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Title of Thesis: "Early Phase Endotoxin Tolerance: A Study of the Cellular Mechanisms Which Underlie a State of Acquired Refractoriness to .Endotoxin-induced Toxic Manifestations."

Name of Candidate: CPT Gary S. Madonna Doctor of Philosophy Degree December 13, 1985

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~'3 s . orz~---CPT Gary S. Madonna, MSC, USA Department of Microbiology Uniformed Services University

of the Health Sciences

i i

ABSTRACT

Title of Dissertation: Earl~ Phase Endotoxin Tolerance: A Study

of the Cellular Mechanisms which Underlie a State of Acquired

Refractoriness to Endotoxin-Induced Toxic Manifestations

Gary Steven Madonna, M.S.; Candidate, Doctor of Philosophy,

Disserta tion directed by: Stefanie N. Vogel , Ph.D., Associate

Professor, Department of Microbiology

Endotoxin, the lipopolysaccharide (LPS) cell wall

component which is derived from Gram negati ve bacte ria, has

been shown to induce a number of to xic manifestati ons which

mimic those seen during syst~mic Gram negative infection.

Hyporesponsiveness to the toxic effects of LPS can be induced

for a transient period by pre-exposure of a normally responsive

individual to a s ublethal dose of LPS. This acquired state of

refractoriness to the toxic effects of LPS has been defi ned as

'' r'1at·l y !7-• ndotox:i.n tol~:-~t·· ancE~··. L . .i.tt.l() i s knt;,t.o..~n about th (::>

cellular mechanisms that underlie thi s phenomenon. In th is

study, an early tolerance system was establi s hed by th e

injection of mice with 25 · ~9 of ~~~b§Li~hiQ ~Qli K235 LPS.

Ma xi mal hypores ponsi veness in res ponse to a challenge injection

was observed 3 to 4 days after the initial injection, a nd

iii

no~mal responsiveness ~eturned by 8 days after the initial

exposure to LPS. Fu~ther experiments demonst rate that the

acquisition and maintenance of the tolerant sta te coincides

temporally with an increase in the numbe r of macrophage

progenitor cells in the bone marrow. Cell sizing profiles of

the bone marrow cells from tolerized mice indicate that there

i s an enrichment of a subpopulation of cells that are

siqnificantlv l~rcer thRr1 ·t:i"IP rP ·L ·l~ 1· 1·1 l,t-li"Ja lll~tt• t·,·)w ~t· ,~o ~,t-~L·t··L·n l·, ~ _ l , • C .::'1 _ ,. ... •. - · . . •. _., ... _ ~;.:_. c_ •• 1... . .. ,I• c_ «.. •• _ •. • .....

from control mice. By den s ity gradient sedimentation, it was

shown that the denser population of cells from toleri zed mice

contained the increased numbers of p~ogen itor cells, which, by

cytology, were immature monocytic cell types. The increased

numbers of macrophage progenitors were sustained afte~ a second

(challenge) injection of LPS , but returned to normal levels

after multiple tolerizing inj ect ions. These res ults indicate

t hat early endotoxin tolerance is associated with an increase

in a population of bone marrow cells that is enriched for

macrophage progenitors and suggests the possibility that the

lack of res ponsiveness observed during the hypo~esponsive

period is related to a failu~e of these immature cell types to

t"" Fc>~5pond to LP~3.

Early endotoxin tolerance was i nduci bl e in inbred mice

~ · ~ · ··~.·.•t "" P_- !.·_,f_~ 1-1 F .. !ticallv athvmic (T cell-deficient) and in inbred ~·llilCil w ., l I

mi ce which weY e deficient in a subpopulation of LPS- r esponsive

13 ct:!lls. In addition, splenectomized mice were capable of

being ~end~red fully tole~ant by th e p~ o tocol established for

nonnal mic<::•. These findings suggest that T cells, Lyb 5

positive B cells and spleen cells do not contribute

significantly to the induction of early . endotoxin tolerance.

The endotoxin tolerance protocol developed in this

study was highly protective against challenge with heterologous

preparations of LPS. However, mice which were rendered

tolerant to ~- ~Qli LPS exhibited a delayed mean time to death

following infection with fSQYdQIDQQ~~ DQtY9i095~- However, the

tolerance regimen used in this study did not alter the P5-

9~~.r.qsirE.:.!~::!•.) LDso .

Lastly, early endotoxin tolerance could be induced with

a non-toxic derivative of Lipid A, monophosphoryl Lipid A

(MPL) . Associated with MPL - induced tolerance wer e all of the

cellular and protective man ifestations observed using intact

LPS. These findings under score th e potential of non-toxic LPS

derivatives to mitigate the toxic effects of endotoxin.

v

EARLY PHASE ENDOTOXIN TOLERANCE:

A STUDY OF THE CELLULAR MECHANISMS WHICH UNDERLIE A STATE OF

ACQUIRED REFRACTORINESS TO ENDOTOXIN- INDUCED

TOXIC MANIFESTATIONS

by

Gary Steven Madonna

Dissertation submitted to the

Faculty of the Department of Microbiology Graduate Program of

the Uniformed Services University of the Health Sciences

in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy 1986

V1

Dt::D I CAT I 01,1

To God be the Glory!

Great thi n gs He has don e :

In rny family _ _ _

In rny 1 .if(·::~ _ - .

v~~

ACKNOWLEDGEMENTS

I l.Jou.l.d like? to ·t.'-1ank, o·· .-.tc:.f · · 1. e 11 v J(' · 1 ·r: Jt· - 11 I 1 • .:> . •. <~trl. - ' • (. ·"f~'. (. <:1. .

has done fo~ me over the last 3 years. She has helped me

advance as a scientist. Her ~ esourcefulness a nd knowledge has

broadened my awareness in the field of i mmunology_ Her

leadership, help, and understanding at crltlcal times will

always be remembered. Thank you for help ing me attain this

great goal and captu~e a new one.

Thank you, United States Army, Medical Service Corps

a nd all thE! ''t .. c·:;~al sold.i..c~)t- ~3'' fot .. allowing me~ to: ''13 e , <:t.l.l that J

can be, in the At· my" .

Thanks to the faculty and my advisory committee for the

great courses, fo~ all your knowledge imparted, and to the

graduate students, all great people.

To our special group in the Vogel lab, I couldn't have

don0 it without you.

Great appreciation g6es to: Dr. Marion Fult z, for all

the help using the FACS II; Eric Kaufman, for his technical

help and friendship; Dr. Cathy Barr, for helping me to set up

the columns for polysaccharide separation (over a nd over);

Nancy Coles, for assistance in learning the mouse burn model;

Robbie Matthai (Litton Bionetics), who taught me to

.J t . . · · r ·:J '- ··· 'I c··, ·t !·1 c::> fTI 11· " n • <'.i 1··1 c:l ·t· .. f.) ·t·.!··, r.·.·~ ~::::;E .. ~ C.: t" F.-~ ·t. <".'i t·· J·. lJ ~-'·.; , SP ... I') nec · .. o/TllZ~J flllCC·:) d 11. rid ': .. ·:.. . .v•·:.:, ..

for the ir encouragement and support ..

Thanks Mom (and Dad) for "tT<'Li.n:i.ng me:~ up in the-':' Wd'/ I

should go ''- Thanks Mom Henry, for your many prayers, special

verses to remember and that special talk that showed me the

Lastly, and most dearly, thanks to my family. Elisa,

for your smiles, pretty pictures and understanding. Todd,

thanks for admiring me; thanks for being my friend. Sharon,

see that, you didn't hold me back after all. We did it

together. The best is yet to be.

~X

TABLE OF CONTENTS

$EGIIQ~_III~~~-ANQ_~AlN_$U~III~~$

INTRODUCTION AND HISTORY

MATERIALS AND METHODS

Mice

Reagents

Measurements of endotoxin sensitivity

Determination of numbers of macrophage progenitor

cells in the bone marrow and spleen

Infection of control and endotoxin-tolerized

animals with a Gram negative organism

Preparation and analysis of fluorescent

antibody-stained cells

RESULTS

Establishment of an early endotoxin tolerance

system

Early endotoxin tolerance is associated with

alterations in bone marrow-derived

macrophage precursor pools

Induction of early endotoxin tolerance ln

other murine models

Infection of control and LPS-tolerized ICR mice

with a Gram negative organism

X

PAG~

1

13

13

14

16

20

23

24

26

26

40

76

95

Induction of early phase e ndotoxin tolerance by

a detoxified Lipid A derivative,

monophosphorvl Lipid A (MPL)

DISCUSSION

BIBLIOGRAPHY

101

118

136

LIST OF TABLES

IA~~E-~U~~~B_A~Q_IJikE

I. Effect of early phase endotoxin tolerance

on clinical endotoxic manifestations

II. Effect of initial exposure to ~-CQli LPS

on CSF induced by s ubsequent challenge with

homologous and heterologous LPS preparations

III. Effect of purified polysaccharide derived f~om

E- CQli LPS on induction of CSF

or early endotoxin tolerance

IV. Effect of LPS adm i nistr~tion on recovery of

38

39

41

thioglycollate-induced peritoneal exudate cells 43

v_ Differential counts of c ytospi n preparations o f

density gradient- separated Peak 2 cell s

VI . Percent suppression of control se r u m CSF response

in mice tolerized with homologous or heterologous

preparations of LPS

59

72

VII. LPS-induced se rum CSF response in LPS-responsi ve

(C3H/ OuJ) and LPS - hypores ponsive (C3H/HeJ) mice

VI I I . FACS a nalysi s of Thy 1.2+ a nd IgM+ cells obtained

from BALB/c euthvmic (ny/+) or

BALB/c athymic (ay/ny) mice 8 5

IX. LPS-induced alterations in bone marrow cell s1z1ng

profiles and M-CFU numb e r s in C3H/HeN and

C3.CBA/N (xid) mi ce 91

X. Infection of control a nd tole ri zed ICR mi ce

99

XI. Induction of serum CSF in I CR mi ce

injected i ntr a venously or

intraperitoneally with MPL 105

XII. Effect of MPL exposur e o n LPS-i nduced LD50

an d symptoms of e ndo toxic ity 111

xiii

LI!3T OF FIGURE !:"l

1. Ti me cour s e of the induction of early endotoxin

tolerance as assessed by LPS-induced CSF

2 . De termination of the optimal LPS concentration

for induction of early endotoxin tolerance

3. 24-hour time course for serum CSF production

in non-tolerized and tolerized mice

4. 24-hour time course for interferon induction

in non - tolerized and toleri z ed mice

5. Effect of a tolerizing injection of LPS on

the number of nucleated cells and macrophage

precursors in th e bone marrow

6 . Effect of a toleri z ing injection of LPS on

bone marrow c ell si z ing profiles

7 . Cell s i z ing profiles of bone marrow cells

obtained at various times after LPS injection

x~v

:so

4')

