Immunopharmacology of endotoxicosis: Proceedings of the 5th International Congress of Immunology satellite workshop, Kyoto, Japan, August 27, 1983 9783111506777, 9783110098877

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Immunopharmacology of Endotoxicosis

Immunopharmacology of Endotoxicosis Proceedings of the 5th International Congress of Immunology Satellite Workshop Kyoto, Japan, August 27,1983 Editors M. K. Agarwal • M. Yoshida

W G DE

Walter de Gruyter • Berlin • New York 1984

Editors M. K. Agarwal, M. Sc.; Ph. D„ M. D. Maître de Recherche au CNRS Scientific Director: Laboratoire de Physik-Hormono-Réceptérologie Faculté de Médecine Broussais Hôtel-Dieu Université Pierre et Marie Curie 15, rue de l'Ecole de Médecine F-75270 Paris Cédex 06 France Masao Yoshida, M. D. Professor Department of Bacteriology Iwate Medical University 19-1 Uchimaru, Iwate 020 Morioka Japan CIP-Kurztitelaufnahme der Deutschen Bibliothek Immunopharmacology of endotoxicosis : proceedings of the 5th Internat. Congress of Immunology satellite workshop, Kyoto, Japan, August 27,1983 / ed. M. K. Agarwal; M. Yoshida. - Berlin; New York : de Gruyter, 1984 ISBN 3-11-009887-3 NE: Agarwal, Manjul K. [Hrsg.]; International Congress of Immunology

Library of Congress Cataloging in Publication Data International Congress of Immunology Satellite Workshop (5th : 1983 : Kyoto, Japan) Immunopharmacology of endotoxicosis. Bibliography: p. Includes indexes. 1. Endotoxins—Physiological effect—Congresses. 2. Endotoxins—Toxicology—Congresses. 3. Immunopharmacology—Congresses. I. Agarwal, M. K. II. Yoshida, M. (Masao), 1925. III. Title. [DNLM: 1. Endotoxins—immunology—congresses. 2. Endotoxins—pharmacodynamics—congresses. QW 6301331983] QP632.E4I54 1983 616.9'2 84-7650 ISBN 3-11-009887-3

Copyright © 1984 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. - Binding: Luderitz & Bauer GmbH, Berlin. - Printed in Germany.

THIS BOOK IS DEDICATED TO

PROFESSOR L, JOE BERRY

FOR HIS CONTRIBUTIONS IN THE FIELD OF ENDOTOXINS SPANNING OVER HALF A CENTURY

PREFACE

Bacterial endotoxins have fascinated researchers for over a century. Their beneficial effects include nonspecific increase in resistance to various sorts of infections, induction of interferon, antitumour activity, adjuvanticity, immunogenicity and radioprotection. Their pyrogenic properties had been exploited for several centuries, but this use has since been abandoned. Equally impressive is their array of noxious properties, which include the Sanarelli-Shwartzman reaction, shock, microvascular coagulation, hemodynamic alterations and the depletion of carbohydrates, to mention only a few. No organ or cell type in the host is immune from the influence of endotoxins, but it is not clear whether these efforts are direct mediated and whether one site is affected or several sites simultaneously. The purpose of this workshop was to bring together researchers from various disciplines working in the field of endotoxins. After surveying the immunopharmacological reactions evoked by the bacterial endotoxins, the influence of various pharmacological agents on endotoxin-mediated host reactions was discussed. Since the mechanism of action of these agents is sometimes quite well defined, it was hoped that insight could be gained into the manner of endotoxins reactivity in the host. This proved difficult, however, due to the diversity of experimental models. Finally, problem-oriented themes were chosen with the aim of arriving at a consensus as to the site and nature of endotoxin reaction. It is hoped that we provided a forum for workers interested in a common problem to thrash out their differences in a con-

VIII

genial and relaxed atmosphere. If the workshop has helped redefine the problem in a clearer perspective, the goal of the organizers will have been accomplished. The Editors December 1983

ACKNOWLEDGEMENTS Special thanks and appreciation are due to Professors Y. Yamamura and M. Hanaoka, President and General Secretary, respectively, of the 5th International Congress of Immunology, for their assistance regarding the opening and the publicity for this workshop. The organizers and editors are especially indebted to Dr. M. Hi rata for his painstaking and capable help in the organization and in the editorial work connected with this book. Thanks are equally due to all the members of the Department of Bacteriology, Iwate Medical University, and to Ms. V. Braymer, Lecturer, Iwate Medical University, for their assistance during various phases. For financial assistance we wish to thank the following: Packard Instruments (France S.A.) Falk Foundation e.V. (Dr. H. Falk) Iwate Medical University (President Pr. K. Obara) The Society for Promotion of Science of the Alumni

Association

of Iwate Medical University ( Chairman Pr. M. Yoshida and Chief of the Academic Section Pr. S. Katsura). Alumni Association of the Bacteriology Department, Iwate Medical University (President Pr. R. Kawana). Members of Ichirokukai, Company of Odashima (President Mr. M. Odashima), and several other

companies also contributed to the project.

Société Française d'Immunologie kindly provided some financial assistance to M.K.A. towards travel to Kyoto. Finally, grateful appreciation is due to all those persons who contributed at different times in different ways although the space does not permit specific, individual mention.

CONTENTS

Dissociation of Tissue Localization of Endotoxin from Endotoxin Lethality George LSzar, Elizabeth Husztik, Susanna Ribarszki and Alexander Pinter

1

Sinusoidal and Parenchymal Cells as Targets of Endotoxin Effects on the Liver Riccardo Utili, Giovanni B. Gaeta, Augusto Andreana and Giuseppe Ruggiero

11

LPS-Induced Hydrogen Peroxide Release from Peritoneal Macrophages of Normal and Immunodefective Mice Hideyuki Kato, Hideo Yaoita, Tatsuo Saito-Taki and Masayasu Nakano

21

Induction of Metallothionein in Macrophages: A Molecular Mechanism for Protection Against LPS-Mediated Autolysis Steven R. Patierno and Duane L. Peavy

39

RES Function and a Role of Plasma HDL in EndotoxinPoisoned Mice Shuhei Sakaguchi, Hiroharu Abe and Osamu Sakaguchi

57

Neutralizing Effects of Anti-Salmonella Re Antibody on Endotoxic Bone Marrow Reactions and Induction of Procoagulant Activity Masao Yoshida, Kazuaki Kudoh, Michimasa Hirata, Katsuya Inada and Masami Ogasawara

77

Murine Immune Responses to the Salmonella Lipopolysachharide Regions Emilio Jirillo, Hiroshi Kiyono, Suzanne M. Michalek, Donato Fumarola and Jerry R. McGhee

93

LPS-Induced Non-Specific Resistance to Immunodefective CBA/N Mice Against Salmonella Infection Masayasu Nakano, Kazuyasu Onozuka and Tatsuo Saito-Taki

..

115

Antitumor ActionFrans of Endotoxin in theandMouse Nanne Bloksma, M. A. Hofhuis Jan M.N. Willers ..

133

The Endotoxin Skin Reaction in Human Cancer Patients Yutaka Katayama, Nozomi Yamaguchi, Masaaki Kanou, Hideo Hayashi, Yoichi Fujita, Masami Oshima, Kenji Ogino and Masashi Kodama

151

XII Effects of Immobilized Lipopolysachharide on the Vx2 TumorBearing Rabbits Tohru Tani, Totaro Oka, Kazuyoshi Hanasawa, Y o s h i h i r o Nakane and Masashi Kodama

169

The A c t i v a t i o n of Complement System by Bacterial Endotoxins and i t s Effect on Malignant Tumor Tissues of Mice Yutaka Katayama, Nozomi Yamaguchi, Shun-ichi Yoshida, Tomoyuki Mizukuro, Masahiro Horisawa and Masashi Kodama ..

177

E l i c i t a t i o n of the Shwartzman Reaction by a Combination of Endotoxin and Agents which Activate the Complement System: Microvascular Events Henry Z. Movat and Clement E. Burrowes

197

Complement in C l i n i c a l and Experimental Disseminated I n t r a v a s c u l a r Coagulation Yasumasa Furukawa, Toshikazu Yoshikawa, Masashi Murakami and Motoharu Kondo

213

Effects of S t e r o i d , Anti-Thrombotic Agents and D e f i b r i n o genation on Endotoxin-Induced Disseminated I n t r a v a s c u l a r Coagulation in Rats Toshikazu Yoshikawa, Yasumasa Furukawa, Masashi Murakami and Motoharu Kondo

221

S e r o l o g i c a l and Immunohistochemical A n a l y s i s on the r o l e of Complement in Endotoxin I n i t i a t e d L e t h a l i t y in Mice Shigenobu Matsuo, Morio Totsuka, Hiroshi Hayasaka and Kokichi Kikuchi

235

Studies on Endotoxin-Like Proper-ties of Several Simple Polysaccharides Takeshi Mikami, Toshihiko Nagase, Shigeo Suzuki and Masuko Suzuki

245

Bone Resorbing Potential of Endotoxin and i t s Immunopharmacological Modulations A. Nowotny, F. Sanavi, A. M. Nowotny, E. Kovats, J. Rothmann, D. S i e g l e r , K. S a l l a y and P. H. Pham

261

Catechol amine-Hyperresponse in Endotoxemia of Mice Kazuo Kuratsuka and Reiko Homma

281

Modulation of Endotoxicosis by Steroids and Diabetogenic Agents i n Responder and Refractory Mice S t r a i n s M. K. Agarwal and G. Lazar

299

Pathogenesis in the Aggravation of Acute C h o l a n g i t i s : i t s r e l a t i o n to the Interplay of Endotoxemia and Host Resistance H i r o s h i Shimada, Gizo Nakagawara, Fumihiko K i t o , Tetsuo Abe, Mamoru Kobayashi and Shuzi Tsuchiya

315

XIII Endotoxemia in Pregnancy Due to Chloramphenicol Administration Hiroshi Irie and Wataru Mori

331

General Discussion

345

Author Index

371

Subject Index

373

DISSOCIATION OP TISSUE LOCALIZATION OP ENDOTOXIN PROM ENDOTOXIN LETHALITY

George Lazar, Elizabeth Husztik+, Susanna Ribarszki and Alexander Pinter Institute of Pathophysiology, Institute of Medical Biology"1", University Medical School, Szeged, Hungary

Introduction Correlations between reticuloendothelial activity and sensitivity to endotoxin are contradictory. The following substantiate the concept that reticuloendothelial system (RES) plays a pivotal role in resistance to endotoxin: 1) Soon after the injection of endotoxin there is deep depression in the reticuloendothelial activity, which persits until death. However, in survivors, RES function recovers and goes on to hyperfunctional state. Similar pattern of reticuloendothelial response was also observed in other types of experimental shock (1). 2) The blockade of the RES with inert, nonmetabolizable, colloidal materials sensitizes to endotoxin poisoning and abolishes the tolerance to the biological effects of endotoxin, including the lethal effect (2, 3). 3) Animals previously injected with one or more sublethal doses of endotoxin, exhibit hyperfunctional RES and increased tolerance to bacterial lipopolysaccharide (LPS) (4, 5). Other studies, however, have indicate that the relationship between host RES activity and endotoxin sensitivity is more complex. It is true that endotoxin tolerant animals have hyperfunctional RES, but the classical RES stimulants such as BCG, zymosan, Corynebacterium parvum, particulate glucan

Immunopharmacology of Endotoxicosis © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

2 do not confer resistance of e x p e r i m e n t a l a n i m a l s to e n d o t o x e m i a , but p r o f o u n d l y increase h o s t s u s c e p t i b i l i t y to e n d o t o x i n (6). P a r t i c u l a t e g l u c a n w h i l e i n c r e a s i n g a l l p a r a m e t e r s of R E S renders the LPS n o n r e s p o n d e r , C3H/HeJ, mice n e a r l y a s responsive a s conventional mice

(7)• S u b s t a n c e s

not a f f e c t i n g R E S a c t i v i t y such as S t r e p t o z o t o c i n ,

poten-

tiates the t o x i c effects of LPS i n e x p e r i m e n t a l a n i m a l s

(8);

d e p r e s s i o n of R E S a c t i v i t y b y m e t h y l p a l m i t a t e , r e n d e r s e x p e r i m e n t a l a n i m a l s h i g h l y resistant to l e t h a l a n d s u p r a l e t h a l doses of endotoxins

(9).

