S 243 P-III-1-39

P-III-1-40

THE EFFECf OF COX-2 INHIBITORS ON PLATELET AGGREGATION. -VISUAL OBSERVATION USING ARIlFICIAL MICROCHANNELSKeiko Hosbi, Naobiro Yamamoto, Junko Yamazaki, Sbinya Nakagawa, Xin-Ping Zhou, Sbiro lino, Yutaka Mizusbima Imtitute of Medica1 Science,St.Marianna University,

School of Medicine. KawasakiJAPAN This study examined the e!Teet of COX-2 inhibitors (nimesulide and NS-398) on plate1et aggregetion induced by collagen using artifical microchannals. Wistar rats were treated with COX-l inhibitors (indomethacin and aspirin,40mg/kg, respectively) or COX-2 inhibitors (nimesulide and NS-398, 200mg/kg, respectively). One hour after medication, blood sampIe was collected and diluted PRP to 500,0001/L1 with PPP. The sampie was flowed into microchannels in a single-crystal silicon substrate and we measured the passage time for 100 J.L I of sampie. Sampie added collagen 0.1 /L g/ml was also measured similarly.

No appreciable effeet of nimesulide and NS-398 was seen on platelet aggregation. In contrast, aspirin and indomelhacin caused markerl reduction in collagen induced platelet aggregation. These results show that COX-2 inhibitors da not inhibit on platelet aggregation hy collagen.

THE INVOL VEMENT OF CYCLOOXYGENASE-2 IN THE PATHOGENESIS OF PREECLAMPSIA. Pravit Akarasereenont, Sirikul Chotewuttakorn, Athiwat Thaworn. Department of Pharmacology, Faculty of Medicine

Siriraj Hospital, Bangkok 10700, THAILAND. The role of COX-2 in the pathogenesis of preeclampsia have been investigated. Human umbilical vein endothelial cells (HUVEC) were obtained from babies born to preeclamptic mothers (pHUVEC; test groups) and normal pregnancy (nHUVEC; control groups). Cells were grown to confluent in 6-well culture plates until use. Then, cell extracts and 24 h incubated supernatant medium from both test and control groups were measured for COX-2 pro tein (using immunoblot) and 6-keto-prostaglandin (PG) F 10 level (using enzyme immunoassay), respectively. The study showed that there are COX-2 protein expressed in pHUVEC but not in nHUVEC. Moreover, the amount of COX-I protein expressed in pHUVEC has not been increased when compared to nHUVEC. Interestingly, 6-keto-PGF lo level of 24 h incubated supernatant medium from pHUVEC is less than from nHUVEC. These results suggested that COX-2, not COX.-I, has been involved in the state of preeclampsia as altering vascular activity by imbalance PGs.

Bronchial Asthma and Airway Inflammation P-III-2-01 EFFECTS OF NEUROPEPTIDES AND GLUCOCORTICOSTEROIDS ON THE FUNCTIONAL ACTIVITY OF MONONUCLEAR PHAGOCYTES

Ingrid M. Garrelds l •2 , Peter Th.W. van Hal\ Henk C. Hoogsteden\ Pramod R. Saxena 2, Freek J. ZijIstra 2 • Depts. of IPulmonology & 2Phannacology, University Hospital Rotterdam & Erasmus University Rotterdam, The Netherlands Inflammation of the airways underlies a major part of the clinical symptoms in asthma and COPD. A great variety of mediators, including eicosanoids, cytokines and neuropeptides, play a Tole in the initiation and perpetuation of this inflammation. It has been hypothesized that in asthma and COPD the production of neuropeptides by NANC fibers is increased as a result of damage to the epithelium, whieh may lead to inereased levels of neuropeptides in the alveolar spaee and modulation of the funetion of the loeal eell population. Mononuclear phagoeytes (MP) aeeount for the majority of

this population.

The aim of this study was to investigate the influenee of neuropeptides (SP, NK-A, NK-B, CGRP) on the produetion of eicosanoids and cytokines by MP. For this purpose the monoeytic eell line U937 was used in separate assays. After stimulation with PMA for 24 hrs, U937 cells were cultured in the presence of LPS (\ and 5 l1g/ml) and/or neuropeptides (\0.", 10·", \0.' and \O~ M) with and without peptidase inhibitors or glueocorticosteroids (budesonide.. fluticasone propionate and predmsolon; \0.7 and w' M) for 96 hours. The productlOn of IL-Iß, IL-6 and

TNF-a was measured by ELlSA; TxB, and PGE, by RIA and LTB, by EIA. After stimulation with LPS the production of IL-Iß, IL-6, TxB, and PGE, steadily increased in time, whereas TNF-a reached its maximum between 6 and 12 hours. LTB, was not detectable. Neuropeptides (hoth in the presence or absence of peptidase inhibitors) did not influence the production of the inflarnmatory mediators. However, glucocorticosteroids completely inhibited both the production of eicosanoids and

cytokines. Conclusion: Tbe observed differences in the kineties of the production of inflammatory mediators stress the importance of time course experiments in studies on the effects of neuropeptides on eicosanoids and cytokines. Our findings in U937 cells will be compared with alveolar macrophages from asthma- and COPD-patients and

healthy volunteers. (The Netherlands Asthma Foundation, grant 93.71)

P-III-2-02 PROTEIN KINASE A IS INVOLVED IN SENSORY NEUROPEPTIDES RELEASE. Harrison S., Spina D. & Page c.P. The Sackler Institute of Pulmonary Pharmacology, Dept. of Respiratory medicine, King's College School Medicine and Dentistry, London

UK.