,'51

8. Density gradient separation and measureme nt '

of M-CFU and HPP activity 54

9. Cell s1z1ng profiles of pooled den s ity gradient-

separated bone marrow cells

10. Effect of a second injection of E- ~gJj LPS

on the numbe r of bone marrow-derive d M-CFU 60

llA . Effec t of repeated inj ecti ons of E- ~gli LPS

on the level of serum CSF following LPS challenge 63

llB. Effect of repeated injections of E- ~gli LPS

on the number of bone marrow-derived M-CFU

llC. Effect of repeate d injections of E- ~Qli LPS

on bone marrow cell sizing profiles

12A. Effect of r e peated injections of f§QYdQIDQQQ~

'/'0

128. Effec t of repe ated i nj ec tion s o f ESQYdQIDQQ~~

l 1 · · r· · 1 ·.·;· ..... ,. 0QC~9iQQ§~ LPS on bone marrow ce .. s1z1 ng pro ·-l .. es -

XV

13. Effect of the tolerizing regimen on the bone marrow

cell sizing profile of inbred LPS-responsive and

LPS-hyporesponsive mice 79

14. Effect of the tolerizing reg i men on LPS-induced

serum CSF response in BALB/c euthymic (oy/+) and

BALB/c athymic (nu/nu) mic e

15. Effect of the tolerizing regimen on LPS-induced

serum CSF response in

C3H/HeN and C3. CBA/N (xid) mice 89

16A. Effect of the tolerizing regimen on LPS- induced

serum CSF in control and splenectomized mice 93

168. Bone marrow cell sizing profiles from

splenectomized and sham-splenectomized

ICR mice f ol lowing LPS admin is trat ion 96

17A. MPL-induction of serum CSF 103

17B. MPL-induction of early phase tolerance to

106

XV~ ,

1 0 u. Interferon induction by MPL and

effect of MPL-treatment on

LPS- induced interferon production io YiYQ 109

19. Effect of a toleri z ing injection of MPL

on the number of macrophage precursors

i n the bone marrow 11 3

20. Effect of tolerizi ng doses of MPL on

bone marrow ce ll sizi ng profiles 116

xvii

INTEQ~UQTION AND HISTORY - ---------------

A common feature of all Gram negative bacteria is the

presence of lipopolysaccharide (LPS) on the bacterial cell

surface. Located exclusively in the outer membrane, this

glycolipid is one of the major components through which Gram

n e gative bacteria interact with their environment and, as such,

represents a major cell surface bacterial antigen. Gram

ne gative lipopolysaccharides shar e a basic common structure: 8

polysaccharide moiety bound covalently to a gl y colipid

( We stphal and Luderitz, 1954 ) . The polysacch a ride componenL

consists of an outermost oligosaccharide polymer or "0-spe c ifi c

chain" wh i ch varies among strains of a giv e n g enus and is

responsible for serotype specificity. This is covalently

coupled to an inner "core region" which is composed of a

branched oligosaccharide. The composition of th e core ~s

common among strains of a giv e n g e nus, but may vnry within

different families of bacteria. The core oligosac c haride is

a ttach e d covalently at a single attachment site ( Gmein e r ~i g .l .

1971; Sidorczyk ~.! ~1· 1983; Rosner ~1 ~1· 1979 ) to an unusual

lipid, Lipid A. In the §~!~~Q~~~i~~i~~~~~. Lipid A consists of

a phosphorylated glucosamine disaccharide substitut e d wi th

ester- and amide - linked long cha i n fatty acids and r e pr e sents

t he le as t variable region of the LPS molecule.

The synthesis and e xpr e ssion of lipopolysac c h a rid e on

th e c ell surface of Gram negative bacteria has be e n shown to

1

2

Play a major role in the pathogenesis of specific enteric

d1 s eases. The ability of shigellae to penetrate the epithelial

cells of the gut, cons idered to be the essential step in the

pathogenesis of shigellosis, is affected by a number of

bacterial surface attributes among which are the chemical

composition of the surface lipopolysaccharide 0-repeat pplym~r

(Gemski gt §!., 1972) and the expression of smooth LPS (Gemski

and Formal, 1975). LPS expression in ~big~ll~ ~QOO~i was shown

by Madonna and Allen (1981) to determine the bacterial

susceptibility to direct serum bacteriolysis and the

opsonification requirements necessary for polymorphonuclea r \

I

leukocyte-mediated microbicidal action. -The loss of sugar

components from the a-antigen was also shown to be accompanied

by an increased sensitivity to the bactericidal action of

antibody and complement in ~?lffiQQ§ll§. (Nelson and Roantree,

1967). Friedberg and Shilo (1970) tested wild- type ~~lillQOQll~

t~ebimY~iYm and cell wall mutants ·wi th deficiencies in their

cell wall lipopolysaccharide for sensiti vi ty to the

bactericidal action of the lysosomal fraction of

polymorphonuclear leukocytes. These investigators found that a

complete lipopolysaccha~ide basal core was essential for

res istance to lysosomal bactericidal act1on, whereas a-specific

side chains of the wild type did not enhance resistance.

Lastly, Medeari s ~1 ~1 - (1968) showed that the presence of

lipopolysaccharide side chains in E ~gli enabled thes e

organisms to ~esist phagocytosis.

Endotoxin has also been implicated in the pathogenicity

of the Gram negative diplococcus, N~!~~~r!9 m~~!~git!9i~· As

in Gram negative rods, the lipopolysaccharide of the

meningococcus resides primarily in the outer membrane

(Zollinger s;! .91·, 1972). Devoe and Gilchrist (1973) showed

that during log-phase growth i~ YitrQ. this organism

over-synthesizes its outer membrane which results in the

release of bleblike structures from the surface of the

organism. The continuous release of these structures and,

hence, endotoxin may play an important rol e in the severe

endotoxic reaction and disease state which ensue. It has <tlso

been found that most meningococcal strains express a "rough"

LPS, (i.e., no 0-antigen; Jennings ~t 91·, 1980), although they

appear to have the same core and Lipid A components as the

It has been

proposed that because meningococcal LPS lacks the high

molecular weight a-antigen, it is more likely to bind with

p latel et s and leucocytes which express LPS-binding substances

than to remain in body fluids (Springer and Adye, 1975 ) .

Although the mechanisms responsible for the production of

p at hologic changes in meningococcal infection have not been

entirely explained, meningococ ca l endotoxln appears to be

responsible for tissue damage, h ypote nsion, vascular collapse

a nd disseminated intravascular coag ul ation ( DIC) s e en in

fulminant meningococcemia (Beatty , 1983).

Experimentally, investigators h ave shown that man y of

the pathophysiologic chan ges associated with Gram negative

sepsis or ca u sed b y injection of liv e or dead Gram negative

3

4

bactet·.ia into expc-?t· imc7:!n ·t.<:tl ·:~·rJl·rn ··l - c· 1 1 1· · t :1 b" •- a ~"', ; an a . ~::: .o :H:? e 1 c 1. ··.e(· ,

injection of purified LPS. Among these are fever

(pyrogenicity), hypotension, hypoglycemia, changes in white

blood cell counts, disseminated intravascular coagulation, and,

if administered in high doses, irreversible (septic) shock and

death (Berry, 1977; Galanos ~1 ~!- 1977; Kadis ~1 §l. 1971;

Morrison and Ulevitch, 1978; Nowotny, 1969). It has been shown

that lipid A is responsible for many of these biological ..

alterations. Luderitz §1 ~l- (1973) and Weinbaum gt gl. (1971)

showed that incomplete lipopolysaccharides from R mutants,

which contain predominantly Lipid A, elicited all the

biological activities induced by intact lipopolysaccharides.

Direct evidence for the biological activity of Lipid A was

p~ovided by Galanos and Luderitz (1975) using electrodialyzed,

triethylamine-neutralized, soluble forms of Lipid A derived

from S~lmgo~ll§ miDO§§Q1§ R345. The activity of this Lipid A

preparation was shown to be comparable to that of intact

lipopolysaccharide as assessed by lethality, local Shwartzman

reaction, complement activation, and stimulation of B

lymphocyte proliferation (Galanos ~! ~l., 1977).

The tenn "endotoxin" was ust:-.'d to dc:?~.:;ct .. ibe tfH? toxic,

cell-associated LPS molecule, and to differentiate it from

classical bacterial exotoxins. However, not all

endotoxin-induced cha~ges are pathologic. Some of the effects

of endotoxins are clearly beneficial and include such

manifestations as immunogenicity, protection aga i nst lethal

irradiation, immune adjuvant effects, and enhancement of

nonspecific resistance to infection or to toxic doses of

endotoxin (reviewed by Nowotny, 1983).

The cellular mechanisms which underlie the induction of

these responses by endotoxin have been investigated using

experimental models of LPS-hyporesponsiveness. One such model

is the C3H/HeJ mouse strain. As a result of a spontaneous

mutation which occurred at Jackson Laboratories sometime

between 1960 and 1965 (Watson and Riblet, 1974; .Glade and

Rosenstreich, 1976), C3H/HeJ mice bear a single autosomal

mutation, ~e§d(Watson gt ~l-, 1978), which renders them highly

refractory to the effects of LPS both in ~i~Q and in Yitrg

(reviewed by Morrison and Ryan, 1979). It 1s interesting to

note that a defect in LPS sensitivity also arose spontaneously

on another inbred background, CS7BL/10ScN (Forni and ·Coutinho,

1978; Vogel ~1 ~l-· 1979), and by complementation studies,

·- - · -· ·t - 1: 1 - -.- ·t ·1 ·- ·t·· ·t 1- -, ·- •· -, · " "" ·t .. · 1 · · ·- - ··-· t·... ' c··'''·l/ IJ:::. J appeat ~ .0 l 8 OC.c:l .e<. d .. . lt'o .::odlllE <,;,1t::nE . . lC. OC.U .:.- d.::- liE·. -·~lr rC•.

defect (Watson Qt ~l., 1980).

The fact that C3H/HeJ B cells did not respond to LPS to

proliferate in YiiCQ suggested a possible receptor defect.

For--r1i and Coutinho (197:::) and 'cout.inho §1 .2.1- (1978) descr-ibed

an antiserum which discriminated between responder and

nonresponder splenocytes . This antiserum was prepared by

injecting rabbits with spleen B cells from C3H/Tif

(LPS- responder) mice (two intravenous injections, two weeks

apart), collecting the serum one week after the final injection

and absorbing the antiserum on C3H/HeJ tissues (pooled liver,

t ... · r -~ - •-lt"'t"l" c c" 1 J ·-1' L!=•:-.-.i tJt" ft"e t:! Li'=1.id A, but not twJ -llYinUS, <:t 1<..1 ~,.., ·:. ::. 1 : : .. - ~.:> • ,.