I n this study, to u n d e r s t a n d the correlations

between reti-

c u l o e n d o t h e l i a l a c t i v i t y a n d s e n s i t i v i t y to e n d o t o x i n , the 51 v a s c u l a r clearance a n d tissue d i s t r i b u t i o n of Cr-labelled e n d o t o x i n a n d e n d o t o x i n s e n s i t i v i t y have b e e n s t u d i e d i n mice f o l l o w i n g treatment w i t h g a d o l i n i u m chloride sodium p o l y a n e t h o l sulphonate

(10, 11),

(12) a n d c a r r a g e e n a n

A l l of these substances, a l t h o u g h h a v i n g different chemical p r o p e r t i e s , s i g n i f i c a n t l y depress

(13)• physico-

reticuloendothe-

lial f u n c t i o n .

Materials and methods CTLP

m a l e s w e i g h i n g 30-35 g were m a i n t a i n e d o n a s t a n d a r d

l a b o r a t o r y diet a n d tap w a t e r , a d libitum. G a d o l i n i u m chloride

(K. a n d K . L a b o r a t o r i e s , P l a i n v i e w , N e w

York) w a s d i s s o l v e d i n 0.85% saline at a c o n c e n t r a t i o n of 2 m g / m l a n d w a s i n j e c t e d i.v. at a dose of 1 m g / 1 0 0 g b o d y w e i g h t 24 h o u r s before testing; k a p p a , lambda, or i o t a c a r r a g e e n a n (Marine Colloids, R o c h l a n d ) w e r e d i s s o l v e d i n b o i l i n g 0.85% saline at a c o n c e n t r a t i o n of 10 m g / m l , a n d were i n j e c t e d i.p. at a dose of 5 mg/100 g b o d y w e i g h t 24 h o u r s before t e s t i n g ; sodium p o l y a n e t h o l

sulphonate

(Liquoid, H o f f m a n - L a R o c h e , Basel) w a s d i s s o l v e d i n 0 . 8 5 % saline at a c o n c e n t r a t i o n of 4 m g / m l , a n d w a s i n j e c t e d i.v.

3

at a doses of 3 or 2 mg/100 g body weight 1 hour "before testing. E. coli 026:B6 lipopolysaccharide B (Difco Lab., Detroit, lot 688839) was labelled with 50 >uCi/mg ^1Gr-sodium chromate (Isotope Institute of Hungarian Academy of Sciences) by the method of Braude et al. (14). For organ-uptake studies, one 51 hour after the i.v. injection of 250 /ug Cr-labelled endotoxin animals were killed and the radioactivities in the blood, liver, spleen, lung and bone marrow were determined in a well-type scintillation detector and the results expressed as a percentage of the injected dose. For measurement of endotoxin sensitivity, mice were challenged i.p. with proportional doses of endotoxin (E. coli 026:B6 lipopolysaccharide B, Difco Lab., Detroit, lot 688839) and the number of survivors was recorded after 48 hours. The significance of differences in the mean response between treatments was determined by the t test. The Chi squere test was used to determine the significance of differences between survivors after various treatments.

Results Data in Table 1 show that animals were sensitized to LPS by pretreatment with either 2 mg (37.5% survival) or 3 mg (15% survival) polyanethol sulphonate per 100 g body weight compared to control (80% survival) treated only with LPS. Stud51 ies with Cr-labelled LPS show that both doses of the polyanethol sulphonate caused the retention of radioactivity in the blood and reduced the hepatic uptake of the injected radioactivity. The uptake of 51Cr-labelled LPS in the spleen and lung was significantly increased (Fig. 1). The effect of carrageenan on endotoxin sensitivity and the 51 distribution of Cr-labelled endotoxin were very similar to those observed in mice pretreated with Liquoid. All forms

4

Table 1 Sensitization to LPS lethality by Liquoid Treatment

Living/total (48 hr)

1. E 2. L 3. L + E

28/35 19/20 3/20 20/20 6/16

4. L 5. L + E

Survival ( % )

Statistics

80 95 15 100 37.5

2 vs 3 vs 4 vs 5 vs

1 1 1 1

p»0.05 pCO.OOl p»0.05 pyabout 1.5 fold in the livers in mice injected with endotoxin plus lead acetate than in those treated with endotoxin alone. This lipid peroxide formation could not be observed in C3H/HeJ mice( non responder strain ) administered with endotoxin only, but these

64 were a little formation in the mice injected with endotoxin plus lead acetate( data oimnited ).

a)

xio,-3

b) _

Xanthine oxidase activity 6 hr

T

Lipid peroxidç- level 6 hr

"3 15000)54J o u a o< 6

0) •o >1 •G II •O

G

T3 C O

D

Fig. 4.

1000-

500-

£L

Effect of lead acetate on super oxide anion generation

and lipid peroxide level in livers of endotoxin-poisoned mice. A: Control mice. B: EndotoxinC6 mg/kg,i.p.) injected mice. C: Lead acetateC50 mg/kg,i.v.) injected mice. D: Lead acetate(50 nig/kg, i.v.) and endotoxin 06 mg/kg,i.p.) injected mice. Mice in each group were starved for the indicated time before decapitation after injection of endotoxin. Each bar represents the.mean + S.E. of 6 mice. Asterisk(*) indicates significant difference from the value of endotoxin-poisoned mice (B) at P < 0 . 0 5 .

[ B ].

Gltathione peroxide ( GSH-Px ) and

superoxide

dismutase( SOD ) activities. GSH-Px is distributed for the destruction of hydrogenperoxide ( ^ 2 Q 2 ^

and

or

9anic

hydroperoxide compounds by

which cellular membrane is greatly damaged. SOD plays an important role as a scanvenger of superoxide and other free radicals in the protection against cell damage. As seen in Figure 5 ( a,b ), GSH-Px in the livers of endotoxin-lead vity

treated mice exhibited about 30% less acti-

than that in the control 18 hr postintoxication ,

whereas the activity in endotoxin alone-poisoned mice

sig-

nificantly decreased

lead

or saline alone

.

than in mice treated with either The nonprotein SH level markedly

eased in the livers of lead plus endotoxin injected 6 hr postintoxication( data are omited portant role in tissues, especially

decrmice

). GSH plays an im-

in the

oxidation-redu-

ction system responsible for the inner tissue metabolism, and also protects SH enzyme and m e m b r a n SH from

free

radicals.

GSH-Px activity T IB hr T 2

-

I

b) 4 _

SOD activity 6 hr

a •S3•u o n Q.

d> e \

2 -

a a

a

D

21

B

•

B

Fig.5. Effect of lead acetate on glutathioneperoxidase(GSH-Px) and superoxide dismutase(SOD) activities in livers of endotoxinpoisoned mice. A: Control mice. B: Endotoxin(6 mg/kg,i.p.)injected mice. C: Lead acetate(50 mg/kg,i.v.)-injected mice. D: Lead acetate(50 mg/kg,i.v.) and endotoxin(6 mg/kg,i.p.)-injected mice. Mice in each group were starved for the indicated time before decapitation after injection of endotoxin. Each bar for GSH-Px activity represents the mean + S.E. of 12 mice, and SOD activity is the result from 6 mice. AsteriskC*) shows significant difference from the value of endotoixn-poisoned mice(B) at P

c o a u>

•

•

+

-P •H

c SB

(0

M CO

a) Ol

+ 1 r • ro

o 1

•

(0 H ai

o

rH rH

•H 0)

1

•

ro •

i id Q

• o

•

• o

o • o

o • o

o •

o

o •

o

• o

tn 3.

CJ LT) h fu

C M •G -p

c 0) rH a C O

-H S

>i P

id g a e -H

i i id Q

-a a) •p o a) •n ß -H a) o -H e

ß a) tu o ß

P

w OH

w

^

1 (d

tó

w cu 1-1 ai

S

A

>i id Q

>1 id Q

>1 id a

102 •a1 • o o < + 1 + 1 co r• TJ • •H CM VO

00 • O + 1 (N • oo

r- i • i o 1 + H i • i vo I

CO • o + 1 un •

rO • « O o + 1 + 1 1— r-

CM • Ï— + 1 (N

co i • i o 1 + 11 un i

co • • o o + 1 + 1 co

CT» • O + 1 CN

ro

r-

CTI

VO 1

(N

00

un • o + 1 oo • co

un • o + 1 VO • r-

CO 1 • i O 1 + 11 r- i • i va 1

(N • • • o O O + 1 + 1 + 1 CTI CN (N • • • in O

• O + oo • io

in oo • • • o o o + 1 + 1 + 1 (N co vo

•si1 1 • i o 1 + 11 oo 1

VO • O + 1 co

co • o + 1 vo

ro • o + 1 CM

CO 1 • 1 O 1 + 11 CM 1

m

in

r-

oo

CM

n

•

(U 1 >1 1 >1 id i d id i id Q o D 1 Q

>1

id

^

O

1 1

a) tu -a Vi 0) 4H -P

rH

1 1 1

UD

O

aj

«

m

o

tf

(U

1

f«i




P.C. Spleen

rTT —

10'

'3 10

• • • •I 10 '

Figure 1. Effect of LPS on the infection of Salmonella enteritidis No.11 strain in (CBA/N female x BALB/c male)Fl mice. LPS (20pg) was ip injected 24 hr before the infection, and mice were ip challenged with 1 x 10^ organisms of the No.11 strain. Viable numbers of the organisms in the peritoneal cavity (P.C.) and spleen were determined by quantitative cultivation at 24 hr after the infection. Results are expressed as the arthmetic mean of 3 mice ± the standard deviation.

mice.

When male as well as female mice were treated with

LPS before the infection, these mice showed an augmentation of bactericidal ability.

The numbers of organisms in LPS-

treated mice were clearly less than those in untreated control mice.

The bactericidal effect induced by LPS was

prominent in their peritoneal cavities rather than in the spleens.