It has been assumed for a long time that protein kinase A (PKA) was involved in relaxation of smooth museIe. It was Ouedraogo (1994) who suggested PKA may be involved in the release of noradrenaline at the nerve terminal. In this investigation we

have looked at whether PKA inhibitor, H-89 can effect the release of sensory neuropeptides. Guinea-pigs were killed by cervical dislocation and bronchial preparations of 2mm in length were removed and suspended between 2 'L' -shaped stainless steel holders, placed in an 8 ml organ bath under a resting tension of 500 mg. Bronchial preparations were incubated in Krebs-Henseleit solution, bubbled with 95% O 2 and 5%

CO, at 37" C in the presence of indomethacin (5 ~M), propranolol (I ~M), atropine (I and thiorphan (10 ~M). H-89 (10 11M) failed to significantly altenuate the eNANC

~)

contractile response induced by electrical stimulation (3Hz, 20 sec, 0.5 ms at max. voltage), (% inhibition of control response 0 ± 0 % vs. H-89 89.8 ± 5.3 %, P > 0.05, n ;; 4), and also the contractile potency of substance P (contral 6.8 ± 0.4 % vs. H-89 6.9 ± 0.4 %; P > 0.05, n ;; 4). In contrast, H-89 significantly attenuated the contractile poteney

to capsaicin (EC50), cyc\osporin A (EC25) and relaxant potency of isoprenaline (IC50) (CAP, control 7.7 ± 0.6 % vs. H-89 5.6 ± 0.2; CsA, control 5.5 ± 0.6 % vs. H-894.5 ± 0.2 %; ISO, control 8.2 ± 0.2 % VS. H-89 7 ± 0.2 %; P < 0.05, n ::: 4). In conclusion. H-89 appears to attenuate the release of sensory neuropeptides.

S 244 P-III-2-03 Systemic administration 01 endotOJ(in induces bronchopulmonary hyperreactivity (BHR) dissociated from inflammation and TNF-a. J. Leiort, M. Singer, D. Leduc, P. Renesto, M. Chignard, M.A. Nahori and B. B. Yargaftig Unite de Pharmacologie cellulaire, Institut Pasteur, 75015, Paris, France. The intra-nasal (i.n.) administration of E. eali endotoxin (LPS) to mice induces neutrophilic inflammation and early augmentation of airways resistance,

followed by BHR to aerosolized methacholine, whieh are in part attributable to TNF-a (aecompanying Abstract and Gon~alves de Moraes et a1., Br. J. Pharmacol., llZ, 1792, 1996). LPS (i.p., 0.3-1 mg/kg) induced a small increase in lung resistance and dose-dependent BHR, both evaluated plethysmographically (Buxco) in C57BL/6 mice. Neutrophils were absent from the bronchoalveolar lavage fluid (BALF), but lungs were enriched in myeloperoxidase (MPO), indicating migration of neutrophils to lungs but failure to cross to the alveolar side. TNF-a (ELISA) was undeteetable from the BALF, and present in blood up to 90 min after the LPS injection. Dexamethasone (5 mg/kg s.c. 1h before LPS) suppressed TNF-a protein in blood and mRNA in the lungs, but failed to modify MPO (no proteetion against neutrophil recruitment). At this dose, dexamethasone suppressed BHR. At lower doses (down to 0.6 mg/kg), dexamethasone continued to suppress TNF-a protein, but failed to reduce BHR, showing thus a remarkable dissociation. mrTNF-a given i.p. failed to modify the lung responses to metacholine and two of its antibodies were only effective against BHR to a very limited extent. Results with the neutrophil-depleting agent vinblastine confirmed the concept that LPS may

P-III-2-06 T CELL RESPONSE IN PERIPHERAL BLOOD DU RING ASPIRIN-INDUCED ATTACK IN PATIENTS WITH ASPIRIN-INTOLERANT ASTHMA

Yo~hinobu Hattori, M~sarni. Tanigu.chi,. KohF A!lzai, Shi~-ich.i Motani, Sumito Isogai, Chlzuko Nak~gawa, K1YO~hi MatsUl, Huoakl Ml~uno, Shigehide Ohkawara, Morihide Ando, Kanehuo Matsushita, Ittetsu Tanaka, Kelko Kako, Maorong Tong, Motohiko Sato, Mitsushi Okazawa, Hiroki Sakakibara, Susurnu Suetsugu (Departrnent of Pulmonology and Allergology, Fujita Health University School of Medidne, Toyoake, Aichi, 470-11 Japan)

T cell activation has been irnplicated in allergen-induced asthma. It is notknown, however, whether the mechanisms of aspirin intolerance depend

on T cells or not. This study was designed to confirm the role of T Iymphocytes in aspirin-induced asthma (AIA). We performed lysine-aspirin (L-ASA) iv and methacholine inhalation challenges in six ArA patients, and analyzed peripheral Iymphocyte populations (CD3, 4, 8, 25 and HLA-DR) before, 30minutes and 24 hours after dosing. The maximum decrease in FEVI afterLASA (ASA 12.5-50 mg) was 32%, and after methacholine was 28%. No AIA patients showed a late response. L-ASAiy: before CD4 (%) CD8 (%) CD4XCD2S (%) CD4XHLA-DR (%)

(' P
45.3 18.9 11.4 4.2

24 h 42.1' 20.0 11.9 4.3

49.8' 16.2 18.5' 6.2'

stimulate BHR in absence of inflammation.