5

other B cell mitogens (lipoprotein and purified protein

derivative of tuberculin), competed with the antiserum for

binding to the B cell surface membrane which suggested a Lipid

A receptor defect. However, when Kabir and Rosenstreich (1977)

14 compared the binding of C-labeled LPS from ~~1~2~~11~

~l~~~~21~ to splenic lymphocytes from C3H/HeN (LPS-responsive)

and C3H/HeJ (LPS-hyporesponsive) mice, they found that spleen

cells from both strains bound LPS equally well. Further, · a

highly lipophilic, polysaccharide- deficient glycolipid from S.

~i~Q~!21~ R595 was shown to bind cells at least 20 times better

than did smooth LPS. This binding was enhanced by trypsin or

neuraminidase treatment. These data suggested to the authors

that LPS binds via a nonspecific interaction between its lipid

moiety and some lipid component of the cell membrane.

Jacobs (1984) used a sensitive hapten - sandwich

immunofluorescence technique and at the single cell level

showed that a positively charged membrane protein is

res pons ib l e for· the initial interact ion of LPS ~'lith murine

lymphocytes. As such, C3H/St (1E! 0) splenic lymphocytes,

pre-treated with pronase or the cross-linking reagent

paraformaldehyde, lost the ability to bind LPS. Additionally,

when the LPS-cell interaction was carried out at pH 4.5 (w hich

increases the number of positively charged imidazol e groups)

the number of LPS positive cells which were detected was

increased. Zimmerman ~.!:: al. (197 7 ) and Kern (1984 ) compared

the binding of iodinated Lipid A to splenocytes from C3H/HeN

and C3H/HeJ mice using an immunofluorescence-autoradiography

6

7

procedu~e . Like Kabir and Rosenstreich (1977), who used intac t

' LPS, Kern found that the strains were ind i stinguis hable on the

basis of binding and also noted th a t spleen cells from the

res ponder strains, but not t he non-responder strain,

proliferated in the presence of Lipid A. Thi s group postulated

that a separation of LPS-binding and LPS-triggering sites co~ld

account for the discrepancies between the finding s of Coutinho

@t ~l- vs. thos e who had measured binding of LPS to cells . "

directly, and that C3H/HeJ mice had retained the capacity to

bind LPS but had lost the capaci t y to be trigge red. \

One of the strongest lines of evidence to suppor t the

hypothesi~ that a membrane defect results in a functional

defect in C3H/HeJ mice comes from the s t udies of Jakabovits et

§l. (1982). Using fu s ogenic membrane vesicles, these

investigator s inserted the membrane components from lymphocytes

which res pond to LPS (~e§0 ) into the membranes of C3H/HeJ

s pl een cells. They found that the latter could now be

s timulated by LPS, and suggested that the inability to respond

was due to the lac k of suitable membrane constituents.

Although the ~e~ gene defect has not been fully

delineated, expe riments us ing CJ H/HeJ mice, in conjunction with

· · . ( n) . I . -J I syngeneic, tully LP~-respons1 ve ~e§ stra1ns, 1ave prov1ae c

strong evidence that the pathophysiologic changes which result

from LPS administration are mediated indirectly by a number of

immunoregulatory factor s [e.g., endogenous pyrogen (Atkins §1

al., 1967), tumor necrotizing factor (Mannel Qi ~l., 1980),

col0ny-s timulating factor (Apte Qt ~l., 19 76; Apte and Pluznik,

1976; Metcalf, 1971; Quesenberry Qi §1., 1972), glucocorticoid

antagonizing factor (Moore Qt ~1-, 1976), and prostaglandins

(Skarnes and Harper, 1972)] produce~ by macrophages in response

tQ LPS. Furthe rmore, agents which activate macrophages, either

in YiYR (Suter~! ~l., 1958; Benacerraf gt §1., 1959) or in

YiirQ (Moore§! ~1-, 1980; Rosenstreich and Vogel, 1980),

increa~e sensitivity to LPS, even in the C3H/HeJ mouse strain

(Vogel ~1 §!., 1980).

· A second model of murine endotoxin hypores ponsiveness

l S a state of endotoxin refractoriness which is ~cquired after

exposure of a normally sensitive animal to a sublethal

injection of endotoxin. The acquisition of such a state of

resistance was first encountered nearly 100 year s ago when

wholt~ bact,?.r ial vaccin,?.s wet·e us~:::>d to induc1? "th,:?rapeutic

fevEH"" in the tr·eatment of cet·tain disl=)ases . It vJas found that

increasing quantities of typhoid vaccine were required in order­

to maintain elevated temperatures (Heyman, 1945). Acquired

resistance to alcohol-fractionated pyrogenic preparations,

initially shown by Centanni (1894), was demonstrated by reduced

fever-inducing activity in animals given repeated inj ect ion s of

these preparations. Centanni (1942) also demonstrated that

thi s pyrogenic substance was stab le to heat and was not protein

in nature. Favorite and Morgan (1942) were the first to use

thf::> ter:rn '' 1?ndotox in toler anC(;')" to des c:r- ib':? this phenomenon.

They also noted that the prese nce of anti-LPS antibodies did

not correlate with such resistance . These findings led them to

postulate that such acquired resistance had a nonimmunologic

8

basis. The development of tolerance to LPS-induced lethality

was also no-ted by Wh.:H·ton and Creech (191+·:)). In tht'::'it .. st.J .. td.iE~~:·::;,

tolerance was showh t6 be induced in mice following a single

injection of endotoxin, to peak three days later, and was shown

not to correlate with anti-LPS antibody titers. Beeson (1946,

1947a' 1 '?'47b) showed that: ( 1) "endotoxin toler anC(,? II could not

be transferred by serum, (2) the acquisition of tolerance was

accompanied by enhanced blood clearance of endotoxin and, (3)

blockade of the reticuloendothelial system (RES) with

thorotrast (thorium dioxide) retarded clearance of endotoxin

and abolished tolerance.

Up to this time~ endotoxi~-induced fever was thought to

be the result of a direct effect of LPS on the thermoregulatory

center of the brain. In 1948, Beeson provided evidence that

f?ndotoxin stimulated "host cc;•lls" to n?.leas~~ an intermt~diatP

factor, endogenous pyrogen (EP), which acted on

thermoregulatory centers in the brain to evoke fever. After

Beeson (1948) reported the extraction of EP from rabbit

polymorphonuclear leukocytes, these cells were considered to be

the major source of pyrogen for the next 20 years. Thus, until

EP was shown to be a macrophage secretory product by sPveral

groups (Atkins gt §!., 1967; Hahn§!§!., 1967; Murphy §1 g! .•

1980), tolerance to the pyrogenic effects of endotoxin was

thought to result from enhanced uptake of endotoxin by the RES

which diverted the endotoxin away from EP-producing cells.

In 1965, Greisman and Woodward provided evidence for

two distinct mechanisms of endotoxin tolerance wbich were

9

dt~scribt~d tf::>mpor-a.l.ly as "c~arly phase·?" and ".l<:ttt~ phase"

endotoxin tolerance. The concept of an early phase of

tolerance was based on experiments in man, in which a

refractory state was observed within hours after continuous

intravenous infusion of endotoxin and, in rabbits, by 24 hours

after a single injection. This type of hyporesponsiveness w~s

shown to be specific for endotoxins as a class (e.g., tolerance

was not induced for other p~rogens, such as staphylococcal

enterotoxin), was not transferable with serum, and was found to

wane over subsequent days. A later phase of tolerance to the

pyrogenic effects of endotoxin could be demonstrated if

endotoxin were re-administered after 5 days. Unlike early

phase tolerance, this late phase was shown to be transferable

with serum and exhibited marked a-antigen specificity. Thus,

the early phase of pyrogenic tolerance was hypothesized to

reflect a decreased capacity of macrophages to release EP, and

late tolerance to be mediated by specific humoral antibodies.

By passive transfer experiments, Greisman and Woodward (1965)

and Greisman ~l §l. (1966) showed that pyrogenic quantities of

endotoxin were present in the serum of tolerized animals.

These findings indicated that early phase tolerance was not

mediated simply by clearance of endotoxin from the circulation.

As with early tolerance to fever induction, an early

phase of endotoxin tolerance for the lethal effects of LPS was

demonstrated in mice 24 hours after a single intraperitoneal or

intravenous injection of endotoxin (Abernathy, 1967; Urbas chek

and Nowotny, 1968; Wharton and Creech, 1949; Williams @l.~l.,

10

1983)_ Early tolerance to . a lethal challenge with LPS also

extends to endotoxins of various Gram negative bacteria. Thus,

no a-antigen specificity was seen during early tolerance.

As in the case of the genetically LPS-hyporesponsive

C3H/HeJ mouse, \ . stud1es of early endotoxin tolerance in animals

have indicated a central role for the macrophage. Dinarello ~

and co-workers (1968) sho~ed that isolated Kupffer cells from

rabbits [shown previously by Braude §1 ~!- (1955) and Cremer

and Watson (1957 ) to be the primary site of endotoxin

localization following i.v. injection] were st imulated by LPS

to produce EP. However, isolated hepatic Kupffer cells from

rabbits r e~dered tolerant by a previous LPS injection were

found to be refra~tory to stimulation by LPS to release EP in

~it~Q. Cellular refractoriness to endotoxin was also s hown in

human blood monocytes. These cells normally respond to LPS

with release of large amounts of colony-stimulating activity

(Sullivan gt §1., 1983); however, within 72 hour~ of a second

exposure of endotoxin, a marked reduction in release of this

factor was observed. In addition, Rietschel Qt §l- '(1982)

showed that peritoneal macrophages from tolerant animals

secreted substantially lower leve l s of prostaglandins (PGE 2 and

PGF za> than peritoneal cell s from control animals. Lastly,

Williams Qi ~l - (1983) suggested that an interaction of

macrophages and splenic nonadherent cells were required for the

early tolerant state.

The goal of this study was to probe further the

cellular mechanisms which accompany a state of early endotoxin

11

tolerance. To this end, a model system, based on that

described by Williams Qi Ql. (1983), was established us ing

highly purified endotoxin derived from ~§~bQLi[bi~ [Qli K235.

Using this model system, a state of early phase endotoxin

tolerance was found to be associated with marked alterations in

bone-marrow derived macrophage progenitor pools. The cellular

mechanisms which underlie the establishment of early tolerance

were analyzed in mice which were genetically

LPS- hyporesponsive, T cell deficient, B cell deficient, or

asplenic. Further experiments were carried out to investigate

whether induced hyporesponsiveness to LPS rendered mice

refractory to infection by a Gram negative organism,

f§§Y~QillQQ~§ §§LY9iOQ§§. Lastly, a non-toxic derivative of

Lipid A, monophosphoryl Lipid A, was assessed for its efficacy

as an inducer of early endotoxin tolerance.