These results suggest that LPS has the ability to

enhance bactericidal activity in CBA/N-defective mice, the same as in phenotypically normal female mice. Dose-effect of LPS on bactericidal activity in the peritoneal cavity and spleen

121

10-

•f

in

e

01

-H c (fl Cn

U O

>4-1 0 ^01

10

ai

« >

•H

103-| LPS(ug) : 20

0.2 Fl male

0.02

none

none Fl female

Figure 2. Anti-bacterial effect of LPS in the peritoneal cavity and spleen of CBA/N-defective mice. LPS was ip injected 24 hr before infection with S. enteritidis No.11 strain (4 x 105 organisms, ip). The viable numbers of infecting organisms in the peritoneal cavity ( O ) an,

, s ai S- 00 1/1 •a l/l >1 ai < • 1—Ö IO s > s fXXJ c • 00 o CM i CTI E

f"

. '' *

i

>

®

/ W

Fig. 6.

'

m

J

3

4

Time(hour)

Fibrinogen and Platelet counts in Experimental DIC in Rats

Fig. 4

Fibrin thrombi in glomerular capillaries. a.(Weigert 1 s fibrin stain, x400) b.(H and E x400)

2 ) Effect of various agents on experimental DIC

M e t h y l p r e d n i s o l o n e . The rats were injected with at 0.1, 1, 10, 30 m g / k g , and i m m e d i a t e l y were infused

with

100 m g / k g of e n d o t o x i n

p r e v e n t i o n w a s n o t e d in all p a r a m e t e r s , 1.0,

10.0 or

30.0 mg/kg

of

methylprednisolone

a f t e r the

injection,they

for A hr. A

significant

in r a t s pre t r e a t e d

methylprednisolone

(Table

3).

with

226 Table 3« Effects of methylprednisolone on experimental DIC in rats infused with 100 mg/kg of endotoxin for 4hr. FDP (Ug/ml) 56.0(8.6

Control3^

Fibrinogen (g/D

84.9+12.1

15

109.4+21.7**

163+11**

67.5+16.5*

8

83.5+10.1**

305+20»

40.1+6.6**

8

59.8+12.0**

343+41**

24.5+ 5.0"

8 8

Fibrinogen (g/i) 5

0 . 7 5 X + 3.68-,

i h Y = 0L88 X + 2.5 9

line of lhr-tabulation was realized over the first three endotoxin

Dose Endotoxin*

il-Lz

doses exclusive of 2000ug dose.

I * 5 Difference

It is

2/20

2000 »a/™

20

6/20

30 Hi0 opo

14/19

17/20

11/19

9/20

58

3/19

B5

45

8/20

evident that the lethal response of 2000ug dose

Fig. 2 Dose-response line (B)

in the lhr-tabulation period was intensely suppressed.

Such a paradoxical

suppression of the response caused by large dose of endotoxin

disappeared

20hrs after the challenge, giving

the linear relation over the four doses inclusive of 2000ug dose.

The paradoxical suppression will be dis-

cussed later. Effect of hydrocortisone, histamine and serotonin on the lethal adrenaline-hyperreaction. It is known that substances such as hydrocortisone, histamine, and serotonin are physiologically released in vivo after endotoxin administration

(15-17).

A pre-

liminary study on the effect of administration of these three substances on the lethal adrenaline hyperreaction was conducted.

Table 3 shows that suppressive effect on

adrenaline hyperreaction was observed for each substance

287

Table 3.

Effects of some biologically active substances on the lethal adrenaline-hyperreaction

Substances

Treatment-period Without Adrenabefore and after treatment line

tested dose/Ms

L P S

Contro1

+2h

Control

aj 3/40**

8/40

11/40

0/40

8/20

5/20

14/20

0/20

mg

b) 1/20****

mg

0/20****

0/20 * * * *

#1 Hydrocortisone

1.25 mg

#2 Histamine

6

mg

10 6

Serotonin

-2h

#: Experimental number. LPS: 40ug dose of E. eoli LPS (W) was intravenously given to mice as a constant dose. Adrenaline: The mice of exp. #1 were challenged ip. with 50ug dose of adrenaline contained 0.1ml of saline 4hrs after LPS administration.

But the mice of exp. #2 were challenged ip. with 65ug

dose of adrenaline. a) Numerator represents number of dead mice challenged with adrenaline; denominator, total number of mice tested. b) Significant difference between the lethal rates of LPS-adrenaline group and of adrenaline control group; Significance level: ** 2.5%, *** 1.0%, **** 0.1%

within the experimental conditions.

These findings will

be discussed later in relation to the paradoxical suppression of the hyperreaction caused by large dose of endotoxin. 7)

Effect of hydrocortisone on the adrenaline-hyperreaction. In the previous section it was shown that hydrocortisone had suppressive effect on the adrenaline hyperreaction. Furthermore, it is known that hydrocortisone is a potent therapeutic agent for endotoxin shock.

Therefore, the

288

Fig. 3 The effect of hydrocortison-pretreatment on the lethal adrenaline hyperreaction

effect of hydrocortisone on the adrenaline-hyperreaction was studied in detail (5).

The effects are not simple

and they are shown schematically in Fig. 3. Hydrocortisone acetate (2500ug per mouse) was given to mice in various periods before LPS administration.

Then

4hrs later, the mice were challenged with 65ug of adrenaline. The effects of a single dose of hydrocortisone are quite different, depending on the pretreatment periods of hydrocortisone.

Suppressive effect was observed in

the case of short pretreatment periods such as less than 2 days.

On the contrary, enhancing effect was noted

when adrenaline was given 1 to 2 weeks in advance.

No

significant effect was found between 3 and 5 days and in much longer periods such as 6 weeks. 8)

Circadian rythms of the lethal adrenaline-hyperreaction. It was noticed that there was some difference in the intensity of responses between the experiment performed in the morning and that in the afternoon.

Then the

289

intensity of the lethal adrenaline-hyperreaction was tested using the same LPS solution administered at different time of the day such as at 11.30, 16.00, 24.00 and 8.00. Fig. 4 shows that the response intensity in the midnight and in the morning is weak but the intensity near noon and in the afternoon is strong.

This figure demon-

strates a circadian rhythm of responses. Therefore the adrenaline-hyperreaction is of interest from the chronopharmacological points of view. 9)

Time-response relation of the lethal noradrenaline-hyperreaction.

Fig. 4 Circadian reiponie of lethal adrenalinehyperreaction

Recently we found that mice in a state of endotoxemia died hyper-

20

reactive death by the administration of sub-

^ B Death rate 1 h after Ad.

$

•

lethal dose of noradrenaline instead of ad-

Death rate 20 h after Ad.

± 10

renaline. The new hyperreaction was named the lethal noradrenaline-hyperreaction which is similar to the lethal adrenaline-hyperreaction mentioned

11.30

16.00

24.00

8.00

Administration timeaf LPS in a day LPS: 40^0.2 mi iv/mouse Ad: 50*%.! mi mouse, 4h after IPS.

above. Table 4 shows the time-response relation of the noradrenaline-hyperreaction, which appears intensely as early as lhr after until

endotoxin

administration and persists

4 to 5hrs and then decreases

or disappears 24hrs thereafter.

in intensity

The time-response

relation of the noradrenaline-hyperreaction is quite

290

Table 4

Incidence of lethal noradrenaline-hyperreaction by LPS Noradrenaline lethality after LPS _ ,, . , . » in following periods (h). b)

rT.„

. . LPS control ug iv

1

40

0/20 a)

40

0/20

40

0/20

2

3 *

8b) 9 *** * * 11 12 *** * * 11 12

****

* *

14

4 9

5 *

***

10

9

*

6

24 h

***

450

600

900 ug

3/20 a)

13

***

„ , , . Noradrenaline ^

11

2

0

2

4

2

2/20 3/20

a)

Numerator represents the number of dead mice; denominator the

b)

Under the columns of noradrenaline lethality, denominators are

total number of mice tested.

omitted and numerators only are shown.

The omitted denomi-

nators are the same as those of LPS control and

noradrenaline

control-groups. •Significance levels; see the legend of Table 1.

different from that of the adrenaline-hyperreaction in that response appears rapidly after noradrenaline challenge.

In addition, as shown in the columns of norad-

renaline controls, the hyperreaction required largeamounts of noradrenaline such as 450 to 900ug per mouse. On the contrary, the adrenaline-hyperreaction required much smaller amount of adrenaline such as 40 to 65ug per mouse.

The facts suggest that noradrenaline may be less

toxic than adrenaline as a therapeutic drug of endotoxin shock. 10)

Dose-response relation (A) and (B) and circadian rythms of response in case of the lethal noradrenaline-hyperreaction. Table 5 shows that a linear dose-response relation (A)

291

Table 5

Dose-response relation.

Varying dose of NA.

LPS constant dose LPS control ug iv 40

0/15

0.4

0/15

Dose of Noradrenaline (ug) 56.2

112.5

0/15

3/15

0/15

2/15

225

450 ug

450

****

9/15

****

9/15

NA control

****

13/15

0/15

****

10/15

Challenge of NA : 4h after LPS administration is realized with varying doses of noradrenaline in serial 2-fold dilutions and certain constant doses of endotoxin such as 40 and 0.4ug per mouse.

The slope of the dose-

response line is not as steep as that after adrenaline. Furthermore, in spite of a 100 times difference between 40ug and 0.4ug of endotoxin, the dose-response relations of the two doses of endotoxin are similar.

This fact

suggests that it may be difficult to obtain a linear dose-response relation

(B) with varying doses of endo-

toxin and a constant dose of nonadrenaline.

This esti-

mation was confirmed by actual experiments. Also,the noradrenaline-hyperreaction did not show any significant difference between the intensity of response in the early morning and that in the afternoon.

The fact

suggests that the noradrenaline-hyperreaction would not have circadian rhythms of response unlike the adrenalinehyperreaction. In summary several different points between the adrenaline and the nonadrenaline-hyperreactions were described.

292

11)

Effect of spinal transsectomy on lethal adrenaline-hyperreaction. Egdahl demonstrated that secretion of adrenaline was increased from the adrenaline medulla of dogs given endotoxin but the secretion was lost from the medulla of dogs whose spinal cord had been transsected at the level of Table 6

Effect of spinal transsectomy on lethal adrenaline-hyperreaction

Operation

-

+

+

-

LPS

40

ug

+

+

-

-

Adrenaline

65U9

+

+

+

+

Death rate after Ad. Challenge 1

h

%

69

6

6

0

20

h

%

75

59

6

0

the 7th cervical vertebra ( 13 ). The result suggested that secretion of adrenaline caused by endotoxin administration was brought about by nervous stimulation Of the target brain cells by endotoxin. Then the effect of spinal transsectomy on the lethal adrenaline-hyperreaction was studied in mice. In Table 6 the first column is LPS control column and the next one shows the column of the experimental purpose. In lhr after the adrenaline challenge, the death rate(6%) of the mice operated surgically was suppressed significantly, as compared with that (69%) of the LPS control. In 20hrs after the adrenaline challenge, however, the suppressed death rate(6%) of the operated group of mice increased to 59% again. Such reincrease of death rate in the 20hrs tabulation was observed only in this group of

293

mice previously given endotoxin,but not in the operation control group of mice that lack endotoxin administration, shown in the third column. Mechanisms of the results obtained are still insoluble but they will be later discussed briefly. In addition, the spinal transsectomy brought about also similar effects on the lethal noradrenaline-hyperreaction.