All parameters were unchaoged after methacholine inhalation. These results indicate that T celis playa role in ASA-induced attack.

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P-III-2-07

Intra-nasal endotoxin-induces airways inflammation and bronchopulmonary

hyperreactivity (BHR) which only partially involves TNF-a. J. Lefort, D. Leduc, P. Renesto, M. Singer, N. Leroy M. Chignard and B. B. Yargaftig Unite

de Pharmacologie cellulaire, Institut Pasteur, 75015, Paris, France Aerosolization of endotoxin (LPS) to mice induces neutrophil migration into

lungs and bronchoalveolar lavage fluid (BALF) and TNF-a production (Gon~alves de Moraes et al., Br. J. Pharmacol., llZ 1792, 1996). We now studied whether LPS-induced lung inflammation is accompanied by BHR, a marker of allergie and non-allergie lung diseases and, if so, whether neutrophils and

TNF-a are involved. BHR was evaluated plethysmographically (Buxco) in non-anesthesized C57BL/6 mice, BALF and blood were collected at different time points for differencial cell counts and TNF-a evaluation (ELISA), after the intra-nasal (i.n.) administration of 0.33 mg/kg of E. cali LPS. TNF-a mRNA and myeloperoxidase (MPO), a marker for neutrophils, were evaluated in

whole lungs. By itself, LPS induced augmented basal pulmonary resistance, with a peak at 3h. TNF-a was only detected in the BALF at 1h, and at 1.5-6h in serum. Neutrophils in BALF were identified from 1.5h on and plateaued at 624h, when a marked MPO was observed in the whole lung. BHR in response to aerosolized methacholine was present at 24h after LPS administration. Two anti-TNF-a antibodies, administered i.n. with LPS and i.p., reduced

neutrophils in BALF, MPO, and BHR. Dexamethasone (5 mg/kg s.c. 1h before LPS) suppressed mRNA and TNF-a pro tein, and inhibited by 50% the neutrophil recruitment and BHR. Thu,s LPS induces, together with neutrophilic inflammation, an immediate augmentation of airways resistance, followed by

BHR to methacholine, which are only in part attributable to TNF-a formation.

GLYCOCONJUGATE SECRETION IN HUMAN AIRWAYS IN VITRO: EFFECTS OF EPITHELIUM REMOVAL Hassania Sosse-Aloui, Carlos Labat, Vincent Thomas d<: Montpreville, Jacques Bara', CharIes Brink 'INSERM U-55 and CNRS ERS-566 at Centre Chirurgical Marie Lannelongue, 133 av. de la Resistance. 92350 Le Plessis-Robinson, France. Glycoconjugate release in human airways in vitro was determined using an ELiSA assay with an anti-human mucin monoclonal antibody (303) which reacted strongly with Leb antigen but also recognized "in vitro" Le' and Le' dcterminents. The amount of glycoconjugate measured in the medium derived from human isolated bronchial ring preparations did not change under control conditions during the course of the experimental procedure (period I; 128 ± 46 ~g/g tissue and subsequently Period 11; 159 ± 48 ~g/g tissue; N= 13 paired lung sampies). In the supematants of airway preparations with an intact epithelium the amount of glycoconjugates detected was 90 ± 38 ~g/g tissue (period I; N=12 different lung sampies) and removal ofthe epithelium did not alter this basal glycoconjugate release (94 ± 60 ~g/g tissue: Period I, N= 8 different lung sampIes). Ouring Period 11 methacholine (100 ~M) induced a 10- and 4-fold increase in glycoconjugate release from airways with and without an epithelium, respective1y. Treatment with atropine (100 ~M) prevented the increase of glycoconjugate release in preparations with an epithelium. Conclusion: The epithelium may not regulate the basal or stimulated release of glycoconjugates from human airways.

P-III-2-05

P-III-2-08

IgG SUBCLASSES IN TRIMELLITIC ANHYDRIDE (TMA)INDUCED PULMONARY HYPERSENSITIVITY IN THE GUINEA PIG. Jean F Re~al, Daniel G. Fraser. Dept. of Phannacology, University of Minnesota, Duluth, MN, USA. Sensitized guinea pigs respond to intra tracheal challenge with TMA conjugated to guinea pig serum albumin (TMA-GPSA) with immediate bronchoconstriction and delayed cellular infiltration into the lungs; responses characteristic of asthma. These responses have been shown to be mediated by IgG I in the guinea pig. The purpose of this study was to determine if TMAspecific IgG2 influeneed the response. Guinea pigs wcre passively sensitized with IgGI andlor IgG2 purified by anionic exchange chromatography. The TMA-induced increase in pulmonary resistanee was signifieantly greater in animals sensitized with IgGI + IgG2 (66.7±11.2%) compared with animals sensitized with IgGI alone (11.6±1.9%) or IgG2 alone (l±1 %). The number of eosinophils in the bronchoalveolar lavage was signifieantly greater after TMA-GPSA eh allen ge in animals sensitized with IgG I plus IgG2 compared to control animals or animals sensitized with only IgG I or only IgG2. These data demonstrate an augmentation of the IgG I-mediated response by the addition of IgG2 and suggest a significant role for both IgG subclasses in TMA-induced pulmonary hypersensitivity. (Supported by NIH ROI ES07546).