12

~IQE -- Outbred, female HSD(ICR)BR mice (ICR) were used for

the majority of the studies described herein and were obtained

from Harlan Sprague Dawley (Indianapolis, IN). For some n d

experiments, inbred C3H/OuJ (Le§ ) and C3H/HeJ (Le§ ) mice were

obtained fro~ the Jackson Laboratories (Bar Harbor, ME). For

experiments which involved the use of athymic (nude) mice,

BALB/c (oy/oy), and their euthymic littermates, BALB/c (oy/+),

were obtained from Harlan Sprague Dawiey (Indianapolis, IN).

T-cell deficiency was verified in BALB/c (QY/QY) mice using

fluorescence activated cell sorter (FACS) analysis as descr1bed

below. Female C3H/HeN mice (Harlan Sprague Dawley,

Indianapolis, IN) were age-matched with female, congenic

C3.CBA/N (xid/xid) mice. The latter were bred at the Uniformed

Services University of the Health Sciences (Bethesda, MD) and

were the kind gift of Dr. James Kenny, Dept. of Microbiology,

Uniformed Services University of the Health Sciences. In an

attempt to minimize potential environmental differences due to

the different sources of these two strains, C3H/HeN and

C3.CBA/N mice were housed in the same room for 2 weeks prior to

treatment.

In one series of experiments, ICR mice were

splen~ctomized. Splenectomies were performed as follows: Mice

were given general anesthesia (Metofane '(Methoxyflurane),

Pitman-Moore, NJ). Each spleen was removed after the arteries

and veins which supply the spleen were tied off with 4.0 silk

13

14

suture. The peritoneal lining was c losed with 4.0 silk sutur e

and the s kin brought together and fastened with 9 mm autoclips

(Clay Adams, Becton Dickinson, Parsippany, NJ). Sham

splenectomies were also carried out in a similar fashion except

spleens were gently . lifted out of the peritoneal cavity with~ut

severing the vessels and were replaced immediately prior to

closure. The mice were allowed to recover from surgery for

seven day s prior to treatment.

All mice were used at 6-8 weeks of age. Mice were

housed in cages with Micro-Isolator Unit tops (Lab Products,

Rockville, MD) in a negative pressure Horsfall I so lator Unit

(Hazelton Systems Inc., Aberdeen, MD), and were fed RMH 3000

mouse chow (Agway, Inc., Syracuse, NY) and acidified water ~d '

Protein-free, ph~nol-water

extracted LPS, from f~~bgLi~bi§ £9li K235 L+OC+ (serotype

Ol:K1:HNM, Barry ~1 §l., 1962), was prepared by the method of

Mcintire ~1 gl. (1967). f~§YdQffiQQ§§ gQtY9iOQ§§ F-D type I LPS

was obtained from List Biological, Inc. (Campbell, CA).

Phenol - water extracted ~§lffiQOQll§ t~ehirnYtiYm LPS and

~~Gt~rgid~~ fr§9ili~ LPS were the kind gifts of Dr. Su zanne

Michalek (Univ. of Alabama, Birmingham, AL). Each LPS

preparation was prepared in pyrogen-free saline at a final

concentration of 1 mg/ml and was put i nto sol ution by

15

sonication (three, 5 sec bursts) using a Sonifier Cell

Disrupter, Model Wl85 (Heat Systems, Ultrasonics, Inc.,

Plainview, NY) with a microtip and a setting of 3 on the output

control.

Detoxified endotoxin (monophosphoryl Lipid A, Lot 375),

derived from a heptose-less mutant of ~- 1~ebimYr1~m G30/C21

(Qureshi Qi §l., 1982), was obtained from Ribi ImmunoChem

Research, Inc. (Hamilton, MT). The monophosphoryl Lipid A

(MPL), 1 mg/vial, was solubilized by addition of 0.5 ml ster1le

' distilled water, followed by swirling of the suspension in a

0 65-70 C water bath for 10-20 seconds, and sonication for 10- 20

seconds. After three to four cycles of heating and sonication,

the solution clarified to slight opalescence. To this solution

was added 0.5 ml of 3.6% NaCl to give a final stock solution of

MPL (1 mg/ml) which was subjected to one additional cycle of

heating and sonication.

Phenol-

water extracted E- £Qli K235 LPS was subjected to acid

hydrolysis [1.0% (approximately 0.17 N) acetic acid, lOCfc, 4

h], centrifuged to pellet insoluble Lipid A, and the soluble

polysaccharide-rich supernatant removed and dried (Galanos i1

l l g71) T~1e pr.1lvsar.:char1de fraction was further separated ~= ·• , . I- 1-

from any remairiing LPS by gel chromatography on a Sephadex

G-2 5-300 (Sigma, St. Louis,· MO) column (60 x 1.5 em) which was

equilibrated in a solution which consisted of 0.4% (v/v)

pyridine (Baker Chemical Co., Phillipsburg, NJ) and 1.0% (v/v)

glacial acetic acid (Mallinckrodt Inc., Paris, KY) in water

(Jansson @t §l., 1981). Those fractions which contained the

polysaccharide were identified by detection of glucose using

the phenol-sulfuric acid method (Dubois ~t ~l., 1956).

Fractions which contained polysaccharide were then pobled and

,::Jt·l·E~.,·.:J ~-·r_·J ·v­-~£YQ- Th~ final polysaccharide preparation was

adjusted by total glucose content to be equivalent to the

glucose content of the phenol-water extracted E- ~Qli LPS

prepared at a final concentration of 1 mg/ml. Mice were

lnJected with 25 ~g/mouse of E- cgli K235 LPS or the dilution

of purified polysaccharide which was equivalent in glucose

content to that of 25 ~g ~- ~Qli LPS.

in_~~[Yill -- After injection of saline or endotoxic

preparations into mice, sera were collected. CSF activity in

the sera was determined using a bone marrow colony assay in

semi-solid agar (Williams @t ~1-, 1983). Blood from mice in

each experimental group was collected from the orbital pl ex us 6

h after injection, pooled. and the serum obtained after clot

formation and centrifugation (1500 x g, 10 min) in a Beckman

Microfuge 12 (P~lo Alto, CA). Serial dilutions of se rum were

prepared in serum-free RPMI 1640 (M.A. Bioproducts,

Walkersville, MD) supplemented with penicillin/streptomycin

(100 IU/ml and 100 ~g/ml, respectively; Gibco Laboratories,

16

Ch<:lqt·in Falls, OH), 1!5 mM HEPES buff(?.r (R(7.!S(?ar·ch Ot .. 9<':1nics In·c .. . ,

Cleveland, OH), 2 mM glutamine (Gibco Laboratories, Grand

Island, NY), and 0.2% sodium bicarbonate (Fisher Scientific

Co.~ Fair Lawn, NJ). Two tenths ml of each serum dilution was

added to each of two tissue culture wells (35 mm diameter) in 6

well tissue culture plates (Costar, Cambridge, MA).

LPS has been shown to interfere with the colony assay

(Moore ~1 ~l-• 1980). For this reason, bone marrow cells for

the CSF colony assay were bbtained from endotoxin

hypon?.spons.i ve C3H/HeJ ( Le~l ) mic(:>, to pn?clude possibl(~

effects of endotoxin carried over in serum samples. Femur s and

tibias were homogenized in ice cold RPMI medium using a mortar

and pestle. Bone fragments and cell aggregates were allowed to

settle in a conical centrifuge tube. The crude cell suspension

was enriched for mononuclear cells by density gradient

centrifugation (400 x g, 20 min, 20°C) in Lymphocyte Separation

Medium (LSM; Litton Bionetics, Charleston, SC) using a Sorvall

RT 6000 refrigerated centrifuge (Dupont Co., Wilmington, DE).

The cells collected from the interface of the LSM gradient were

washed twice in RPMI medium supplemented w{th 10% fetal calf

serum and were enumerated using a Coulter Counter Model ZM

(Cou~ter Electronics Ltd., England). Bone marrow cells were

diluted to a final concentration of 1 x 1J5

cells per ml in

molten colony assay medium (RPMI 1640 supplemented with

glutamine, HEPES buffer, sodium bicarbonate,

penicillin/streptomycin, 15% fetal calf serum (M.A.

E) . - r· •"()l::ll.l(" ·t···· ) :=trtd () .... ··-)·%., B 2(("_: t·.c-·-,"'.l9•"'-"lt" ( 1)1" f c: 0 Labor .. ;:;t tor-ies' De t t .. oi t., , 1 o,.l, . · ·--~· , ~ - ..- 1.

17

MI)). One milliliter of this suspension was then added to each

of the wells which contained 0.2 ml of serial serum dilutions,

mixed by swirling, and allowed to solidify. The cell cultures

were then incubated at 37°C (5% co 2) for 7 days, at which time

bone marrow .colonies (>50 cells/colony) were counted under an

inverted microscope at 40X magnification. Serum CSF activity

is based on the linear part of the dilution curve and is

expressed as colony forming units (CFU) per ml of serum.

To mt·?as1 .. a··~~ thP

lf?.Vel of intt':t"ff?t'on in St·?r um <:tftt~r· administration of salinf~~ Ot"

endotoxic preparations, blood samples were collected and

processed as described for the collection of se rum for CSF

activity. Interferon activity was measured by an antiviral

assay (Rubinstein @t ~1., 1981) modified as d~scribed elsewhere

(Vogel @1 ~l., 1982). Two-fold serial dilutions of serum

samples were prepared in a final volume of SO pl of assay

medium (Minimal Essential Medium Eagle (M.A. Bioproducts,

Walkersville, MD) supp lemented with antibiotics, HEPES buffer,

glutamine and 10% fetal calf serum) in flat-bottomed, 96-well

Falcon culture dishes (Becton Dickinson, Oxnard, CA). To each 5

well was added 10 L929 fibroblasts in a volume of 50 ul. The

I . . ·- 'l" ..,,...p ("' ',- o/ supernatants were aspirated after a 24 1 lncubatlon d, ~, J ~oM

co2 ) and the? cells infectt;:d with 100 u 1 PGt- ~H3ll of Vc~s.iculat"

Stomatitis Virus (Indiana strain) in medium ' containing 10%

fGtal calf serum at a multipli city of infection of

approximately 0.1. At 24-48 h post-infection, complete

cytopathiri effect was obsGrved microscopically in infected

18

19

medium-treated control cells. The cells were then washed with

cold Earle's Balanced Salt Solution (Gibco Laboratories, Grand

Island, NY) and fixed for 10-30 min with 5% formaldehyde

(Fisher Scientific Co., Fair lawn, NJ). The cells were stained

for 5 min with o;os% crystal violet (Sigma Chemical Co . , st.