Discussion The mechanisms of the lethal adrenaline-hyperreaction is not understood.

Some workers reported the release of adrenaline

in vivo by endotoxin administration (7 - 13).

Such a release

of adrenaline may be involved also in endotoxemia.

The ex-

ogeneous addition of a sublethal dose of adrenaline to the possibly increased adrenaline contents in blood in a state of endotoxemia may result in the shock-like death of mice because of an untolerable amount of adrenaline.

In the lethal

adrenaline-hyperreaction the death rate of mice within lhr after adrenaline challenge must correspond to the shock-like death of mice.

As shown in Fig. 1, the dose-response line is

realized in narrow range of adrenaline such as in serial 1.3-fold dilutions. steep.

Consequently the slope of the line is

The fact suggests that the change of challenge dose

of adrenaline could be a critical factor in this hyperreaction and that fine quantitative analysis of the adrenalinehyperreactivity induced by endotoxin may be possible with such a dose-response relation.

In case of noradrenaline-

hyperreaction, similar dose-response line was observed in narrow range of noradrenaline doses such as in serial 2-fold dilutions.

Consequently the slope of the line is relatively

steep but not as steep as in case of adrenaline-hyperreaction.

It may safely be said that the change of noradrenaline

could also be a critical factor in the noradrenaline-hyper-

294

reaction.

On the other hand, the dose-response line with a

constant dose of adrenaline and varying doses of endotoxin, as shown in Fig. 2, is realized in wide range of endotoxin doses in serial 10-fold dilutions such as 2 to 2000ug per mouse.

The fact suggests that the adrenaline-hyperreacting

activity of endotoxin preparations could be estimated singly or by parallel line assay method using such a dose-response line. Furthermore, Fig. 2 shows that a paradoxical suppression of the hyperreaction due to large sublethal dose of endotoxin such as 2000ug per mouse was observed in the lhr-tabulation after adrenaline challenge but such a suppression disappeared in the 20hr-tabulation.

The fact suggests that there are

some differences between the mechanisms of the early lethality such as death of mice within lhr and the mechanisms of the later one tabulated

20hrs

after adrenaline challenge.

Large sublethal dose of endotoxin such as 2000ug per mouse may release large amount of physiologically active substances such as hydrocortisone, histamine and serotonin which have in fact suppressive effect on the lethal adrenaline-hyperreaction, as shown in Table 3.

In consequence the large dose of

endotoxin could induce such a paradoxical suppression.

In the

lethal noradrenaline-hyperreaction, however, linear doseresponse relation could not be observed in wide range of endotoxin doses in serial 10-fold dilutions, as suggested from Table 5.

The paradoxical suppression observed in adrenaline-

hyperreaction action.

was

not

seen with of noradrenaline-hyperre-

These facts and the different time-response, as

shown in Table 4, suggest that there are differences in the mechanisms concerned between two kinds of the hyperreaction but the real reason remains to be solved.

Especially, the

noradrenaline-hyperreaction was recently noticed and is still under investigation. As shown in Fig. 4, the lethal adrenaline-hyperreaction demonstrates a circadian rhythm whose response intensity is weak in

295

the midnight and in the morning, and strong near noon and in the afternoon.

Such a circadian rhythm may depend largely on

the function of adrenal cortex of nocturnal aminals like mice. We suppose that glucoccorticoids play a role in this circadian rhythm.

Without knowing the exsistence of the cir-

cadian rhythms, it would have been impossible to obtain reproducible results for the hyperreaction experiments. As for the effect of hydrocortisone on the adrenaline-hyperreaction, the effects are quite different depending on the pretreatment periods of a single large dose of hydrocortisone acetate such as 2500ug per mouse (see Fig. 3).

Suppressive

effect in case of short pretreatment periods of less than 2 days is reasonable judging from the preliminary results as shown in Table 3.

However, the enhancing effect in case of

1 to 2 weeks pretreatment periods ical.

would seem to be paradox-

The reason could be that large dose of hydrocortisone

brought about hypofunction of the adrenal cortex and resulted in lack of secretion of glucocorticoids,

in other words,

in an "exhausted state" of cortex function.

Consequently the

hyperreaction might be enhanced by the lack of suppressive substances like hydrocortisone.

This enhancement of the

hyperreaction would be restored to the original state due to the normalization of cortex function by 6 weeks after the hydrocortisone administration. As for the effect of spinal transsectomy on the adrenalinehyperreaction (Table 4), the results in lhr-tabulation are consistent with Egdahl's study (14), that endotoxin did not directly act on adrenal medulla to release adrenaline but via central nervous system.

However, the suppressed death

rate in lhr-tabulation increased again in the 2 0hr-tabulation. The reason is not clear at present. As shown in Table 1 and 4 and in Fig. 1, the fact that noradrenaline is less toxic than adrenaline for mice in a state of endotoxemia would be worthy of notice in clinical use of adrenaline or noradrenaline in endotoxin shock.

The results

296 in Fig. 3 suggest that administration of glucocorticoid preparations may give beneficial effect to the untoward response of adrenaline in endotoxin shock.

Summary Catecholomine-hyperresponse in mice induced by endotoxin included the lethal adrenaline and noradrenaline-hyperreactions of which characteristics were compared in the following points:the time-response relation, the dose-response relation with a constant challenge dose of the catecholamines and varying doses of endotoxin and vice versa, the paradoxical suppression of response due to large sublethal dose of endotoxin, the circadian rhythms of response and the effect of spinal transsectomy.

Acknowledgements We are grateful to Mr.Y.Shimazaki for his helpful assistance and Miss H.Kohama for her preparing and typewriting of the manuscript. References 1.

Kuratsuka,K.,Homma,R.,Shimazaki,Y.,Funasaka,I.:in

Animal,

Plant,and Microbial Toxins, ed.by Osaka,A.,Hayashi,K., Sawai,Y. vol. 1, 521-534, Plenum Press, New York, London (1976) 2.

Kuratsuka,K.,Shimazaki,Y.,Funasaka,I.: Japan. J. Bact.31: 110 (1976)

3.

Kuratsuka,K.,Shimazaki,Y.,Funasaka,I.,Watabe,Y.: Experientia 34: 1483-1484 (1978)

4.

Shimazaki,Y.,Funasaka,I.,Kuratsuka,K.: Japan. J. Bact.35: 224 (1980)

297

5. 6. 7. 8 9. 10. 11. 12.

13. 14. 15. 16. 17.

Kuratsuka,K.,Shimazaki,Y.,Funasaka,I.,Homma,R.: Japan. J. Bact. 35:225 (1980) Kuratsuka,K.,Shimazaki, Y. ,Funasaka, I. ,Homma,R.: Japan. J. Bact. 36:249 (1981) Boquet,B.,Izard,Y.: Proc.Soc.exp.Biol.Med. 75:254 (1950) Thomas,L.: Ann.Rev.Physiol. 16 :467 (1954) Rosenberg,J.C.,Lillehei,R.C.,Moran,W.H.,Zimmerman,B.: Proc.Soc.exp.Biol.Med.: 102:335 (1959) Heiffer,M.H.,Mundy,R.L.,Mahlmare,B.: Am. J. Phsiol. 198: 1307 (1960) Alican,F.: Am. J. med. Sci. 244 (1962) Schayer,R.M.: in Bacterial Endotoxins, ed. by Landy,M., Braun,W., 182-186, Rutgers Univ. Press, New Brunswick, (1964) Jacobson,E.D.,Mehlman,B.,Kalas,J.P.: J. clin. Invest. 43: 1000 (1964) Egdahl,R.H.: J. clin. Invest. 38:1120-1125 (1959) Hinshaw,L.B.,Jordan,M.M.,Vick,J.A.: Am. J. Physiol. 200: 987 (1961) Hinshaw,L.B.,Jordan,M.M.,Vick,J.A.: J. clin. Invest. 40: 1631 (1961) Kobold,E.,Katz,W.,Thai,A.P.: Fedn.Proc. 22:430 (1963)

M O D U L A T I O N O F E N D O T O X I C O S I S BY S T E R O I D S A N D D I A B E T O G E N I C A G E N T S IN R E S P O N D E R A N D R E F R A C T O R Y M I C E S T R A I N S .

M. K. A g a r w a l a n d G. Lazar^ C e n t r e N a t i o n a l de la R e c h e r c h e S c i e n t i f i q u e and L a b o r a t o r y of P h y s i o - H o r m o n o - R é c e p t é r o l o g i e , U n i v e r s i t é P i e r r e et M a r i e C u r i e , 15 rue de l'Ecole de M é d e c i n e , 75270 P a r i s Cedex 06, France. ^ I n s t i t u t e of P a t h o p h y s i o l o g y , M e d i c a l Szeged, Hungary.

S c h o o l of

Szeged,

I n t r o d u c t ion Two d i s t i n c t sets of h o s t p a r a m e t e r s h a v e c l a s s i c a l l y related to e n d o t o x i n l e t h a l i t y in e x p e r i m e n t a l a c t i v i t y of the r e t i c u l o e n d o t h e l i a l

system

(RES) was

to p l a y the p r i m o r d i a l role in this r e s p e c t n e a r l y d e c a d e s ago subsequent

been

animals.

The

believed

four

(1) b u t this v i e w u n d e r w e n t d r a s t i c r e v i s i o n s studies

in

(2-3).

N e g a t i v e c a r b o h y d r a t e b a l a n c e and h y p o g l y c a e m i a are

consis-

t e n t l y a s s o c i a t e d w i t h e n d o t o x i c o s i s a n d some six d e c a d e s it w a s b e l i e v e d that they m a y be c a u s a l to e n d o t o x i n (4). H o w e v e r , g l u c o c o r t i c o i d h o r m o n e s can p r o t e c t endotoxin lethality without restoring c o n t r o l level

in n o r m a l m i c e discovery,

against

these parameters

per

(6-8).

the n o n - r e s p o n d e r

C3H/HeJ mouse

has b e c o m e a p o t e n t tool to p r o b e the site a n d n a t u r e endotoxin action

to the

(5), and e l e v a t e d g l y c a e m i a ,

se, does not l e a d to p r o t e c t i o n Ever since the

ago

lethality

(9). D a t a r e p o r t e d h e r e e x p l o r e the

ship b e t w e e n the two a f o r e m e n t i o n e d

sets of h o s t

of relation-

parameters

in n o r m a l

and r e f r a c t o r y m i c e s t r a i n s w h o s e s e n s i t i v i t y

bacterial

endotoxins had been modified by streptozotocin

a d i a b e t o g e n i c a g e n t , a l o n e or in c o m b i n a t i o n w i t h a g e n t s s u c h as g l u c a n , a n d s t e r o i d

hormones.

Immunopharmacology of Endotoxicosis © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

to (SZN),

RES-active

300 Materials and Methods Male, C3H/HeJ and C3H/eb mice animals

(CNRS, O r l é a n s , F r a n c e ) a n d OF.,

( I F F A - C R E D O , France) w e r e h o u s e d w i t h free a c c e s s

p e l l e t f o o d a n d w a t e r ad l i b i t u m . A n a u t o m a t i c s w i t c h 12 h o u r s of d a r k n e s s a n d 12 h o u r s of light and h u m i d i t y c o n t r o l l e d Streptozotocin

(SZN:

to

assured

in a t e m p e r a t u r e

environment.