EFFECTS OF PEPTID-LEUKOTRIENE RECEPTER ANTAGONIST, PRANLUKAST ON EOSINOPHIL COUNTS IN PERIPHERAL BLOOD AND SPUTUM IN PATIENTS WITH CHERONIC ASTHMA Surnito Isogai, Masami Taniguchi, Yoshinobu Hattori, Shin-ichi Motani, Kohji Anzai, Chizuko Nakagawa, Kiyoshi Matsui, Hiroaki Mizuno,

Shigehide Ohkawara, Morihide Ando, Kanehiro Matsushita, lttetsu Tanaka, Keiko Kako, Maorong Tong, Motohiko Sato, Mitsushi Okazawa, Hiroki Sakakibara, Susumu Suetsugu

(Department of Pulmonology and Allergology, Fujita Health University School of Medicine, Toyoake, Aichi, 470-11 Japan) Aim:

Recent reports suggest that bronchial inhalation with peptid-

leukotrienes (pLTs) provoked eosinophil accumulation in bronchial rnucosa

and sputum in patients with asthma. We examined the effect of Pranlukast on eosinophillia in peripheral blood and sputum in chronic asthmatics. Melhods: Twenty seven adult asthmatics (age 21-58) received pranlukast (225 mg) twice a day for 8 weeks. All patients used ßMDl and theophylline, but did not use systemic steroids. Results: The peripheral eosinophil count significantly decreased (12.4->8.7% , p<0.01), and sputum eosinophil number also decreased (score 2.6->1.6, p
S 245 P-III-2-09

P-III-2-12

REGULATION OF EXPRESSION OF PLATELET-ACTIVATING FACTOR RECEPTOR ON EOSINOPHILS IN ATOPIC ASTHMA ~eishi Kisllimoto', Takayuki Sugiura', Takashi Izumi', Takao Shimizu', Takeshi Fukudal , Sohei Makinol , Keizo Waku' 'Faculty of Pharmaccutical Sciences, Teikyo University, Kanagawa, Japan; 'Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; IDepartment of Internal Medicine and Clinical lmmunology, Dokkyo University School of Medicine, Tochigi, Japan We studied the regulation of the PAF receptor expression on eosinophils in allergie inflammation. When peripheral blood eosinophils obtained from healthy nonnal subjects were treated with 5 ng/ml recombinant human interleukin-5 (rhIL-5), the specific binding of PHjWEB 2086, a specific ligand for PAF receptor, to the cells and the amount of PAF receptor mRNA in the cells were significantly higher than those to untreated cells at 12 to 18 h. The PAF-induced increase in intracellular calcium ion concentration in eosinophils was also markedly augmented by exposure to rhIL-5. Furthennore, we found that the levels of surface expression and mRNA of PAF receptor in eosinophils from atopie asthmatic patients were significantly higher than those in normal subjects. Thc cffect of 12-h exposure to rhlL-5 was not apparent in eosinophils from the patients, whereas rhlL-3 and rhGM-CSF increased the number of the receptor in the patients as weil as in normal subjects. Conclusion: These data suggest that, in atopi.: asthma, thc expression 01' functional PAF receptor in eosinophils is enhanced by exposure to IL-5 in peripheral blood. Refer~!!.~: Kishimoto, S. ct al. (1996)1. lmmunol., 157,4126.

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P-III-2-10 SUBBIOACTIVE DOSES OF PGE INHIBIT ASPIRIN-INDUCED ATIACKS IN PATIENT WITH ASPIRIN-SENSITIVE ASTHMA

Kiyoshi Matsui, Masami Taniguchi, Yoshinobu Hattore Shin-ichi Motani, Sumito Isogai, Chizuko Nakagawa, Kohji Anzai, Hiroaki Mizuno, Shigehide Ohkawara, Morihide Ando, Kanehlro Matsushita, Ittetsu Tanaka, Keiko Kako, Maorong Tong, Motohiko Sato, Mitsushi Okazawa, Hiroki Sakakibara, Susumu Suetsu~u (DiVIsion of ~Y~hl~~~b~fi janpda~llergology, Fujita Health University School of Medicine, Toyoake,