Louis, MO) in 20% ethanol, and subsequently washed with tap

water. Optical density was measured at 595 nm using an ELISA

reader (Biotek, Inc., Burlington, VT) . Three controls were

included for each assay: (i) medium-treated, uninfected L929

cells (cell control); (ii) medium-treated, ·virus-infected L929

cells (virus control); and (iii) a titration of NIH mouse

fibroblast interferon reference standard (G-002-904-511;

Research Resources Branch, National Institute of Alle~gy and

Infectious Diseases, NIH) adjusted to 100 U/ml. The first

dilution of a sample which exhibited an optical density equal

to that of the virus control wells (i.e., no interferon

protection) was defined as the endpoint. Reciprocal titers

(expressed as units per milliter) were calculated based on the

titration of the NIH standard.

D~!~rmio~tigo_gf_~Q%_l~!b§l_dg§g_iLD 50 ) -- Mice (20

· 1 ) l·r, .l·E• .. ct.P_c:l 1· r1traoeritoneallv wi~h either saline, m1ce group were _ _ ~ •

E- ~Q!i K235 LPS (25 p9/mouse), or MPL (100 p9/mouse) on day 0.

Three days later, five mice from each group were challenged

i.p. with either 3000, 1500, 750, 375, or 187 pg of E- ~gli LPS

in 0.5 ml of pyrogen-free saline. Deaths were recorded daily

for 3 days. The LDso for each experiment was calculated by the

method of Reed and Muench (1939).

~§§~§§IDQD1_gf_go~gtg~io~io~y~g~-~~me1gm~ - - Mice were

assessed for visual manifestations of endotoxicity as described

by Pier gt §l. (1981). Accordingly, mice were observed for

ruffled fur, conjunctival discharge, and diarrhea 6 h following

administration of 25 ~g/mouse of endotoxin.

QEIEB~l~~IIQ~_QE_~y~~ER~_QE_~~QBQE~~QE_EBQQE~IIQB_QEL~~-I~_IUE

~Q~E-~~BBQ~-A-~Q-~fhE~~

Er~e§t§1iQO_Qf_bgog_m§rrg~-~Qll~ -- Groups of 5 mice

were injected with either saline, LPS, or MPL as indicated.

Five femurs per group (one per mouse) were homogenized using a

mortar and pestle with 3 changes of ice cold (4°C), serum-free

RPMI 1640 (supplemented with antibiotics, HEPES buffer, and

glutamine)_ The cell suspension was collected in a 15 ml

conical centrifuge tube and brought to final volume of 15 ml.

The suspensions were vortexed and were incubated on ice for 10

min to allow for settling of bone fragments and cell

aggregates. The top 10 ml of cells were then tran s ferred to a

second tube and kept cold (~C) until cultured. A sample of

this cell suspension was used to obtain a nucleated cell count

and cell sizing profile using a Coulter Counter Model ZM and

Coulter Channelyzer ClOOO equipped with a Coulter X-Y Recorder

4 (Coulter Electronics, _ Inc., Hialeah, FL), which was

20

calibrated according to the manufacturer's instructions.

fr~e~[~1iQQ_Qf_§el~~Q-~gll§ -- , Group~ of mlce were

injected with saline or LPS as indicate<:!. "1'rgl l 'l l .::> . I_ f? ce . sp. f?en

suspensions were prepared from five spleens per experimental

group by mascerating spleens on 40 mesh stainless steel

screens. The cells were resuspended in ice-cold, serum-free

RPMI 1640 (supplemented with antibiotics, HEPES buffer, and

glutamine). A sample of each cell suspension was used to

obtain a nucleated cell count employing a Coulter Counter Model

ZM. These spleen cell suspensions were then used for

determination of the number of macrophage progenitor cells.

macrophage progenitor cells (macrophage colony forming units,

M-CFU) in each bone marrow or spleen cell suspension was

determined by the method of MacVittie (1979) using a double

laver, semi-solid agar colony assay in 60 mm x 15 mm giided

tissue culture dishes (Nunc, Roskilde, Denmark). The bottom

layer in each culture dish was comprised of approximately 7000

U of macrophage-specific colony stimulating factor (CSF- 1) in

2.5 ml of 0.5% Bacto-agar (Difco Laboratories, Detroit, MI) in

medium composed of tryptic soy broth and CMRL 1066 supplemented

with fetal bovine serum, horse serum, an antibiotic/antimycotic

mixture, serine, L-asparagine, sodium pyruvate and sodium

bicarbonate as described (MacVittie, 1979). The CSF-1 used was

partially purified to insure removal of contaminating

21

interferon (Warren and Vogel, 1985). The upper layer contained

r.:: 1" 4 l 11 ~ x U nuc . eated bone marrow ce s from each cell suspension

in 2 ml of 0.33% agar in medium. Duplicate cultures were

incubated at 37°C (5% C0 2) for 10 days and colonies (>SO cells)

coun tc:!d.

To test for the presence of more primitive macrophage

precursors which exhibit high proliferative poteniial (HPP),

the bottom layer contained CSF-1 and 10% human sp l een

conditioned medium (HSCM). Human serum (20%) was included in

both top and bottom layers. HSCM provides a source of

"Sync:!rgistic Act i vity" (:3A), s hmm by Bt·adlc:!y and Hodgs on

(1979) to be essential for HPP development. HSCM was the kind

gift of Dr. Thomas MacVittie (Armed Forces Radiobiology

Re~earch Institute, Bethes da, MD), and its preparation has been

described elsewhere (Metcalf and Johnson, 1981). HPP colonies

fail to develop in the presence of CSF alone (Bradley and

Hodgs on, 1979), but in the presenc e of b6th CSF and SA will

produce very large macrophage colonies (> 2 mm diameter) which

are readily distinguishable from M-CFU by size and are counted

after 12 days of incubation.

B£e§r~1igo_gf_bgo~_m9~LQW_~~ll§_b~-~§D§i1~-gr~diDoi

§£0iill£O!§!!QO -- In some experiments, ICR mice were injected

with either saline or C- ~Ql! LPS and after 3 days th e ir bon e

marrow cells collected and separated by unit gravity

s edimentation (Bertoncello ~1 ~l., 1981) in a 60 ml separatory

funnel. One x 10 7 cells from gnJups of fiv<:'! mice (,J C:-'?.t"(~

22

~esuspended in 10 ml of RPMI 1640 (supplemented with glutamine,

HEPES buffe~. sodium bicarbonate, antibiotics and 10% fetal

calf serum) and layered onto a 50 ml gradient. The gradient

was poured stepwise in four 12.5 ml volumes (which consis~ed of

30%, 25%, 20%, and 15% fetal calf serum in RPMI medium) laye~ed

onto a 10 ml cushion of 50% fetal calf serum in RPMI 1640

supplemented as indicated above. Cells were allowed to 0

sediment at 4 C for 3.5 h. Th~ee ml fractions were collected

dropwise f~om the bottom of the gradient, centrifuged, and the

cell pellets ~esuspended in 0.5 ml of 10% fetal calf serum in

RPMI 1640 medium supplemented as indicated above. Nucleated

cell counts and cell sizing profiles were then obtained from

each fraction and the number of M-CFU or HPP in individual or

pooled fractions was determined as described above.

0 overnight at 32 C in Trypticase Soy Broth (TSB) (Difco

Laboratories, Detroit, MI). Four tenths ml of this overnight

cell suspension was used to inoculate 65 ml of TSB which was

fur-t:hQt' incub<:tt.f?.d at ~52°C ~"ith shak .ing. 13act(·)t-i <:tl \elt· owth was

monitored every 0.5 h using a Spectronic-20 (Bausch and Lomb,

l~ochestm·, f'.IY) at OD 660 • 1\t. OD 660 ::: 0.~:~5, the lat.(·:~ lo(;~ phase

1 · -~ -- n ·· < ... ,_ "'' cells were washed and resuspended in sa 1ne toLD 660 - .• b 'J

23

8 x 10 viable bacteri~/ml). Ten-fold dilutions were then made

in saline for injections of mice and appropriate dilutions

plated on Trypticase Soy Agar (Difco Lab6ratories, Detroit, MI)

to obtain viable cell counts.

Ioj~g1igo_erg1gggl -- Mice were injected with saline

(control) or LPS (tolerized) on day 0. On day 2, the lower

back of each mouse was shaved. On day 3, each mouse was

anesthetized with Metofane (Methoxyflurane, Pitman - Moore, NJ) I

and a third degree burn inflicted by igniting 0.4 ml of 95%

alcohol on a 2.5 x 2.5 em area of exposed bare skin (the area

exposed in an asbestos template) (Stieritz and Holder, 1975;

Pavlovskis ~1 §1., 1977). After 10 sec, the flame was

extinguished and 0.5 ml of each 10-fold dilution of bacteria

injected subcut~neously at the burn site (5 or 10

mice/bacterial dilution). Deaths were recorded daily for one

week.

Individual spleen cell suspensions from five athymic

BALB/c (oy/uy) mice and five euthymic BALB/c Coy/+~ mice were

prepared in Hank's Balanced Salt Solution (without phenol red;

Gibco Laboratories, Chagrin Falls, OH) supplemented with 10%

newborn calf serum (Gibco Laboratories, Grand Island, NY) and

0 . 4% (w/v) sodium azide (Matheson Coleman and Bell, Norwood,

OH). Following ly s is of erythrocytes with an ammon1um chloride

24

lysing solution (AKC-lyse, M.A. Bioproducts, Walker s ville, MD),

aliquots of each cell suspension were treated with

fluoresceinated (FITC) chicken anti-mouse ~ polyclonal

antibody, FITC-anti-Thy 1.2 antibody (a monoclo~al rat IgG2b;

Becton Dickinson, Sunnyvale, CA), or a control antibody,

FITC-CBPC-101 (a monoclonal IgG2b that does not react with 6

murine cells) at a final concentration of 1 ~g antibody/10

cells. Both the FITC-chicken anti-mouse ~ and FITC-CBPC-101

antibodies were the kind gifts of Dr. Fred Finkelman, Dept. of

Medicine, U.S.U.H.S. Fluorescence analysis was carried out

using a fluorescence-activated cell sorter (FACS II; Becton

Dickinson, Mountain View, CA) in the laboratory of Dr. Lyn

Yaffe (Dept. of Pathology, Navy Medical Research Institute,

Bethesda, MD). Cells were assigned to fluorescenc0 intensity

channels ranging from 1 to 1024 according to the amount of

surface-bound fluorescein label detected. For each sample, the

percentage of cells which stained with a specific antibody and

the median fluorescence intensity (MFI) of the staining were

determined by a computer program of curve subtraction and

integration as described previously (Mend ~1 ~l., 1980).