2-deoxy-2(3-methyl-3-nitrosoureido)-D-

glucopyranose) was obtained from Upjohn, Kalamazoo,

Michigan;

this a n t i b i o t i c w a s d i s s o l v e d in c i t r a t e d s a l i n e , p H i m m e d i a t e l y p r i o r to i n t r a p e r i t o n e a l

4.5,

(ip) i n j e c t i o n in a 0.5

ml v o l u m e c o n t a i n i n g the d e s i r e d a m o u n t of p r o d u c t . Particulate

glucan

(Standard B r a n d s , N e w Y o r k , lot IF 5500)

w a s s u s p e n d e d in s a l i n e p r i o r to i n t r a v e n o u s in 0.2 ml v o l u m e s c o n t a i n i n g

(iv)

injection

1 m g of the p r o d u c t . G l u c a n w a s

i n j e c t e d o n d a y s 7 a n d 5 (total of 2 mg) p r i o r to ip

challenge

on D a y 0 w i t h d i f f e r e n t d o s e s of S a l m o n e l l a t y p h i m u r i u m W (Difco, lots 6 4 4 8 0 9 and 654477) in 0.5 m l s a l i n e . The w e r e r e c o r d e d 24 and 48 h Triamcinolone

acetonide

later.

(TA) w a s d i s s o l v e d

i n j e c t e d ip in 1 m g a m o u n t s

in s a l i n e

and

in a v o l u m e of 0.5 ml in all

c a s e s . T h i s s t e r o i d w a s o b t a i n e d f r o m S i g m a in the

non-radio-

a c t i v e form w h e r e a s the t r i t i a t e d p r o d u c t w a s p u r c h a s e d New

England

from

Nuclear.

The f u n c t i o n a l

a c t i v i t y of the R E S w a s a s s e s s e d b y the

clearance test deatiled previously carbon

LPS

survivors

(10). B r i e f l y ,

20 m g / m l

(Gunther W a g n e r , H a n o v e r , G e r m a n y , C 11/1431

physiological

saline containing

carbon

a)

10 U / m l h e p a r i n w a s g i v e n

in 0.2 ml v o l u m e s . B l o o d s a m p l e s of 50 yl from the

of

in iv

retro-

o r b i t a l p l e x u s w e r e w i t h d r a w n 2 a n d 7 m i n later and l y s e d

in

4 ml of 0.1% s o d i u m c a r b o n a t e . The q u a n t i t y of c a r b o n w a s m e a s u r e d at 675 n m a n d c o m p a r e d a g a i n s t a s t a n d a r d c u r v e . and organ electronic

(liver a n d spleen) w e i g h t s w e r e d e t e r m i n e d o n an balance.

Body

301 Blood glucose

levels were quantitated on heparinized

samples

from the r e t r o o r b i t a l p l e x u s c e n t r i f u g e d to o b t a i n c l e a r m a . S a m p l e s of 20 yl w e r e m i x e d w i t h 2.5 m l of the Biotrol reagent

Gluci-

(Biotrol, Paris) c o n t a i n i n g p h o s p h a t e

buffer,

p H 7.5, A s p e r g i l l u s n i g e r g l u c o s e o x i d a s e , h o r s e r a d i s h xidase, amino-4-antipyrine, procedure, D-glucose peroxide,

and h y d r o x y b e n z o a t e .

is t r a n s f o r m e d

in p r e s e n c e of g l u c o s e o x i d a s e , w h i c h is

The i n t e n s i t y of the stable p i n k c o l o u r

pero-

In this

into g l u c o n i c a c i d

c o n v e r t e d to a c o l o u r e d c h r o m o g e n in p r e s e n c e of

and

thereafter

peroxidase.

is m e a s u r e d at 510 nm

and compared with a standard D-glucose curve calculated grams per

plas-

as

liter.

Liver g l y c o g e n w a s d e t e r m i n e d b y h y d r o l y s i s subsequent

transformation

into g l u c o s e

into h y d r o x y m e t h y l f u r f u r a l

t a t e d w i t h a s t a n d a r d g l u c o s e curve at 520 nm. B r i e f l y , 100 m g liver t i s s u e w a s h o m o g e n i z e d

and

quanti50-

in 5 m l of 5'0 T C A - 0 . 1 1

s i l v e r s u l p h a t e , h e a t e d in a b o i l i n g w a t e r b a t h for 15 m i n , a n d c e n t r i f u g e d at 5000 g for 15 m i n

(4°C) . A l i q u o t s of 1 ml

w e r e m i x e d w i t h 6 m l of 36 N H 2 S O 4 , h e a t e d in a b o i l i n g b a t h for 6 m i n , c o o l e d in an ice b a t h , a n d q u a n t i t a t e d spectrophotometer. milligram

water in a

The g l y c o g e n l e v e l s are e x p r e s s e d as

percent.

H o r m o n e r e c e p t o r a s s a y s w e r e p e r f o r m e d on 0.5 ml a l i q u o t s the 1 0 5 , 0 0 0 g liver s u p e r n a t e incubated

in 0.01 M T r i s - H C l , p H

(60 m i n , 4°C) w i t h the r a d i o a c t i v e

of

7.4,

steroid. The

free

r a d i o a c t i v i t y w a s r e m o v e d b y the a d d i t i o n of 0.2 ml of a 50 mg/ml

s u s p e n s i o n of a c t i v a t e d c h a r c o a l

(10 m i n , 4 ° C ) , and c e n t r i f u g a t i o n w e r e c o u n t e d in 10 m l U n i s o l v e

(Sigma),

incubation

(3000 g). 0.2 m l

(Kochlight).

samples

Protein was

d e t e r m i n e d b y the B i u r e t m e t h o d . A l l r e s u l t s are e x p r e s s e d C P M / m g p r o t e i n , as p r e v i o u s l y d e s c r i b e d The standard deviation,

(11-13).

the S t u d e n t t test, a n d the Chi

test,

w e r e u s e d to a n a l y z e r e s u l t s . F u r t h e r d e t a i l s are p r o v i d e d appropriate

legends.

as

in

302 Results D a t a in table

1 show that OF^ m a l e m i c e are s e n s i t i z e d

to

e n d o t o x i n l e t h a l i t y b y SZN in a d o s e d e p e n d e n t m a n n e r . T h e 5 mg dose of this a n t i b i o t i c permitted 40% toxin which,

survival after

the

in c o n t r o l a n i m a l s , p r o d u c e d 781 s u r v i v a l .

This

5 m g dose d i d not s t r e s s m i c e in a n y a p p a r e n t w a y a n d w a s c h o s e n for all s u b s e q u e n t Table

1. D o s e d e p e n d e n t

studies.

s e n s i t i z a t i o n to e n d o t o x i n

by Streptozotocin

in OF., m a l e

lethality

mice.

Streptozotocin

Living/ T o t a l 48 h

1. N o n e

47/60

78

2. 1 m g

14/20

70

t 2 vs 1 : N.. S.

3. 3 m g

11/20

55

3 v s 1 : N.. S.

4. 5 m g

8/20

40

5. 10 m g

2/20

10

4 vs 1 : P < 5 vs 1 : P
401 >, E SO l IO * o td Í. 1- o Ec 3 O ai S < > >3 S O > a, a; ai ai ai u_ t/i o « +-> O < J et to S- o ce •— 1 ai s. i-. m in a. L-:

O

to ai

>,

o c o ai .c ai cl in

— r

to ai tai o 4J >, Cl O IO O -C — i o. 3 o c s•0 o S- IO o E

T D ai c c l/l 10 IO ai — f X E Cl o O IO -O l/l E O 00 O 00 O S>, s- -C —IQI .—

m — r •i- t/1 -C cl ai o— . s- ai +J 4-> 3 io ai — r z CL

u

— r• C ai Cl o c 3 E E«

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348

Jirillo. The C3H/HeJ mice are a potent tool to assay the purity of various endotoxin preparations. When an LPS preparation produces a mitogenic response in this strain, that endotoxin should be discarded since proteins associated w i t h lipid A or the polysaccharide will also stimulate a similar response. Endotoxin from Bacteriodes fragilis used by Michalek et al (see this volume) does not contain KDO and also differs in the sequence of fatty acids. This lipid A is less toxic than other, conventional endotoxin preparations but it stimulates the C3H/HeJ mice. When spleen cells are isolated, in order to remove macrophages and leave only B cells, this Bacteriodes LPS is unable to stimulate mitogenicity of polyclonal activation in the C3H/HeJ refractory strain. Thus, carbohydrate moiety is extremely important in stimulating macrophages which in turn induce mitogenic effects on lymphocytes. The F 1 hybrids of the C3H/HeJ X C3H/HeN backcross gave an intermediate response to this endotoxin confirming a single lps gene for antibody production to LPS. Agarwal. Since you are studying only one or two responses that are very closely related, a single gene m a y control However, the entries listed in the table I above,

are all

functioning at a very low level in the C3H/HeJ strain; how would you envision a single lps gene protein

them.

so

controlling

all of them. Jirillo. In C3H/HeJ mice there is very little expression of the Fc receptor on the macrophage and this can be reversed by interferon. Not all aspects have been explored in this

strain.

May be there are different genetic alterations for each tissue and organ and it is very difficult to answer your question at this point.

349 Nakano. I think that the lps gene controls many responses of endotoxins but we presented evidence for another gene as well. The CBA mice have some defect in the response to endotoxin which may be due to X-linked recessive genes and the whole problem should be analyzed more extensively. Bloksma. The induction of interferon by endotoxins is completely abolished by prior administration of alpha-adrenergic agents. The adrenals of C3H/HeJ mice do not appear to synthesize glucocorticoids normally. In addition, the adrenergic or the sympathetic nervous system is also involved. Agarwal. The glucocorticoid receptor levels are influenced by endotoxins and there is just no evidence of any linkage between the lps gene and the glucocorticoid receptor gene. So I feel convinced that we have to entertain the involvement of more than one gene or foci in overall LPS action. Matsuo. The unresponsiveness of the C3H/lieJ mouse is a B cell defect, primarily concerned with LPS mitogenicity, but its relation to endotoxin lethality is not clear. Several defects become evident after injection of endotoxin in C3H/HeJ mice, such as prostaglandin synthesis inhibition, and the event (s) causal to LPS lethality remain unknown. Hirata. I would like to talk about four other parameters induced by endotoxin. First, endotoxin induced cytotoxic action

in bone marrow is mainly mediated by granulocytes and

monocytes; second, endotoxin induced increase in procoagulant activity and in tissue thromboplastin, in bone marrow cells* is generated from monocytes; third, endotoxin induced decrease in nucleated cells in the marrow

is due to the migration of

the former into the circulation; fourth, endotoxin induced the production of FDP after activation of the fibrinolytic system which is an important parameter for the genesis of DIC.

350

None of these reactions could be induced in the HeJ mice. In fact, the HeJ mice were 100 times less responsiveness

than

conventional mice in the elicitation of the procoagulant activity.