Aim: The severity of NSAlD-induced attacks is correlated with cyc\ooxygenase-inhibitory power. We hypothesized that endogenous PGE2decrease is a trigger for NSAID-induced attacks. In this study, we examined the effects of low-dose PGE infusion of methacholine, allergen and aspirin challenges in aspirin-induced attacks (AIA). Methods: Ten AlA patients were assessed, and five of them also suffered from mite-allergy. Methacholine, mite allergen and aspirin(lysine-aspirin iv)challenges were performed at two week intervals during low-dose PGE or placebo infusion, in a double-blind crossover fashion. PGEI (Prostandin, ONO, Japan) was infused at 2 ng Imin Ikg during the challenges. ResuIts: There was no difference in FEV1, PC20MCh and mite-induced bronchoconstriction between placebo and PGEI infusion. Low-dose PGE1, however, significantly protected against aspirin-induced bronchoconstriction (P
KL-6 AS A NOVEL MARKER OF COLLAGEN DlSEASE-ASSOCIATED lNTERSTITJAL PNEUMONlA Hiroshi Nakajima, Masayoshi Harigai, Masako Hara, Sadao Kashiwazaki Institute 01 Rheumatology, Tokyo Women's Med. Coll., Tokyo, Japan The diagnosis and the evaluation 01 disease activity 01 interstitial pneumonia (I P) associated with collagen diseases are important lor the treatment 01 the patients. In this study, we investigated the diagnostic value 01 KL-6, a mucinous glycoprotein expressed on type I1 pneumonocytes, lor IP in the sera 01 various collagen diseases (n= 140). Result: The numbers 01 the patients with positive serum KL-6 level ( ~500U/ml) were aslollows; rheumatoid arthritis (RA) with IP 12/22, RAwithout IP 0/35, systemic sclerosis (SSc) with IP 13/23, SScwithout IP 1/23, polymyositis/dermatomyositis (PM/DM) with IP 9/12, PM/DM without IP 0/7, systemic lupus erythematosus (SLE) with IP 0/1, SLEwithout IP 0/17. Themean KL-6 level 01 patients with active IP was signilicantly higher lhan that 01 the patients with inactive IP. The levels 01 KL-6 increased with the deterioration 01 IP, while the successlullreatment 01 IP resulted in signilicant decrease 01 i1. Conclusion: Serum KL-6 level is an uselul marker tor the differential diagnosis 01 pulmonary involvement and the evaluation 01 disease activity 01 IP associated with collagen diseases.

This

result suggests that endogenous PGE2 is a protective autacoid, especially in AlA patients.

CHARACTERIZATION OF 10 T CELLS WITH PRO-INFLAMMATORY ACTIVITY IN MURINE PLEURISY MODEL

lHenriaues. M.G.M.O., lRosas, E.c., ISampaio, A.L.F. 2Mengel, 1. and lRibeiro

dos Santos, R.. 'Applied Phannacology, FarManguinhos, Fiocruz, RJ, Brazil, 21041-250 and 'Dept. ofimmunology, USP, SP, Brazil We have recently demonstrated an involvement of yo T cells in eosinophil accumulation induced hy LPS and mononuc1ear cell hy carrageenin, in mouse pleural cavity. Using carrageenin mouse pluerisy model we characterised the y6 T subset involved in inflanunatory process. Intrapleural injection of carrageenin (300 ).lg/cavity) induces a biphasic plasma extravasation that peaks between 48-72 h. IITIrlltmophenotype analysis, revealed a significant increase in the number of y6+

CD4-/CDS- T Iymphocytes at 24h, preceding a massive infiltration ofmacrophages at 4Sh. The specific depletion of 10 T Iymphocytes (by 3AIO mAb) completely inhibited the macrophage accumulation and plasma extravasation at 48 after

carrageenin whereas the depletion of CD4 subset (by GKl.5 mAb) did not. Flow cytometric analyses of cells recovered from BALBtc mice pleural cavity 24h after carrageenin show that the total numher of y8 T lymphocytes average more than tenfold higher in pleral cavity of carrageenin-stimulated mice. The majority of 10 +T cells suhset are from 84 suhset and almost no increase in the numher of y2 or y3

subset was noted. Only II % of yo +T cells are CD69+ but 99% are L-selectin

negative. These results suggest a pro-inflammatory role for a y8 T cell from 84 subset.

Supported by: CNPq

P-III-2-11

P-IlI-2-14

THI AND TH2-TYPE CELLS IN PERIPHERAL BLOOD IN PATIENTS WITH ATOPlC AND NON-ATOPlC ASTHMA

ALLERGIC AIRWAY INFLAMMATION IS IMPAIRED IN YÖ T CELLDEFICIENT MICE, C. Zuany-Amorim*, P. Pereirat , C, Ruffie*, RJ!. Vargaftig* and M. Pretolani*. Unite de Phannacologie cellulaire and tUnite d'lmmunobiologie, Institut Pasteur, Paris, France. yö T cells reside mainly in epithelia-rich tissues and they are believed to play a role in the onset or the resolution of inflammatory immune processes, We examined the antigen-induced cellular recruitment, cytokine release and antibody titers in ovalbumin (OVA)-sensitized yö T celldeficient mice and in control BALB/c littermates. Mice were sensitized systemically with OVA and challenged 3 times via the intra-nasal route with 20 ~g OVA or saline, 3 days apart. Three days after the last challenge, BALB/c mice showed bronchial hyperreactivity (BHR) to inhaled methacholine, a rise in the levels of OV A-specific 19E and IgG 1 in the serum, interleukin (IL)-5, but not interferon-y release in the bronchoalveolar lavage (BAL) fluid and a massive eosinophil, CD4+ and CD8+ T cell accumulation in the BAL fluid and bronchial tissue. In contrast, yö-T -cell deficient OV A-challenged mice showed very low numbers of CD4+ and CD8+ T cells and of eosinophils in BAL fluid and tissue, practically no release of IL-5 and dimished levels of OVA-specific IgE and IgGl. BHR, however, was only minimally affected, We concIude that yli-T -cells play an important role in the initiation of primary immune response and of airway inflammation folowing antigen immunization and challenge in mice.