25

FSTABI ISHMFNT OF AN FAPLv FNDOTOXIN TOI FPtNrF 0 Y0T[' ~1 =~--~=~~--~---~-----~~~~~-=-~~-~-----~~~~~-~~-~-~-~L

Previous stud1es have shown that injection of LPS into

mic e resulted in a rapid ri se in serum CSF which r each ed a

maximum leve l a .. ·t. r.-LI~o~oxJ·. rn~t: e_lv ~--·· ~ ~J -t 1·- 1·~ t " 'M t lf ,. ' ~• r 11 ,.t .. s .-- ll c cC". lOn ~ C:;!"':ca .. ,

1971). In a recent report by Will iams @1 gl .. (1983), thi s

manifes tation of e ndoto xin res ponsiveness was found to be

mitigated in mice challenged with LPS 3-4 days after an initial

s ublethal exposure to LPS. The fir s t se ries of experiments

described herein sought to optimize conditions under whi ch t hi s

sta t t-=~ of ''t·?a r.ly E'ndotox.in tolt·?.r·ance '' \oJ <:ts .induc .i.bl<·;).

expe rime nt was carried out in which groups of outbred IC R mi ce

wer e injected intraperi tonea lly with e ithe r ~- ~gli K235 LPS

(25 ~g/mouse) or saline. Thi s dose o f LPS was chosen based on

previous studies by Williams Q1 ~1- (1983) in which E-£Q1i

055: B5 LPS was used to induce ea rly e ndo to xin tolerance in thi s

mouse s train . On s uccess ive days following an initial

injection, individual groups of mice were challenged with ~-

cgli K235 LPS (25 ~g/mouse ) and bled 6 h later for se rum which

wa s subsequently tested for the pres ence of CSF . Th e ability

of mice ·to res pond to a second (challenge) injection of LPS was

26

27

most suppressed at 3 and 4 days after the initial exposure to

LPS (Figure 1). Afte r t hi s time , tolerance (hyporesponsiveness

to LPS) gradually waned, as indicated by the r eturn of

near-normal CSF production by day 8. Groups of mi ce injected

with saline on day 0 responde d to an injection of F £Qli LPS

with h igh l evel s of CSF throughout the 8 day time period.

These results confirmed those of Williams gt ~1- (1983).

Mainta i nin g a 3 day period between the initial a nd

c hallenge doses of LPS, th e concentrat ion of LPS used for the

initial injection was varied to determine th e optimal amount of

LPS which rende red the mice most hyporesponsive to a subsequent

LPS c ha llenge. Figure 2 illustrates that tolerance, as

assessed by a decreased capacity to produce se rum CSF, was

100 pg/mouse) but optimally at 50 p9/mouse.. However,

50 ~/mouse was occasionally lethal, so a lower dose of

25 p9/mouse was used i n all s ubseque nt experiments. The level

of CSF induced by one in jection of LPS (25 pQ/ mouse) was the

same if administered intraperitoneally or i ntravenously (i.p. -

/~ ., ~520 ± 5(.5 v :::; . 4,5~:20 ± 20.1. Cf-=-Ujm.l serum). t.~.i.th t"E~ ~::.:p1:::oct to

tolerance induction (as assessed by supp r essed CSF ac t ivity),

little difference in the degree of s uppression induced was

J • • :1 • t . t J J ( O •.::Ju/ ob:::;l? t" V(~d if th 1·?. L PS ~o.J ~~ t .. P a c rn 1 n J. :::.: t (:> t" t::-'i. .l n · ·. r ape~ t· 1 ··. on (~a - · . '/ . '·--' .. · / u

s uppress i on ) or intravenously (82% s uppr ession ). Thi s

conf irmed findings whic h wer e pr e vious ly repor ted by Williams

To i ns ure that the 6 h ti me poi nt used in tn lt lal

28

Figu r e 1. Iiwg_~QYL~Q_gf_tbg_iodYc!iRQ_Qf_Q§LlY_QQ~Q1Q~io

iQlQL§Q~Q-~§-~5~Q~§Qd_~y_LP3=indY~Qd_Q~E -- I CR mice were

injected i.p . with either s aline orE- ~Qli LPS (25 u gjmouse).

At interval s of 1 to 8 days, mice (5 mice/group) were

challenged i.p. with a second injection of E- CQli LPS

(25 ug/mouse) . Six hour s later, the mic e were bled and their

se ra pooled a nd assayed for CSF activity as desc ribe d in the

Materials and Methods section. Each po i nt r e pr esents the

arithmetic me an ± one standard deviation of duplicate

'de terminations.

5000

4000

-e ' ::I u.

3000 ~ u. en (.)

:::IE 2000 ::I a: 11.1 en

1000

0

E. coli LPS CONTROL

SALINE CONTROL

2

D SALINE DAY 0

rzl E. coli LPS DAvO

NUMBER OF DAYS BETWEEN INITIAL INJECTION AND CHALLENGING DOSE OF LPS

29

30

Figure 2. Q~1QLIDiD§1iQQ_Qf_1b§_Qe1im§l_LP~-~QD£QD1L~1iQD_fQL

iD~Y£1iQD_Qf_g~r1~_god91Q~io_iQ1Rr~DfQ -- ICR mice were

injected i_p _ with sal ine or varying concentrations of E. £Qli

LPS. At 3 days post-injection, mic e (5 mic e /group) were

challenged i.p. with a second injection of E- ~9li LPS

(25 ~g/mouse). Six hour s later, the mice were bled and their

sera pooled and assayed for CSF activity as described in the

Materials and Methods sect1on. Each point represents the

arithmetic mean ± one standard deviation of duplicate

determinaticins.

u.. (J)

0

:E

10000

8000

6000

~ 4000 w (J)

2000

Saline Saline day 0

days 0/3 LPS day 3

t----LPS (25p..g/mouse) DAY 3

6.25 12.5 25 50 100

CONCENTRATION OF LPS INJECTED

ON DAY 0 (.,u.g)

31

32

experiments was indeed optimal for the produc tion of serum CSF

in respon se to the particular preparation of c. C9li K235 LPS

use d in this study, a 24 h time course was additionally

performed. Groups of 5 mice were injected on day 0 with s aline

or ~- ~Qli LPS (25 ~g/mouse) i.p. Three days later, these mic e

were challenged with 25 ~g LPS and were bled at various times

(0-24 h) thereafter. The result s in Figure 3 confirmed the

previous finding s of Metcalf (1971 ) that 6 h pos t-injection of

LPS was optimal for detection of serum CSF in control (sa line

pre-tr e ated) mice. In tolerized mice, i.e., those which had

received LPS on day 0, the CSF r esponse was reduc e d through ou t

the 24 h time period following the challenge (day 3 ) injection.

The experiments descri bed thus far utilized CSF

production as an indicator of endotoxin responsiveness io yjyp .

To insure that the apparent induction o f endotoxin tolerance

was not unique or specific for this particular manifestation of

endotoxi n sensi tivity, th e serum samples assayed in Figure 3

for CSF activity were also assayed for the presence of

interferon (IFN). Previous studies have s hown that

administration of LPS to endotoxin-responsive animals res ult s

in a sharp ri s e in serum IFN which reaches maxi mum l e v e l s at

a p p ro xima tely 2 h post-administration (Youngn e r a nd S tinebri ng ,

1965 ). The r esults in Figu~e 4 illustrate t hat control mic e

(i.e. , those that received sa line on day 0) responde d normally

to LPS with peak IFN levels at 2 h pos t-inj ec tion o f LPS .

Serum from tolerized mi c e, howeve r , yielded no detectable

] J ··f II- J·I ·J·.·. r'1t-(JJ. •c.,_ I"J(-_ll .t ·t. ·t.I-, F.3 24 h time oeriod following _eve .s 0 . -~ ·~ - r

33

i.p. with sa line or~- ggli LPS (25 ~g/mouse) . After 3 days,

the mic e (5 mi ce/group ) were challenged with f- ~Qli LPS

~ g/mouse) and bled at the indicated times. Sera from each

group were pooled and assayed for CSF activity as described in

t he Materials a nd Methods sec t ion. Each point represents the

arithmetic mean ± o ne standard deviation of duplicate

determinations.

u. Ill (J

== ;:) a: "' Ill

0 2 3 8

TIME AFTER SECOND INJECTION (HOURS)

D Saline on day o LPS on day 3

LPS on day o LPS on day 3

... 8 24

34

35

Figure 4. 24=bQYL_1iillQ_CQYLSQ_fgr_iDt2LfQLQD_iDdYcti~J_in

DQD=1Ql0ri~§~-~od_tglgrii0d_mi£0 -- Pooled serum samples ,

obtained from mic e treated as described in the legend of Figure

3, we re assayed for the presence of interferon as described in

the Materials and Methods section. Results repr esent the

antiviral activity contained in serum pools obtained from 5

mice per treatment g r oup_

> 1-

> ;:: u < z 0 a: Ill ... a: Ill 1-~

TIME AFTER SECOND INJECTION (HOURS)

D Solin• on dey 0

LPS 011 doy 3

LPS 011 dey 0

LPS oft dey 3

36

37

challenge.

Further confirmation of the efficacy of this toleri z ing

regimen was established using LPS-induced lethality and

s ymptoms as a measure of endotoxin sensitivity_ Exposure to an

initial dose of 25 ~g LPS/mouse more than doubled the LD 50

(414 ~g non-tolet·ized vs. 994 ~g tolerized) a nd essentially

eliminated overt symptoms of endotoxicity (i.e., diarrhea,

ruffled fur, and conjunctival di~charge) which are normally

observed by 6 h after c hall e nge with a sublethal dose of LPS

(25 pgjmouse). These data are shown in Table I.

Since one of the characteristics of early endotoxin

tol e ra~ce is that it is inducible with unrelated LPS

preparations (i.e., it is not a-antigen-specific; Greisman and

Hornick, 1976), groups of mice were injected with saline orE-

~gli LPS (25 ~g/mouse) on day 0, but c hallenged on day 3 with

25 pg LPS/ mouse of either homologous E- ~Qli LPS or

heterologous LPS preparations der ived from P§QYdQffiQQ0§

Table II shows that an initial exposure on day 0 t o ~- ~gli LPS

markedly reduced the level of CSF which was induced by

challenge with any of the LPS preparations 3 davs later.

previous studies had indicated that Lipid A, the biologically

active moiety of the LPS molecule, was capable of inducing a

state of e ndotoxin tolerance (Staber, 1980), it had not been

38

Svm ::>torn~/ -··· ...•..•.. - ·- --· .... -· -- ..... -· ·- -· ................ r .... ... L ..... ,.,,. ..... "·'· .......... ..... ·-· __ ..... .... .. ....... .... __ .................. ..

F: X PE' t" .i.. mtJn t.<:t 1 b

LDso ( JJ <:;J) Dia;· ;-hE')a nufflc~d fur .. Conjunctival

___ 9LQYR-~---------------------------------- -------~isgb§L9~--Non-tolerized 414±55 9/1,::. 1 :')/ 1r:.

Toleri zed 994±92 0/16 Cl / 16 0/ 1(,

a Mice were i njected i.p. on day 0 with sal ine

(non-tolerized) or E .. CQli K235 LPS (25 JJ g/mouse; tolerized) ..