As to

the FDP production, different

preparations, some containing even in very high doses

endotoxin

10. 41 protein (Boivin type),

(500 yg)

did not produce an increase

in FDP. Thus, I think that neither endotoxin nor lipid A associated protein (LAP) has

any effect on blood coagulation

in HeJ mice. It is therefore likely that in our experiments all these responses are under the influence of the lps gene. Of course I do not know whether all endotoxin responses are regulated by the one lps gene or several. Jirillo. How

about the butanol extracted LPS which also

contains protein. Hirata. We only used Boivin type of endotoxin. Jirillo. Which strain did you use. Hirata.

We used E. coli 0111:B4, for LPS extraction.

Yoshida. The defect in the C3H/HeJ mice is cellular since humoral factors such as the complement system are normal. To my knowledge, the coagulation system, Hageman factor and so on have not been analyzed. Dr. Agarwal mentioned the receptor systems. Such examples indicate that the whole question is very complicated and at the present time we do not have precise mechanisms of action. Agarwal. The moral of the story is that we have good hard work to do back home and come w i t h fresh data in three years from now to Toronto for a more meaningful discussion of the uniqueness or multiplicity of genes involved in LPS action.

351

B. The Relative Importance of Lipid A and other Constituents in Endotoxin Toxicity. Agarwal. At one time lipid A was said to be causal to the toxicity provoked by endotoxins. In recent years the carbohydrate moiety has been gaining ground, like the Shinkansen. So we could have some discussion on the relative importance of these two constituents of the LPS in endotoxicosis. Niwa. In so many interesting presentations today different endotoxin preparations were employed by different

investiga-

tors. So it is important to elucidate the relationship

between

chemical structure and biological activities of endotoxins. Last November, during an international symposium at Susono City, Dr. Westphal et. al. stressed that lipid A was the most important component of endotoxins. Almost all the endotoxic activities could be attributed to lipid A. However, many problems remained unanswered. For example, some synthetic analogues of lipid A were synthesized by Dr. Shiba of Osaka University which elicited adjuvant activity but, as far as I know, no pyrogenicity or limulus positivity. Some biological activity was found in the polysachharide portion such as bone resorption (Nowotny), colony stimulation and so on. Today, Dr. Suzuki presented endotoxin like activities of mannan which is very weak; poly I:C and muramyl dipeptide also have weak endotoxin like activity. Since polysachharides other than endotoxins m a y contribute to x i n poisoning

deeper understanding of endoto-

I would call upon Dr. Suzuki to tell us some

more about the biological activity of mannans. Suzuki. Our present study is limited to the biological

effect

of simple polysachharides and does not include lipid A or other lipoidal material. The neutral mannan fraction (WNM) is highly toxic in mice and induces anaphylaxis The

LDJ-Q

like syndrome.

of WNM in ddy strain of mice was 12 mg/kg

(iv). It

consists largely of a-1,6 linkage and very small side chains.

352 Synthetic mannan consisting of a-1,2 linkage shows very high LD 5 Q values. The 1025 fraction is the same as WNM but also contains phosphate, glucosamine and protein. This acidic yeast mannan has an LD^-Q of more than 200 mg/kg. The artificially phosphorylated WNM, containing 1.2% phosphate, also shows small LD^Q values. Dextran T-2000 and other dextrans from Leuconostoc mesen.teroides also show high LD^Q values. So it occurred to us that the mannopyranosyl residue is an important factor in the lethal activity; dense branches consisting of a-1,2 and a-1,3

linked residues are also

important. Non reducing terminals with phosphate groups decreased LD^Q

values.

D-mannose and N-acetyl-D-glucosamine was effective in preventing death. The toxic effect of this mannan is attributable to blood coagulation caused by hyperactivation of arachidonic acid metabolism since aspirin decreased lethality. These findings constitute examples of the biological effects of polysachharides which does not contain lipid A. Jirillo. The induction of antibody response requires polysachharides as a first factor and lipid A as the second factor. Free lipid A, intraperitoneally, is able to induce anti-Lipid A response in C3H/HeJ mice. Suzuki. In the responder C3H/HeSL strain, LD,.Q of WNM was 9.5 mg/kg whereas in the C3H/HeJ nonresponder it was 75 mg/kg. Yoshida. Is there some hydrophobic part in your mannans? Are they chemically heterogeneous? Suzuki. Yes, generally, polysachharides consist of heterogeneous species. We could not detect lipoidal material during gas chromatography or alkali treatment. Yoshida. Activity of lipid A might depend upon heterogeneity.

353 Niwa. The next problem is the importance of protein moiety of endotoxins. Nakano. We used Boivin antigen which contains lipid A but which is very potent in eliciting hydrogen peroxide. Lipid A and lipid A - BSA complexes were used in some of our experiments; the former was not effective due to solubility problems. Lipid A - BSA complexes are not very potent in eliciting nonspecific resistance in mice. In contrast, alkali detoxified LPS has very strong protective activity. We do not have enough data about the protein moiety but I think that the carbohydrate portion of LPS is very important for nonspecific protection against Salmonellosis. Agarwal. Can you get hydrogen peroxide production by either the protein or the carbohydrate preparations? Nakano. We have not checked that yet. Yoshida. I would like to mention two points. We have to take care of the physical status of lipid A for solubility. It is very difficult to control the solubility of lipid A uniformly, even in the LPS of R-forms. Some samples are easily soluble while others are not. Some say that polysachharides have lipid A activity but this could just be contamination. It is possible to assay for picogram amounts of LPS by colorimetric procedures or the limulus lysate test. Niwa. Dr. Homma of Kitasato Institute found interferon induction, and sometimes tumour regression, by the protein fraction of LPS. Jirillo. Oral administration of Salmonella renders the C3H/ HeJ mouse responsive to LPS. This may be a novel finding that could be exploited in future to solve many dogmas.

354

Suzuki. We treated WNM with 100 mM NaOH at 7° C for 3 h. Alkaline treatment of WNM resulted in the loss of gelation activity but lethal activity was retained completely. McGhee. Dr. Suzuki, what tests have you done to eliminate endotoxin contamination in your carbohydrate

preparation.

Suzuki. We used gas chromatography, alkali treatment, and limulus lysate tests. Our polysachharide was reactive to limulus lysis. Nowotny. In view of tremendous heterogeneity of endotoxin and lipid A preparations, it is absolutely essential to know how did you obtain lipid A and how did you purify it. Nakano. I just followed the method of Galanos et.al. Agarwal. Dr. Jirillo, you mentioned three regions of LPS; are they recognized by the same lps gene? Did you use carbohydrate

preparations?

Jirillo. All three regions of LPS are recognized by the same lps gene. We did not use carbohydrates but crossover sometimes clarify the specificity of the lethal

studies

response.

Niwa. I am wondering what part of endotoxin is responsible for bone resorption. Have you tested Re-glycolipid, Dr. Nowotny. Nowotny. Re-glycolipid is also active but less so than the smooth type endotoxin and this is not due to solubility problems. Niwa. Your polysachharide may contain some degraded lipid A. Nowotny. No fatty acids were present though we detected phosphorous and glucosamine.

355

Niwa. Do you think that any polysachharide may be active or just the core polysachharide. Nowotny. Polysachharide rich fraction, which is a horrible mixture of all partially degraded hydrolytic products in the supernatant after you have precipitated all lipid, is active. McGhee. Can you distinguish the toxic effects of lipid A from bone resorption. Nowotny. Yes, that is quite clear. McGhee. Have you done any other biological tests for mitogenicity and so forth for Eikenella. Nowotny. Mitogenic response due to Eikenella LPS was normal. McGhee. What about free lipid A. Nowotny. We do not stick out our neck very far for the omnipotence of free lipid A which certainly seems responsible for the toxic effects of LPS; other components from Gram negative bacteria that contaminate LPS may be responsible for the beneficial

effects.

Agarwal. How do you explain the beneficial effect of antibiotics on LPS activity. Nowotny. Very good question. Bacterial penetration

into

gingival tissue seems to be of prime importance in our model. When we apply a ligature and two injections of Cytoxan, the bacteria form an artifical pocket around the ligature. As long as they remain there nothing happens but damage is produced if they move downwards and this latter step is prevented by the antibiotic.

356

McGhee. The LD^Q of your Serratia LPS was only 100 ug for a rat of 200 g or so which m a y be a good model for LPS action. Agarwal. Have you tried Gram positive infections since a lot of native flora in the mouth is really Gram positive. Nowotny. Do you m e a n the monoinfected rat? Agarwal. Yes. Nowotny. Other authors have tried that but bone resorption is not restricted to Gram negative organisms in the germ free model.

357 C. Humoral Factors in Endotoxicosis. Agarwal. Bacterial endotoxins elicit a very large variety of mediators from different cell types all of which can find their way into the blood stream. Thus the problem of humoral factors is really very complex. Since Pr. Yoshida has taken so much trouble in the organization of this workshop perhaps he can take some more and lead the discussion in this area. Yoshida. I would like to divide this theme into two sections. First we could discuss the role of complement and second that of neurotransmitters, histamine and so on. I now ask Dr. Katayama for his viewpoint. Katayama. Although endotoxin is toxic to the host, it also has many beneficial effects. My major concern is the antitumour activity of endotoxins. In fact, LPS is the strongest antitumour agent in nature and can cause destruction of the tumour tissue without affecting normal cells. In other words, endotoxins recognize tumours and the latter recognize LPS or its derivative. Therefore, we can use endotoxins for analyzing the nature of the neoplastic cell. Since endotoxin does not accumulate in the tumour, its action is derived by mediated substances such as interferon, DNAse, interleukins etc. all of which are stimulated by LPS. Our conclusion is that the major effect is mediated by the complement. If we treat normal mouse serum, or serum from a tumour bearing mouse, with protein A coupled to Sepharose, both sera have antitumour activity. The influence of protein A is not to remove something but to produce something new. It has not been proven at this time but complement may be a candidate for this effect of protein A. Bloksma. Tumour bearing C3H/HeJ Mice injected with endotoxin do not show tumour necrosis although they have a normal complement system. These are in conflict with the general

358 role assigned to complement and its activation in LPS induced tumour necrosis. I think that a high amount of activated complement is so vasoactive that it can induce tumour damage although it may not mediate factors in tumour bearing mice t

injected with endotoxin. Yoshida. Some feel that complement is very important in endotoxicity whereas others doubt that. Bloksma. It may be an additional factor but it is not the major factor. Furukawa. I emphasize that complement plays an important role in pathogenesis of endotoxin induced DIC. In decomplemented rats treated with CVF, DIC was inhibited. Yoshida. The studies of Drs. Furukawa and Yoshikawa emphasize the significance of complement in DIC. Yoshikawa. Complement is not the major factor in the antitumour activity . Many immunopotentiators such as levan, BCG, PSK, OK432, activate the complement system. Complement is not the major mediator since it alone can not kill the tumour. The AKR mouse, defective in C5, easily die of leukemia; tumour growth is inhibited when C5 is administered into the AKR mice. So complement is necessary for anti-tumour activity of LPS. Yoshida. In bone marrow reactions, there is decrease in nucleated cells due to leukocyte migration into the circulation; red blood cells in the marrow are increased due to hemorrhage; marrow cells are cytotoxic. All these three reactions occurred in the AKR or A mice defective in complement

components.

Matsuo. In our studies, the complement system in itself is sufficient for the lethal effects of endotoxin.