Kohji Anzai, Masami Taniguchi, Yoshinobu Hattori, Shin-ichi Motani, Sumito IS06"ai,

Chizuko Nakagawa, Kiyoshi Matsui, Hiroaki Mizuno, Shigehide Ohkawara, Morihide Ando, Kanehiro Matsushita, Ittetsu Tanaka, Keiko Kako, Maorong Tong, Motohiko Sato, Mitsushi Okazawa, Hiroki Sakakibara, Susumu Suetsugu (Division of Pulmonology and Allergology, Fujita Health Uruversity School of Medicine, Toyoake, Aichi, 470-11 Japan)

In order to clarify the differences in THI ITH2 cell balance between atopic and non-atopic asthmatics, we measured the lL4-and INFY-producing T cells in peripheral blood. Five atopic and five non-atopic adult asthmatics who had received no steroid therapy were assessed. All atopic subjects showed mite allergy and high IgE levels (>1000 lU/ml), and the non-atopic group showed negative skin tests with common allergens and low IgE levels «lOOIU Iml). The distributions of Thl and TH2 cells were analyzed by flow cytometry. Peripheral white blood ceIls, taken during the asyptomatic state, were stimulated with BFA, PMA and ionomycin for six hours. Cells were fixed and stained with FITC-or PE-conjugated antibody specific to each cytokine in combination with the surface marker, CD4. The percentage of IL4 (Th2 cells)or INFT (THI cells)-producing cells were analyzed among CD4-positive populations. Atopic asthma Non-atopic asthma P 24.1±8.5 Thl cell (%) 23.2±4.2 NS Th2 cell (%) 6.1±1.4 3.0±1.1 <0.01 4.0±1.2 TH1/Th2cell 9.5±5.7 <0.1 There were no difference between non-atopics and healthy subjects.

This data suggests atopic asthmatics have higher systemic Th2 cell levels.

S 246 P-III-2-15

P-III-2-18

IN ENDOTHELINS PARTICIPATION OF ENDOGENOUS INFLAMMATORY REACTIONS 'A.L.F. SAMPAIO, 'G.A. RAE, 'P. D'ORLilANS-JUSTE and 'M.G.M.O. HE.ffi.IQUES. Lab. 1Applied Pharmacology, FarManguinhos, Fiocruz, Rio de Janeiro, 7;041-250; 'Depts. Pharmacology, UFSC, Florian6polis, 88015-420, Brazil and Fac. Med., 'Univ. of Sherbrooke, Sherbrooke, Canada Jl H 5N4. ET -A receptor antagonists inhibit paw edema induced by carrageenan (CAR) or by ovalbumin in sensitized mice (Sampaio et al., J Cardiovase Pharmacol. 26 (suppl 3), S416, 1995). This study assesses the effects of BQ-123 (ET-A antagonist) and BQ-788 (ET-B antagonist) on leukocyte reeruitment and plasma extravasation indueed in the pleural cavity of mice by OVA (12.5 J.lg/cavity), LPS (250 nglcavity), CAR (300 J.lglcavity) or zymosan (ZYM, 500 J.lglcavity). Treatment with BQ-I23 (1.5-150 pmollcavity; 5 min before) inhibited OV A-induced recruitment of mononuclear cells (from 3.8 ± 0.5 to 2.1 ± 0.4 x 106 cells) and especially eosinophils (from 1.04 ± 0.2 to 0.1 ± 0.05 x 106 cells), but not neutrophils, measured 24 h later. BQ-123 also reduced LPSindueed neutrophil and eosinophil infiltration assessed 24 h (but not 4· h) after injeetion, whereas it failed to affeet the reeruitment of neutrophils and eosinophils triggered by CAR or ZYM, respeetively. BQ-788 (150 pmolteavity) failed to modify reeruitment triggered by any of the inflammatory stimuli. Plasma extravasation was inereased by CAR and ZYM at 4 h but neither antagonist influeneed these responses. Therefore, endogenous endothelins, acting upon ET-A reeeptors, may weil be involved in leukoeyte recruitment to the pleural cavity triggered by allergie stimuli and LPS. Support: CNPq (Brazil) and MRC (Canada).

Immediately suppressive effect of KSR-591, a Dovel glucocorticoid, on the rat preurisy model. Yohsnke Imai, Masahiro Hiratochi, Shuniehi Tsukamoto, Fumiaki Itoh and Masami Kojima. Phannacological Lab., KISSEI Phannaceutieal Co. Ltd., 4365-1, Kashiwabara, Hotaka, Nagano, 399-83, Japan. Glucocorticoid, whieh acts to modulate inflammatory and immune responses, TCquires more than several hours to show the suppressive effeet on inflammatorie reaction. KSR-592 is a structurally novel dihalogenated corticosteroid under development as an anti-asthma steroid for the inhalation therapy. In the present study, we examined the immediate effeet of KSR-592 on plasma exudation induced by antigen and zymosan in pleurisy of rats. Male Wistar rats were passively sensitized with anti-DNP As mAb. One day after, the rats were intrapleurally injected the DNP As. Zymosan-induced pleurisy was caused by intrapleural injeetioD of 2% zymosan. KSR-592 was injeeted s.c. or locally into pleural eavity 30 min prior to both inducers. Aeute plasma exudation was suppressed by s.c. treabnent of KSR592, dose dependently. Local administration of KSR-592 suppressed zymosaninduced aeute plasma exudation without effeeting histamine content released simultaneously. Furthermore, INFo. production in the pleural fluid was suppressed by KSR-592, dose dependently. These data indicated that KSR-592 demonstrates immediate inhibitory effeets. At least, these effeets is not due to inhibiting the histamine release. Other meehanism of the effeets ineluding the role of INFo. detected in &eute exudates needs to be studied further.