Three days later, the m1ce were challenged with E .. fQli LPS for

LDso determination and assessment of symptoms as desc r ibed in

th e Mate rials and Methods section ..

b .. , .. ~- r::.. I f.\ 5 0 ") ·:: ..... ~ (": ''[ ., r: I ll '0l ·t·. p (~ I") '.1 ·t-.1-1 F-• rn p '[".1"1 n ;j cTf·' ) I '··· ·- .~ V" c..l .. ~ ~ <.. • .. _ .,, <.. • ..• ... I ~. 1 . ~· - . - .. - Hc-::~r:.-) d and Muench

(1939). Results represent the arithmetic mean ± one s tandard

deviation of two separate LD 50 experiments ..

c The propor t ion of mice which clea r ly exhibited the indicated

symptom 6 h after the injection of 25 JJ9 LPS on day ~-

·rab.l~~ II

QQ_Q~E_indY~~d-~Y-~YbSQ9Y9Ql_~b~ll909~

Wi!b_bgmglQ9QYS-~Q0_bQt9CQlQ9QYS_lP$_er9B0L0!i2Q§ a

T t- r:? a t rn(-:-' n t

Sal i.ne :5 ., oo:s ± /~50 :s, 050 ± :-=.;(,J.

190 ± ?Cl 0

--~P$ __________ ______________ ____________ __ ___________________ _

a G~aups of 5 ICR mice we~e inj ected i.p. with eithe r E. ~Qli

LPS (25 ~g/mouse) or saline on day 0 and aga in on day 3 with

eith er homologous E- ~gli LPS o~ a he t e r ologous LPS p r eparation

(25 pg/mouse) a s ind i cated. Si x hours after the day 3

injection, t h e mice were bl e d and se ra assayed for CSF . The

res ults represent the arithmetic me an ± o ne s tandard deviation

of duplicate determinations.

39

demonst~ated previously that the polysaccharide moiety was

inactive as a tole~ogen. This issue was particularly ~elevant

~;i nc(~ ''put'" i. f i ~ =~ cl '' poly •.::;ac chat· idE) d1::~t- i ved f r· om 1 .... PS u~:; ing th(:>

procedu~e of Freeman (1942) had been used to induce certain

LPS-mediated responses, including the induction of CSF

(Urbaschek Q1 ?l-, 1980). In this study, the polysaccha~ide

was prepared by mild acid hydrolysis of the phenol -water

extracted ~- ~9li K235 LPS and was separated from residual LPS

by gel chromatography, as described in the Materials and

Methods sec tion . The concentration of polysaccharide was

adjusted by total glucose content to be equivalent to the

glucose content of 25 p9 ~- ~9li K235 LPS (70 nmoles). When

administered to mi ce on da y 0, it was found that t he

polysaccharide moiety was non-toxic, unable to induce se rum

CSF, and failed to induce tolerance to LPS as assessed by serum

CSF or symptoms of endotoxicity (Table III).

~AB~Y-~~QQIQ~I~_IQ~~BA~Q~-l~-A~5QQl~I~Q_WIIH_A~I~BAIIQ~$_lN

~Q~E-0ABBQ~=PEBI~~R-MAQBQPHAg~_EBEGWB~QB_tQQL~

Effg~t_gf_Q?tl~_Qndgtgxin_tglg~gn~Q_QQ __ tbg_gli~ilgtigo

gf_PQLitQDQ0l_QXY0?tQ_ffi0~LQPb09Q§ Previous studies have

indicated that macrophages play a central role in endotoxic

reactions through the production of soluble immunoregulatory

mediators suc h as Interleukin 1 and CSF (reviewed in

Rosenstreich and Vogel, 1980; Vogel and Mergenhagen, 1982).

Since it had been shown that: (1) relative LPS sensitivity

40

41

Table' III

Pi~Y.. .. . !J.

::;aJ i. nE~ Sal irH:~ CJ

3al.inH LF'S :·:.; ' 490 ± .J.•;".Jo;:;

.L.Y~3 LP!3 600 ± .1.:=.::7

!:>a l .in1:~ p::; Cl

p c~ ,,., p~~~ Cl

r' '-~ ~ ~~> LP!:> l.~ ' .100 ± ::~:·:;:) .1.

with either saline, E. ggli LPS (25 ~g/mouse), or

polysaccharide (PS) derived from E: ~Qli LPS. LPS and PS were

equilibrated by total glucose content as described in the

Materials and Methods section.

b,.,. J I ~1x hours after the day 3 injection, the mice were b.ed anc

the sera assayed for CSF activity. The results repr e sent the

a~ .. i thme t. i c mean ± on(:·~ ~.;tan da r·d dPv ia t. ion of dup.l .i.e <:t t.c:~

correlated with the differentiation s tate of the macrophage

(Suter C't a.I . .. , lo:"'~:;::::. BF'n<'tc:(:."t"rnf F:•t ;::t] , l.:"'S9· i"lnnrf''·' e>t <::t 1, --· ..... -... ,_, . . - ·- ';" - -· - .. ... .. _";, .. .:. .:: .. :.::. .. . . . .. . :• - ... ... _-:;, ,_: ..:: .. :..::. .. '}

1980; Rosenstreich and Vogel, 1980; Vogel §i 01., 1980) and (2)

macrophages from endotoxin-tolerized mice were refractory t o

LPS stimulation iD ~itLQ (Dinarello Q1 ~1., 1968; Rietschel et

~l-, 1980), we had originally planned to examine the

diffe r entiation state of peritoneal exudate macrophages from

endotoxin-tolerized mice. When thioglycollate was admini s tered

to mice between the initial and challenge LPS injec tion s

(Williams ~t 01., 1983), the peritoneal exudate cells recover e d

from mice which had been injected twice with LPS . were

strikingly reduced in the proportion of macrophages which were

elicited in control mice (Table IV).

It has been established that mononuclear phagocytes originate

from precursors in the bone marrow (Volkman and Gowans , 1965).

Therefore, the reduction in peritoneal exudate macrophages in

mice injected twice with LPS s uggested that such mice migh t be

'J I I :1 . b .. OC .(aC:·?~ C ln macrophage development. A blockade in development

could result i n an accumulati on of immature pr e cur s ors in the

bone marrow. To test thi s , an 8 day time cour se was carr i ed

out. Groups of mice were inJected with either saline or ~-

ggli LPS on day 0, and, on suc cess ive days, the pooled bone

c.:ells from the femur s of each group were e numerated and lll<::l t" t" OI.AJ

plated i n an excess of CSF- 1. CSF-1 has been s hown to be

42

43

:3d1. ine con t~·· o.I. :::t.~ 10 1

( ::;<:t 1 in(·3,/::>a.l. intO?)

7(. 1 1

( 3a 1 ine/I...P3)

1. .1

( LP:3 j L. p ;:; )

a . M1c e were treated on day CJ a nd on day 3 as i ndi ca ted by the

o r der o f treatment s i n parentheses. Thioglycollate was

admi niste r ed on day 1 and peritoneal exudate cells c ol lec ted by

lavage on day 3 , 3 h after th e sec ond i nj ection as described by

~·J .ill i ams ~;~t ·~1 - ( 1 '):3::.;) -

b Cy t ospi n preparations wer e f ixed in methanol and were stained

using a mod ified Wr ight 's stain. Differenti al counts were

performe d on 200 cells per slide under o i l i mme r sion. Results

represen t t he arithmetic mean of differential counts de r ived

fro m t wo separate ex periments_

44

specific for cells of macrophage lineage and to give rise

exclusively to macrophage colonies in culture (Stanley and

Guilbert, 1980). Those precursors which respond to CSF- 1 to

form colonies are referred to as macrophage colony forming

un i L:; ( t-1--CFU) .

A single injection of LPS on day 0 resulted in an

initial decrease in the total number of nucleated cells per

femur on days 1 and 2 post- injection, followed by a return to

normal levels over the next 3 days (Figure 5A). Figure 5B

s hows that the number of macrophage progenitors (M- CFU),

adjusted for the tcital nucleated cell count fro m eac h g r oup,

was greatly increased by the third day after LPS injection and

returned to normal by day 8 . Injection of sali ne on day 0

produced no signif icant change in the number of M-CFU per femur

injected i.p. with 3 ml/mouse 0f the sterile inflammatory

I . J l'J t .. , J.og _yeo .. at~-::~, and the bone marrow cells cultured for

M-CFU on day 3, showe~ no such increase in the number of

macrophage progenitors when compared to saiine-injec t e d mice

( s aline- injected = 2.1 x 10 4

± 3 .9 x 10 3 M-CFU/femur vs.

thioq.lyco.llat.t::~·-injc::'ct.c::'d ::: 2.7 x 10 4 ± t.:,. _::;:: x 1.03 1"1 .. ·-CF'U/fc•JnUt").

A more primitive population of macrophage progenitor s

with a higher proliferative potential (HPP) than M-CF~,

responds to CSF-1 only in the presence of a s ynerg is tic

co .. ·-factot .. , t"C!f(·:.,•rt"f:)d to as '' ~3ync·~rqistic Activity'' (Bt"<:tdlc)y and

Hodgson, 1.975). The in s e rt in Figure 5B shows that, like the

more mature progenitor population (M- CFU), t he number of HPP

45

46

m0LLQ~ -- ICR mice ( 5 mi ce/group ) were in jecte d i.p . on da y 0

ma rrow cells from each g roup were collected and pooled at th e

indicated t imes t he r eafter .. Nucleated ce ll counts for each

bone marr ow cel l s us pe nsion wer e obtained us ing a Coulter

Counter (Figure SA) . f.... v 'l(''l4 l . i" -1ve A . _ ce .. l s · rom C·'.'!dC h c c::.' l.l

s us pen s i on we r e th e n pl a ted i n duplicate i n an excess of CSF- 1

a nd the:::· n u rn b c? t" of t·1 ·- CF~u dc:! t E~t- m:i.n ed (F:·i q:.t r E! SB) a~::; dc:!sct· .i.bc~d 1 n

t h C·' t1 a t f} t" :i. a 1. s a.·. rh.-1 t'l H ·t-.. 1"1 c .. l f .. · I:·.'···; :_.:; c=~ r_·: ·t .. ·.:. c.·.l r·1 . c~ .; rTll.l ·1 ·t .. . ,. 1.. ·~ -, 1 l, .... ·1 11 ··- \/ ·1 rJ4 _ _ ·-' -'· ... . .. <.t It:. C.· ... :.> ... , , •. ) -" .. -

cells wer e plated in a n excess of CSF-1 plus human sp l een

conditioned med i um (as a source o f s ynergi s tic ac t iv ity) f o r

determ ination of t he number of HP P colon ies (Figur e SB,

ol.n ~:.::c·t · t) .

"' ::> :If ... ... ' ..

I

2

Cll ,.j

-'

"' ..., " ... .. .. "' ,.j

u ::> z

!5 :E

"' ...