359 Yoshida. Can you provide some evidence for the role of complement in endotoxin lethality. Matsuo. In our recent studies, FUT 175,which is a protease inhibitor, reduced endotoxin lethality. Administration of CVF prior to endotoxin challenge reduced LPS lethality in the rit. Yoshikawa. In our experiments, DIC was also significantly decreased in decomplemented rats. Although FUT 175 is anticomplementary, it also has other effects such as antiplasmin, antithrombotic, and so on. Thus, the protective role of FUT 175 is not necessarily due to its anti-complementary activity. If one could find an agent capable of inhibiting complement pathways, a definitive answer may become feasible but at the moment such a material does not exist. Matsuo. FUT 175 inhibits not only complement but also other proteases such as trypsin, plasmin. FUT 175 has stronger inhibitory effect on complement activation than other protease inhibitors, although it may not be specific. CVF is an inhibitor of the alternate pathway but has an effect of weakening animals; therefore CVF is not very good in studying endotoxin lethality. Hirata. I would like to emphasize that many kinds of anticomplement drugs are being used by different authors. Are they specific on the complement system? In endotoxin induced DIC, cell derived thromboplastin has an important role. So one should check the efffect of drugs on leucocytes or other cell types. Niwa. The antitumour activity of endotoxins is very impressive but can we expect this in clinical use in the near future. I am rather pessimistic about it.

360 Katayama. There is a long history of using mixed bacterial vaccines, consisting of Streptococcus and Serratia strains, which was said to have cured over 200 patients suffering from inoperative cancer. However, purified LPS is not as good as crude vaccine. Agarwal. Can anyone tell me whether the CBA animals are cmplement deficient. Dr. Nakano reported that CBA mice are 10 fold more sensitive to endotoxin, as compared to normal mice, and if they are defective in complement the latter may not be important in endotoxin lethality. Yoshida. The interaction of complement and endotoxin is very confusing in vivo. The interaction between these two materials in

vitro is very important to analyze the molecular

mechanisms of endotoxin action. Agarwal. Would anyone like to comment upon the endotoxin detoxifying component which was very fashionable at one time/ and the role of complement in that system. Yoshikawa. Complement levels decreased after either LPS or the cobra venom factor (CVF); we can protect against LPS induced DIC by CVF. Have you tried to administer CVF before endotoxin injection. Matsuo. We did not use CVF; there are conflicting reports as to the protective role of CVF in endotoxin lethality. Yoshikawa. Do the catalases or scavangers of free radical protect mice against endotoxin toxicity. Sakaguchi. 50 mg/kg trypan blue had a very good effect on the relationship between free radical formation and the scavanger system.

361

Yoshikawa. AKR mouse is complement deficient. Did you try this strain. Matsuo. No. Niwa. Is there any difference in thrombocytopenia after LPS in the responder versus the nonresponder mouse. Natsuo. We do'nt know. McGhee. Would you speculate as to how the macrophages become activated in CBA mice. Nakano• Some macrophages are directly activated by the LPS. In some cases low serum antibody titer may affect phagocytic ability and intracellular killing but we think that direct activation of macrophages is more important for protection against Salmonella infection. Niwa. Besides complement, blood coagulation factor is very important in LPS lethality. Matsuo. Ulevitch and Cochran have reported that C3H/HeN mice exhibit low complement titer after LPS but DIC, thrombocytopenia, or thrombosis was not observed in C3H/HeJ animals. McGhee. Is it possible to induce resistance in the CBA animal and can that be transferred by either cells or polyclonal antibodies into normal recipients. Nakano. We did not assay for antibody and did not attempt passive cell transfer. O'Brien has already showed that these animals have low antibody levels.

362 Movat. The studies described by Dr. Yoshida bring up an important point with respect to the procoagulant activity in the blood. As you know, in man it has been established that monocytes, other than polymorphonuclear

leucocytes,

are responsible for the procoagulant activity and this has been confirmed in rabbits. Semeraro, not long ago, showed that unlike the rabbit, in rats no procoagulant activity could be elicited with endotoxin. Can you comment upon the differences between rodents. Yoshida. We have no experience with other experimental animals; I think that in human DIC the cells may be different from those in laboratory animals. Movat. Did you perform two stage clotting assays to see

if

your procoagulant activity is a tissue factor like material. Hirata. We only performed one stage assays and showed that procoagulant activity is tissue thromboplastin. McGhee. You mentioned that your antibody may be acting in possibly two different ways. Since both IgG and IgM work, it does not appear to be a complement mediated antibody effect. Yoshida.We have no experience concerning the complement. Both antibodies are however important. McGhee. Have you ever looked at immunocomplexes of endotoxin and the anti-Re antibody injected into the animals for their effect on the bone marrow. Yoshida. No. Agarwal. Have you tried protease inhibitors like leupeptin

363 and e-aminocaproic acid etc... to see if they would have some protective effect. Hirata. We have used one protease inhibitor, trans AMCHA, which has antifibrinolytic activity. It also inhibited the bone marrow reaction, especially the bone marrow reaction and decrease in nucleated cell counts in the marrow. The inhibition was however partial, not complete. Agarwal. Dr. Movat, you mentioned platelet accumulation. Do they have an eventual role in Shwartzman. Movat. The overall reaction is very complex. Although the monocytes, macrophages, etc... produce the procoagulant activity, the hemorrhage seems to me to be primarily polymorphonuclear or neutrophil mediated. Yoshida. Next we should like to discuss the neurotransmitter. I would like to have the comments of Dr. Bloksma on the presentation of Dr. Kuratsuka. Bloksma. I found his data very interesting and they fit very well with my hypothesis. The toxicity of adrenaline seems to be mediated by the a-adrenergic receptor. Endotoxins are capable of S-adrenergic blockade and facilitate the action of a-adrenergic receptor.

Concomitant administration of

B-adrenergic receptor blockers together with adrenaline increases the toxicity of the latter. So, it may be that endot oxin increases the toxicity of adrenaline by its 3-blocking action thereby facilitating the a-adrenergic effect which is related to the toxicity of adrenaline.

364

Kuratsuka. I wish to ask Dr. Bloksma about the histamine receptor. More than 10 years ago I tested the histamine sensitizing activity of antitumour drugs isolated from Japanese fungi. The product, called Lentinan, is now commercially available and has potent histamine sensitizing in mice. Can you comment upon the histamine

action

sensitizing

activity, or histamine receptor, in the antitumour

activity

of drugs or of endotoxin. Bloksma. Very difficult question. We found that the H1 receptor blocking agents, given prior to endotoxin,

diminish

the antitumour action slightly. Administration of H2 receptor blocking agents somewhat potentiates the antitumour

effect

of endotoxin. In m y opinion, histamine itself has weak antitumour activity against certain sarcomas. I think that the effects mediated by the H1 receptor are mainly vasoactive effects whereas those mediated by the H2 receptor are the activation of H2 suppressor cells. The slight increase in the antitumour activity of endotoxin, when it is combined w i t h H2 receptor blockers, is due to enhanced H1 receptor mediated by the vasoactive component of endotoxin

activity

induced

tumour necrosis along with the blockade of activation of T-suppressor cells which are switched on by H2 agonists. I believe that the administration of histamine has a facilitating effect and an inhibitory effect because it switches on H2 receptor positive T-suppressor

cells.

Hirata. I am very encouraged by Dr. Bloksma's data because not many reports emphasize the role of histamine in endotoxicosis. About 10 years ago we reported that, in the bone marrow reaction, the cytotoxicity, decreased nuclear

cells,

and marrow hemorrhage, could all be inhibited w i t h antihistamines and an H1 antagonist - diphenhydramine.

Histamine

alone could also lower nucleated cells and provoke

hemorr-

hage. The activity of histamine alone does not persist for a long time though it was very similar to endotoxin activity.

365 Therefore, I think that not only the histamine

released

after endotoxin, but also nascent histamine, or intrinsic histamine formed from histidine

by the action of histidine

decarboxylase, is also involved in endotoxin action. What do you think about the role of intrinsic

histamine.

Bloksma. As far as I know, endotoxin stimulates the decarboxylase so the endogenous histamine is very important. May be the release of histamine from mast cells could add to the antitumour effect of endotoxin. I think that the capacity of endotoxin to potentiate histamine action, together with the increase in histamine production, are all involved in the antitumour action of LPS. Movat. Dr. Nowotny do you think that endotoxin acts directly on osteoclasts or via a host derived mediator. Nowotny. Osteoclast activating factor is released from the macrophage after the latter interact with endotoxin. We do not think that this is the exclusive mechanism.

In recent

experiments, chick bone osteoclasts, which are only 90% pure, exposed either directly to LPS or indirectly through an intermediate membrane, do a beautiful job of absorption of calcium labelled bones. Bloksma. Do you have any evidence for Interleukin 1 in bone resorption. Nowotny. Not yet. Agarwal. Have you tried materials that modify host response to LPS to assess their influence on bone

resorption.

Nowotny. We have only tried indomethacin which had no effect on bone resorption in vitro.

366

D. The Reticuloendothelial System and Endotoxin Poisoning. Agarwal. The history of the involvement of the RES during endotoxicosis dates back to at least 1947. Contrary to the initial premiss, it now appears that enhanced RE function may actually be harmful to the evolution of endotoxin poisoning. Dr. Bloksma can perhaps help us decide as to the true contribution of the RES in host response to endotoxins. Bloksma. It is indeed a dilemma that RE-activation as well as RE-inhibition increase susceptibility to bacterial I wanted to ask Dr. Sakaguchi whether RE-activation

endotoxins. could

influence HDL production since RE-inhibition by trypan blue and lead acetate sensitizes to LPS and decreases HDL levels. Sakaguchi. This relationship is not yet very clear. I think that apo serum amylase protein increase may be responsible for the detoxification of endotoxin. Shimada. A good crrelation appears to exist between DIC and RES. CH50 levels correlate well with K values and platelet counts. High complement levels m a y be necessary for the removal of endotoxin from the circulation. Bloksma. Is the removal of endotoxin from the blood, as a result of elevated K values, enough to ensure against LPS m e d i a ted damage. Agarwal. I think that release of mediators from RE cells has to be considered. Glucocorticoids may be inhibiting this latter parameter and thereby protecting against LPS mediated damage. Yoshikawa. The relationship between complement, DIC and endotoxin shock is not clear.

367 Jirillo. The role of complement in clinical situations is especially difficult to determine. Matsuo. Does endotoxin poisoning involve an actual decease in the synthesis of complement or an accelerated

consumption.

Shimada. I think that low CH50 levels are an expression both of increased utilization and diminished production. Matsuo. Within 30 m i n of endotoxin administration,

complement

and C3 concentrations dropped greatly but recovered to normal within 1 h with an increase above control by 24-48 h. Thus, small doses of LPS m a y increase, and larger doses decrease, the levels of complement in vivo. Bloksma. Do different mice strains vary in this regard. Nakano. The three strains of mice have phenotypic

similarities

but different underlying mechanisms. Bloksma. What are these mechanisms. Nakano. I do not know but some of the differences are due to different experimental

systems.

Bloksma. Is there any correlation between the resistance of C3H/HeJ macrophages to LPS, their susceptibility to Salmonella infection, and their capacity to produce f^C^. Nakano. f^C^ production, alone, can not explain all this. Bloksma. Since prior endotoxin administration

stimulates

production, can other macrophage activating agents do the same.