P-III-2-19

P-III-2-16 THE BALANCE BETWEEN NEUTROPHIL SERINE PROTEINASES AND ANTIPROTEINASES IN A MURINE MODEL OF ACUTE LUNG INFLAMMATION INDUCED BY BACTERIAL LPS. M. Chignard V. Balloy. Unite de Pharmacologie Cellulaire, Institut Pasteur, Paris, France.

Acute lung inflammation is characterised by an influx of polymorphonuciear neutrophils (PMN) which are at the origin of tissue injury. Serine proteinases such as elastase, released from activated PMN are

THE

EFFECTS

OF

COMBINATION

THERAPY

WITII

INHALED

CORTICOSTEROIDS AND MACROLIDE IN BRONCIDECTASIS AND DIFFUSE PANBROCHIOLITIS. F. Sonoda T. Ii H. Arnano K. Oishi T. Nagatake and K. Matsushirna. Dept of Int Med, Tagawa Municipal Hospital, Dept of Int Med, Institute ofTrop. Med, Nagasaki Univ, Japan. Dept of Pharmacol,Inst

of Cancer Res, Kanazawa Univ, Japan.

It has been shown that low-dose, long-term macrolide(erythromycin, clarithromycin)

susceptible to playa role. Intra-nasal administration of E. coli lipopolysaccharide (LPS) to mice triggered a time- and concentration-dependent recruitrnent of PMN in the lungs as measured in bronchoalveolar lavages (BAL). A maximal influx was observed at 330 J.lg/kg, with a peak at 48 hours followed bya decrease to the basal level by 120 hours. This was accornpanied with a 24 hour delay, by a 3-

inhaled corticosteroids and macrolide in diff11'ie panbronchiolitis and brochiectasis patients

fold increase of total protein concentrations in the airspaces, an index of the

bronchial

injury of the alveolo-capillary barrier. This rnight result from elastase released from PMN since an elastase enzymatic activity was recovered frorn the same BAL, with a similar peak at 48 hours. In situ activation of PMN in the airspaces by a specific agonist (fNLP), increased this elastase enzyrnatic activity. Nonetheless, there was ro delayed increase in total protein concentrations. In fact, LPS administration affects the antiproteinase screen of

the lung. Indeed, the elastase inhibitory capacity recovered frorn the BAL increased up to 48 hours (8-fold increase) and then declined to its basal level within 120 hours. Thus, the lack of apparent injury in spite of elastase release may be explained by an increased concentration of elastase inhibitor(s).

P-III-2-17 AEROSOLIZATION OF LlPOPOLYSACCHARIDE: A MODEL SCREENING OF POTENTIALANTlINFLAMMATORY DRUGS.

brochiectasis.In the present study, we investigated the effect of combination therapy with who had no effect with treatment of macrolide alone. Furtheremore, we evaluated the inhibitory effect of a combination oi erythromycin and corticosteroids on Ilr8 production in human epithelial

cells

in

vitro.

Treatment

corticosteroid<.(600J.lg/day,beclomethasone

with

dipropionate)

a

combination and

of

inhaled

clarithromycin(oral,

400mg/day) for CADpatients signiiicantly reducedsputum volume andpurulency. The number ofneutrophils and Ilr8levels ofBAL fluids significantly decreased. In vitro, treatment with a combination oi dexamethasone(10-6 M) pl11'i erythromycin(51l g/ml) suppressed Ilr8 production by human bronchial epithelal cells (BET-IAcells) in response to the supernatants

oi sonicated P. aeruginosa or lNFa Dur study suggest beneficial effects oi combination therapy with inhaled corticosteroids and

macrolide in CAD patients. However, further studies to clarify the effects of combination therapy in larger number of patients with CAD are required.

P-III-2-20 FOR

Vera Lucia G. de Moraes* and Sonia S. Costa. *Depto. de Bioquimica Medica, 'NPPN, UFRJ, 21941-590, Rio de Janeiro, Brazil.

We developed an animal model to mimic the local inflammation caused by bacterial particles present in the inhaled air. One hour after inhalation of lipopolysaccharide (LPS) only alveolar macrophages .ware foulld in the bronchoalveolar lavage fluids of BALBtc mice. Three hours after inhalation there is a significant lung neutrophil infiltration, peaking at 24 h after inhalation. Using immunocytochemical, EIA and ELiSA assays, our data show that alveolar macrophages are the target celfs for both the induction of the lung inflammatory response to LPS and its control by systemic treatment wnh cAMP-elevating agents (Moraes et al, submilted). Searching for potential news drugs to block lung inflammation we found that the systemic treatment of mice wnh 8 mg of a Brazilian natural product extracted from the plant Kalanchoe brasiliensis was able to block almost completely the neutrophil recruitment induced by aerosols of LPS, even when n was measured 24 h after inhalation. In the same concentration this product also impaired the mixed leukocyte reaction in vivo and in vitra, strongly indicating it as an antiinflammatory product. *Financial support by CNPq and FINEP (Brazil)

therapy is effective in chronic airway disease(CAD) such as diff11'ie panbronchiolitis(DPB) and

