is in line with the evidence that putative

mediators of bronchial hyperresponsiveness such as leukotrienes (Creese and

Bach, 1983) can also potentiate the contractile activity of different

vasoactive compounds on vascular tissues (Omini et al., 1985). Moreover, it

is interesting to underline that the hyperreactive phenomenon correlated

well with the development of the inflammatory process. In fact, A hours

after the croton oil injection, when no detectable inflammatory exudate was

present, the hyperreactivity was not demonstrable, indicatin E that indeed

the inflammatory cells and their mediators are involved in the

hyperresponsiveness. These data are in line with others obtained using

different proinflammatory agents, where PMN infiltration is required in

order to develop a bronchial hyperreactivity (Fabbri e£ al., 1984; Marsh et

al., 1985; HoEE et al~, 1985). on the other hand it has also been

demonstrated that neutrophil depletion with cyclophosphamide does not

inhibit the induction of bronchial" hyperresponsiveness by ozone (Murlas and

Roum, 1985). In addition, in guinea-pigs exposed to cigarette smoke the

increase in bronchial reactivity.precedes the migration of P MN into the

epithelium (Hulbert et al., 1985) indicating that parallelism between

hyperreactivity and PMN infiltration is not a general feature.

As far as the involvement of the different leukocytes is concerned, our data

Pharmacological Research Communications, Vol. 20, No. 11. 1988 991

do not allow a definite correlation between eosinophil presence into the

inflammatory exudate and the development of the hyperreactive phenomenon. In

fact, pleuritic hyperresponsiveness was associated to eosinophil

infiltration into the pleural fluid even if the percentage of these cells

over the total amount of PMNs was rather small. In addition, in sensitized

animals, in spite of the larger increase in eosinophil number, a greater

hyperreactivity in PS was not observed and even a slight decrease of H

activity in TH was present, indicating that the hyperresponsiveness may also

be due to the proinflammatory role of different cells other than the

eosinophils. On the other hand, active sensitization "per Be" increased the

reactivity of TH but not PS to H. In this regard it is known that

eosinophils characterize the "allergic" asthmatic pathology (Dahl and Venge,

1982) and our data support this hypothesis since a greater percentage of

these cells was observed in the inflammatory exudate of sensitized animals.

Our results seem to indicate that the hyperreactivity obtained with active

sensitization behaves differently from that induced by acute inflammation.

In fact, the former experimental condition seems to involve meanly the large

airways, whereas in the latter all the pulmonary structures are affected. In

addition, the two induced hyperreactivities are not addictive, possibly

because they involve different mediators or the same mediators but released

at different times. In this regard, the possible involvement of circulating

inflammatory mediators, as suggested Dy Cummings et al. (1984), are now

under investigation.

In conclusion, our data show that a localized pleural inflammation may be a

good model for pulmonary hyperreactivity which is different from that

obtained in allergic models and may be more similar to such asthma

pathologies which are often present in clinic and that have not an allergic

anamnesy.

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