368

Nakano. C3H/HeJ mice could respond to muramyl dipeptide but CBA/N mice and beige mice could not. McGhee. What might be the relationship between the lack of macrophage t^C^ production and the ability of the animal to handle an intracellular

infection with Salmonella.

Kato. The injection of LPS into immunologically

defective

mice improved bacterial clearance and antibacterial

activity.

Nakano. CBA mice are really susceptible to Salmonella tion but t^C^ production alone can not explain

infec-

susceptibility.

McGhee. Would you speculate that the macrophage defect is more important in the susceptibility of these mice to infection. Nakano. I think that there is partial

relationship.

Movat. Do you know if the ability to produce t^C^ in the resistant mouse can be restored by BCG, lymphokines, or interferon. Nakano. We have never checked that so far but we would like to try it. Yoshida. Did you examine the activity of macrophages

in

vitro after streptozotocin, Dr. Agarwal. Agarwal. No, we only studied the carbon clearance, the spleen and the liver weights. Yoshida. Is it possible that Streptozotocin may change the character of macrophages such as BCG does.

369 Agarwal. I do'nt know. However, to my knowledge BCG does not totally reverse the resistance to LPS lethality in the C3H/HeJ strain as was possible by a combination of zymosan and streptozotocin.

I am wondering whether Dr. Movat used

soluble or particulate zymosan for his studies on the Shwartzman reaction. Movat. Particulate zymosan was used in our studies. Agarwal. Do you know if soluble zymosan is effective. Movat. I do'nt know. Katayama. Zymosan was claimed to have no activity to provoke Shwartzman but you showed the contrary when zymosan was given intravenously and the route is important. Although zymosan has complement activating property, even very large amounts, given intraperitoneally, had no effect on mouse tumour. Thus, either it should be injected

intravenously

or the activated serum should be diluted for neal

intraperito-

injection.

Movat. Thank you. Zymosan activated plasma was

injected,

or infused, over a 30 m i n period to a total of 30 ml of zymosan activated serum

per rabbit.

McGhee. Do'nt you think 30 ml/kg was a large dose. Movat. Not over a 30 m i n period. Animals were normal. Agarwal.Did you try leupeptins in your Movat. No we did not.

system.

370 Yoshida. The number of Kupffer cells is very flexible and this should be kept in m i n d when we talk about RE functional modifications. Agarwal. I am afraid that time is running out and I pass on the floor to Pr. Yoshida. Yoshida. I would like to thank everyone for participating and I declare this workshop closed.

AUTHOR INDEX Abe, H. Abe, T.

57

Kovats, E.

261

315

77

Agarwal, M. K.

299

Kudoh, K. Kuratsuka, K.

Andreana, A.

11

Lazar, G.

1

Bloksma, N. Burrowes, C. E.

133 197

Matsuo, S.

299 235

Fujita, Y.

151

Fumarola, D. Furukawa, Y.

281

93

93

McGhee, J. R. Michalek, S. M.

213 221

Mikami, T. Mizukuro, T.

245

Gaeta, G. B.

11

Mori, W.

331

Hanasawa, K.

169

197

Hayasaka, H.

235

Movat, H. Z. Murakami, M.

Hayashi, H.

151

Hirata, M. Hofhuis, F. M. A.

77 133

Nagase, T. Nakagawara, G.

Homma, R. Horisawa, M.

281

Nakane, Y.

177

Nakano, M.

Husztik, E.

1

Inada, K.

77

Nowotny, A.

Irie, H.

331

Nowotny, A. M.

261

Jirillo, E. Kanou, M.

93

Ogasawara, M.

77

151

Ogino, K.

151

177 21

Oka, T.

169 115

Katayama, Y. Rato, H. Kikuchi, K. Rito, F.

93 177

213 221 245 315 169 21 115 261

235

Onozuka, K. Oshima, M.

315

Patierno, S. R.

39

151

Kiyono, H. Kobayashi, M.

93

Peavy, D. L.

39

315

261

Kodama, M.

151

Pham, P. H. Pinter, A.

169

Ribarszki, S.

1

177

Rothmann, J. Ruggiero, G.

261

Kondo, M.

213 221

1

11

372 Saito-Taki, T.

21

Totsuka, M.

235

115

Tsuchiya, S.

315

Sakaguchi, 0.

57

Utili, R.

11

Sakaguchi, S.

57

Willers, J. M. N.

133

Sallay, K.

261

Yamaguchi, N.

151

Sanavi, F.

261

Shimada, H.

315

Yaoita, H.

21

Siegler, D.

261

Yoshida, M.

77

Suzuki, M.

245

Yoshida, S.

177

Suzuki, S.

245

Yoshikawa, T.

Tani, T.

169

177

213 221

Subject

Index

Acute fatty

331

liver

Acute inflammatory

response

261

Adrenaline

133, 281

Allergy

77

Alternate

pathway

235

Antibiotics

261

Ant ibody

77

Ant i genie

77

Anti-Re

77

serum

Anti thrombotic

agents

Antitumor activity of LPS Ascitic

221

1 77 133

tumors

Aspirin

221

Bacteremia

261

Bacterial invasion

261

microvesicles

261

products

261

sonicates

261

Batroxobin Bone marrow

221

reactions

Bone resorbing

agents

77 261

Bone resorption in vitro models

261

in vivo models

261

C3H/HeJ mouse

133, 235, 299

C3H/HeN mouse

133, 299

Cancer patients

151

Carrageenan

1

CBA/N mouse

115

CH50

235

Chloramphenicol

331

374 Cholangitis

315

Circadian rhythm

781

Complement

177, 197, 213, 315

Concavalin A

133

Corticosteroids

39, 299, 311

Cytolytic factors

133

Cytostatic factors

133

Cytotoxic factors

133

Defibrinogenation

221

Dextran

245

DIC

77, 213, 221, 315

Dipyridamole

221

Direct hemoperfusion

169

Endotoxemia

315, 331

Endotoxin

1, 11, 39, 57, 93, 133, 1 51 , 1 69 , 1 77 , 1 97 , 21 3 , 235 , 261 , 281 , 299 , 31 5, 331

Fibrin deposition

133

Fibrosarcoma

133

Gadolinium chloride

1

Germ free rats

261

Glucan

304

Glucocorticoid receptor

311

Glucocorticoids

133, 311

Grossed immunoelectrophoresis

235

H

2°2

2 1

Hemmorhage

77

HDL

57

375 Immune

suppression

by Cytoxan

261

by radiation

261

Immunodefective mice

21

Immunomodulator

133,

Immunotherapy

1 69

Inhibition of LPS toxicity

39

Interferon

133

induction

Kupffer cells

11,

Lead

57

acetate

Lethal

hyperreaction

Leukocyte

77 261

Lipid

lysate

peroxide

Lipopolysachharide

245 57 39, 177,

Liver plasma membrane

11

Macrophage

21 ,

Mannan

245

Metallothionein

39

Methylprednisolone

221

Neutralization

77

Non-specific

115

resistance

Noradrenaline

281

Opsonin

315

Periodontal

disease

261

Phagocytos is

11

Plasmacytoma

1 33

Plasma

315

fibronectin

235

281

Ligature model Limulus amoebocyte

169

93 ,

1 1 5,

235, 39

245,

1 33 , 311

1 51 ,

376 Poly I:C

133

Pregnancy

331

Procoagulant

77, 133

Pyrogenicity

245

Reticuloendothelial Salmonella

system

infection

1 15

Salmonella minnesota Ra

(R60)

93

Rb

(R345)

93

Re

(R595)

93

Salmonella

typhimurium

Shwartzman

reaction

Sodium Spinal

93 133, 177, 197, 331

deoxycholate

Sodium polyethanol

1, 57, 310, 331

245 sulphonate

transectomy

1

281

Streptozotocin

299

Ticlopidine

221

Tissue distribution of LPS

1

Trypan blue

57

Tumor necrosis

133

Vx2 tumor

1 69

Vasoamines

133

Vitamine E

221

Warfarin

133

X-linked

immunodeficiency

115

Z inc

39

Zymosan

133

w DE

G M. K. Agarwai (Editor)

Walter de Gruyter Berlin-New York Hormone Antagonists i 7 c m x 24 C m. IX, 734 pages. Numerous illustrations. Hardcover. DM 180,-; approx. US$82.00 ISBN 3110086131

1982

The volume deals with the subject of hormone antagonism both in basic research and in clinical medicine. It groups together antagonists for those hormones where antagonism has been documented specifically and with a certain degree of certitude. It is felt that the book represents a major new reference source and involved research workers will find the volume of much interest since it provides data hitherto unpublished.

M. K. Agarwai (Editor)

Principles of Recepterology 1983.17 cm x 24 cm. VII, 677 pages. Numerous illustrations. Hardcover DM 220,-; approx. US$100.00 ISBN 311 0095580 The past two decades have seen a literal explosion in the field of hormone receptors. The present volume was planned as a first attempt to synthesize this exciting and important field. A single authority in each hormone class was asked to digest all information in his/her area and thereafter attempt an overview both for the specialist and clinician. This book may be taken as a reference source for most of the important concepts and methodologies.

E. M. Spencer (Editor)

Insulin-Like Growth Factors/ Somatomedins Basic Chemistry • Biology • Chemical Importance Proceedings of a Symposium on Insulin-Like Growth Factors/ Somatomedins, Nairobi, Kenya, November 13-15,1982 1983. 17 cm x 24 cm. XIII, 664 pages. Numerous illustrations. Hardcover. DM 240,-; approx. US$109.25 ISBN 311 0095629 This book presents an up-to-date, lucid comprehensive review of all aspects of insulin-like growth factors (somatomedins) - a family of polypeptide hormones that regulate cell growth - and their current status. These hormones mediate the growth-promoting action of growth hormone and are genetically related to insulin. Each section contains a review of our present understanding followed by the most recent research and hypotheses. The volume is compiled in such a way that it can serve as a text for insulin-like growth factors as well as presenting all the latest research information.

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Edited by Thomas Scott and Mary Brewer 2nd printing with corrections. 1983.14 cm x 21,5 cm. VI, 519 pp. Approx. 650 illustrations. Hardcover. DM 59,-; US $29.90 ISBN 3110078600

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The Concise Encyclopedia of Biochemistry, with more than 4,200 entries, is the foremost collection of current information in this rapidly expanding field. The contents are complemented by numerous structural formulas, metabolic pathways, figures and tables. All those interested in or working in the field of Biochemistry and Biology (Life Sciences), will profit from the information contained in this encyclopedia. This truly remarkable book is an essential reference for Biochemists, Clinical Chemists, Clinical Biochemists, Clinicians, Medical Researchers and Experimental Biologists. It will also serve as a very useful source of information for students.

W G DE

WALTER DE GRUYTER • BERLIN • NEW YORK Vèrlag Walter de Gruyter & Co., Genthiner Str. 13, D-1000 Berlin 30, Tel.: (030),260050, Telex 184027 Walter de Gruyter, Inc., 200Saw Mill River Road, Hawthorne, N.Y. 10532, Tel.: (914) 747-0110, Telex 64 6677