Evaluation of antiinflammatory and anti-histaminic profile of Barlaria prionitis Linn Surender Sineh Department of Pbarmacology College of Pharmacy (Univ. of Delhi) Pushp Vihar. New Delhi- 110017, INDIA. Barlaria prionitis Linn (Acantheceae) popularly known as "Kala bansa". In folklore medicine and in other ancient system of medicine , various parts of the plant has been widely used in different body ailments. The juice of leaves administered in catarrahal infection accompanied with fever, bleeding teeth, otitis, boils, and in glandular swelling. However, survey of literature reveals that no detailed pharmacological screening has been carried out. In the present study, the antiinflammatory activity against carrageenan and histamine induced paw edema in rats and antihistaminic/antiasthamatic activity against histamine and acetylcholine induced bronchospasm in guinea pigs has been investigated.The 50% ethanolic extract of leaves of ];!.prionitis showed significant antiinflammatory activities with both phlogistic agents and protection against histamine and acetylchOline induced bronchospasm in a dose dependent manner. The results were compareable to that of standard drug(s) used. Since mediators released during carrageenan induced edema, the beneficial effect of extract on both the phases probably shares a common mechanism.

S 247 P-III-2-21 BIOCONTROLLED ELECTROSTIMULATION IN CORRECTION OF THE INFLAMMATORY PROCESS A.N.Yolobuev, V.A.Soloviov, E.L.Ovchinnikov. N.N.Krukov, P.I.Romanchuk. Samara State Medical University, Samara. Russia The realization cf a electrjstimulation on thc biological active points at consecutive stages inflammation (alteration and exudation) permits to influence on main seetiaDs cf the inflammatory process. Tbc researches were conducted on the staiidard equipment Lasper-CS 306 (Japan). A wide spectrnm of diseases in a stage cf the aggravatuom was chosen: chronic bronchitis (8 patients), chronic adnexitis (6), chronic pancreatitis (3), bacterial asthma (8). Were used the extrameridional biological aclive points (shue-mjanj-sjue). The capacity of effect was reduced in comparison with standard of the therapy. The frequent mode was chosen individually. The irritation of the biological active points was conducted at a level of weak sensations. Decrease emission cf thc mediatops inflammation (gystamine, bradikinine), improvement cf the blood rheoproper~ties, norrnalization vessels penneability microcirculatoring channel. In our opinion, thc decrease cf intensity thc inflammatory process is connected with more exact regulation cf the positive feedback in the system of the formation bradikinine. The effect of the system complement improves regulation of function gystamin's cells, tlmt conducts to nonnalization bloodstream. Conclusion: Use of the standard stimulator in the reflextherapy opens wide prospects in correction of the inflammatory process.

Autoimmune Diseases P-III-3-01 CYTOKINES REGULATE T CELL ACTIVATION IN MULTIPLE SCLEROSIS VIA DISTINCT CALCIUM SIGNALING PATHWAYS Gianvito Martino, Fabio Grohovaz*, Elena Brambilla, Franca Codazzi*, Emilio elementi *, Massimo Filippi, Giancarlo Corni, Luigi M.E. Grimaldi Dept. of Neurology and *Dept. of Pharmacology, University of Milano, San Raffaele Scientific Institute, Milano, Italy Demyelinating inflammatory infiltrates characterising central nervous system (CNS) lesions in multiple sc1erosis (MS) contain few CNS antigen-specific autoreactive T cells and a multitude of pathogenetic non-antigen specific rnononuc1ear cells. We found that IFN-y, TNFa, IL-2, .nd IL-6, but not myelin antigens, synergize to induce non-antigen specific proliferation of T lymphocytes from MS patients by generating a persistent increase of cytosolic calcium concentration via two independent mechanisms: a transmembrane IFN-y-activated calcium influx and a TNFalIL-2 IIL-6-induced inositol phosphate-mediated cytoso1ic calcium mobilisation. The two calcium mobilising pathways were coexpressed in 57% of peripher.1 (m.inly CD4-memory) cells from MS patients but in only 10% of cells from healthy controls (p
P-III-3-02

INDICA TION FOR A ROLE OF FAS ANTIGEN IN THE PATHOGENESIS OF COLLAGEN TYPE II-INDUCED ARTHRITIS IN MICE Jiro Hoshino, Elke Westhäuser*, Annette Hauschild : Deutsches Rheumaforschungszentrum Berlin, * Robert Koch-Institute, BerIin, Gennany In the present work we used mice with collagen type II-induced arthritis (CIA) as an alternative model to the well-documented autoimmune Ipr/lpr animals for studying the role ofFas Ag in the pathogenesis ofhuman RA. Thymocytes of CIA mice were less sensitive to the apoptotic death induced in vitro by monocIonaI anti-Fas Ab. As a probable cause of this desensitization we found a marked decrease in the number ofFas Ag expressing cells in the thymocyte population of CIA mice relative to the nonarthritic control. Furthermore, whereas almost a1I ofnonarthritic mice were killed by the i.p. injection of anti-Fas Ab within 46 h, CIA mice survived this lethaI effect in a CIA severity-dependent manner. ConcIusion: A1though CIA is not genetically prone as is the case with Ipr/lpr mice, its development could also be attributable to the impairment of Fas Ag-dependent apoptotic deletion of autoreactive Iymphocytes.