A

3

2

,_,

. ..___, ___ ... ------· 0-----o-o~"-~

/ 0

·~/ •

~LPS ON DAY 0

SALINE

ON DAY 0

D~--~----~----~----~----~----~----T-----~----------0 2 3

B

7

e

5

5 e 7 •

/\ / . ~"' \ ""--- ...... "X-=><.~· o• on o

- ~.":·

SALINE

ON DAY 0

!:..s.2!! LPS ON DAY 0

OL---~----~---,----,----,----,---~----,----------0 2 3 5 e 7 . 8

DAYS AFTER INJECTION

47

48

precursors was also increased by day 3 after injecti on of LPS

and the kinetics of their appearance (and disappearan ce) was

s1m1lar to the observed alterations in M-CFU numbers.

The number of M-CFU in spleens from tolerized mi ce was

also greatly increased. In the spleens of mice inj e cted 3 day s

previous ly with ~-~9li LPS, the number of M-CFU was

approximately 14-fold greater than in control mi c e

t ... ... ·1 .l· - - l. " . . - t· I 'I v 1 ···· 3 tvt I"'I"" LJ ' ·1 .. . . ' , ~,; d . . .. 1 1 €:!·-- .. 11 J ec · .. oc ., ,:.. /\ u ,., ....... , ... 1 s p . c;~E~n ; l... P ~::> .. ·· J. n J (·?! C t .E:-!CI .,

~ioglp_ioi9~tiQO_Qf_Lf~ - - Cell sizing profiles of the va ri ous

bone marrow cell s uspension s wer e obtained us i ng a Coulter

Chan nely zer. Wh e n control bone marrow cells (day 3,

post-saline) wer e a na l yzed, a bimodal dis tr i bution of cells was

observed (Figure 6) _ Control Peak 1 accounted for 35 - 40% of

the total cells analy zed and consi s ted of smaller c e lls

(average diameter= 7.5~) relative to those in Peak 2 (average

diameter - 10~) which accounted for the r e ma i ning 55- GD% of

cells. In contrast, a ma rked reduction in the relati ve number

of bone marrow cell s in Peak 1 (20% of total) occurred in

toler iz ed mice (day 3, post- LPS ) a nd t here was a compen s atory

increase in. the number of cells within Pea k 2 (8 0%). In

addition, the Peak 2 cell s from tol e ri zed mic e were l a r ger than

th o:':-:;(:;• .in th G Cc)n t t" o .1 F'r::::·a 1-<. ~_:~ (aver-a qE-' d .i. d fll(':·' t e t" === 1.1 .. /1. ~ ; <':1 ~:o;

indicated by the shift to t he righ t on the si z ing profil e ) .

.... . .. ., . ' : - .. ... .. -· ... , ..... ·t· ·t· •.. .. ... .., ,.. .' , ... : ·t· .' .. .. ·:: .. ,.-( '1 ""'·" <:' yf:: '[" \" .'l <::' " · ::.) '] '[ 1·- .1CJU.t" C· I .11"1 Cl..l. t.: d L.t:~ '-'·' t.. I d . . I I(:} d C. ·-IU ..t . . C.' .L .. 1 Ul l < .. lll "•" . . (_, ,_,, ,_,, L .· I " .,_, !,. L .. ...

49

Figu~e 6. Cff~ct_gf_~_lulgrizios_ioJgctiuo_uf_L2~-uo_bQD@

m~rrgw_~§ll_siziog_Qrufilg~ ICR mice (5 mice/group) were

injected i.p. on day 0 with either saline or ~- culi LPS

(25 ~gjmouse) . At 3 days post-injection, the mice were

re-i njected with saline or C- culi LPS (25 pg/mouse) and

sacrificed immediately. Bone tnarrow cells we~e then pooled

from eac h group and cell sizing profiles obtained us1 ng a

Coulter Channelyzer.

50

100 CELL SIZING PROFILE r ,

en 80 _, _, w 0 ~ 0 en 80 a: w m ::E ;:) z 40 w > .... cC _, w 20 a:

20 40 60 80 100

RELATIVE SIZE OF CELLS

51

Figure 7. Q~ll_§iZiD9_Et9filg§_Qf_~QQ~_illDLtQW_£~ll$_9bl~io~d

dt_~~riQYS_!iillD§_Dfl?t_Lf~_iojg~tigo -- ICR mi ce (5

mice/g r oup) were injected i.p. with ~- CQli LPS (25 ~ g/ mouse)

a nd sacrificed at t h e indicated times after injection. Cell

sizi ng profiles of bone marrow cells pooled from eac h group

were obtained us i ng a Coulter Chann elvze r. The arrow i ndicates

the initial position of Peak 2.

52

100 DAY 0

10

10

40

20

100 DAY 1

80

80 Ill ... ... Ill 40 (,)

u. 0 20 a:: Ill Ill :I = z Ill 100 DAY 2 > ~

"' 80 ... Ill a:

10

40

20

100 DAY 3

10

ao

40

zo

zo 40 eo 80 tOO 20 40 60 80 100

RELATIVE SIZE OF CELLS

s1z1ng p~ofile correlated t e mporally with t he acqui sition,

maintenance, and loss of tolerance (see Figu~e 1).

!1 ~~~ n ~:;; ;L l z .... 9. r. <'~ ~1 }, ~,~, o. t ... ~::~ s~ P. ~~~ L ~~-~ :t; J. u n. ··- 9. L .... m ~1 1J ;;;; ~~ .... !;;~ 9 u ~ :::~ .... m <;;\ r. r. q 1:'-J ... (. u l .J. ~~;

To deter mine whether a re la t ionsh i p existed between the

cell sizing profile in toleri zed mice (day 3, post-LPS) and the

increased number of M-CFU also seen at this time, Peak 1 was

separated f rom Peak 2 by density gradient sedi mentation a nd th e

numbe r s of M-CFU 1n individual fractions de t er min e d (Figu r e 8) ..

Al l of the M- CFU activity was r es tr icted t o th e den se

fractio ns (i.e., Pe ak 2) in both saline- a nd LPS-injected m1c e .

Based on two separate experiments, th e to t al numbe r of M-CFU

(obtained by addi ng t he M-CFU values de t er mi ned for individual

densi ty gradient fractions) fro m tolerized mice was >2.5 t imes

g r eate r than obtained fro m contro l mi ce . To verify th e

r elative posi tions of Peak 1 an d Peak 2 after density gr adient

sedime nta ti on . pooled fraction s we r e r e-analyzed by Coulter

Channelyzer (Figu re 9). Thus, the results o f Figur es 8 and 9

indicate that al l ot t he M- CFU act ivity was found in Peak 2,

and that th e i ncrease observed in unfrac tionated, tole r ized

bone ma r row cells was r ef l ected in t he separate d fra c t ions. I n

addition, the distribution of HPP precursor s wa s also measu r e d

in individual density gradi e n t f r ac tions a nd al so correlated

positionally with Peak 2 as we l l as wi th M-CFU activity (Figu r e

when diffe r en tia l counts of cytospin preparations of

t he pooled, density g r adient-separated Peak 2 cells were

53

54

00ct_UEE_gctiYilY -- ICR mic e (5 mice/group) were injected i.p

day 3, bone marrow cells were collected and 1 X 107 cells from

each group were separated by dens ity gradient sedimenta tion as

described in the Material s and Methods section. Fractions (3

ml/fraction) wer e collected from th e bottom of the gradient

after 3 .5 h. Cells from each fraction were enume rated and th en

!. (

.l I . cu .... turuc 1n the presence of CSF-1 or CSF-1 plus human spleen

conditioned me dium ( as a sourc e of sy nergi s tic activity) to

determine the numbe r s of M- CFU and HPP, res pective ly. The

first fractions collected had to be pooled i n order to obtain

adequate numbers of cells for culture . M-CFU and HPP

determinations were carried out in duplicate as des cribed in

the Materials a nd Methods section.

i 0

., ... ... Ill u

Ill 0

0

SALINE ON DAY 0

1 3 12

~LPS ON DAY o

12 11 10 • FRACTION NUMBER

7 1•4 POOL

.. c

55

56

Fi gure 9 . Qgll_siziou_ergfil£s_gf_eggJgd_~gosit~

gr~0i?D1=SQ2~r~tgd_bgog_m~rrgw_cgl ls -- Cells from eac h

f raction (se e Figur e 8 ) were sized usi ng a Coulter Chann elv ze r

an d then pooled according to profile similarities with

un s eparated Pe ak 1 vs. Peak 2 . Cell s izi ng profiles were then

ob t <:t :i. n Qd on th (~ pool c:·~d f t .. action s . Th E~ a r· t .. ct,•J :i.nd .i .. r:: <:t t t:0::·:.; the

pos ition o f c on tro l Peak 2 . Cell s f rom the pooled fractio ns

were e nume rated a nd c ultured i n th e pres e nce of CSF-1 a nd th e

nt .trnbe t .. of !·l ····CFU dc•tc::,t·· rn in e d i n dup .l icato a ~::: de~':.; ct" ibr~d l.n the'

Material s and Methods s ection .

(/) .... .... w t.)

II. 0 a: w = :E :::::1 z

100

80

150

40

20

~ 100 j: < iii 80 a:

80

40

20

20

Saline on Day 0 Peak 1 Pooled traction• 11-14

15 M-CFU I 10 5 cella

E. coli LPS on Day 0 PUk1 Pooled fraction• 10-14

35 M-CFU I 10 5 cella

40 60 80 100 20

RELATIVE SIZE OF CELLS

40

Saline on Day 0 Peak 2 Pooled fractions 1-6

235 M-CFU I

10 5 cella

60

E. coli LPS Oii"'Day 0

Peak 2

Pooled fraction• 1-7

80 100

57

performed (Table V), cells derived from tolerized mi ce were

enriched for the number of large mononuclear cell types. Thes e

latter cells contained a lat·ge nucleus (nuclear:cytoplas mic

ratio >1) and the cytoplasm was strongly basoph ilic, suggesting

the monoblast-promonocyte stage of macrophage development

(Sumner Ql gl., 1972; Gaud Ql ~l., 1975).

QbQQ9Q~_in_thQ_QYffibQc_gf_~=GEW_QflQL_Q_~QGQQd_iuJgcti9D

gf_LP~ -- Based on the observation that a si ngle injection at

~- ggli LPS resulted in profound changes i n the numbers of bone

marrow-derived macrophage progenitors which correlated

temporally with maximum tolerance i nduction, the effect of a

second (challenge) injection of LPS on M-CFU levels was also

investigated. Groups of mice were injected with eithe~ saline

or LPS on day 0 and again on day 3. After th e second

injectiori, bone marrow cells from each group were collec ted at

0, 3, and 24 h post-injection . Figure 10 demonstrates that 1n

mice which received LPS o n day 3 for t he first time, a marked

decrease was observ~d in both the number of nucleated cells

(data not shown; refe r to Figure SA) a nd M-CFU within 24 h

post-LPS (refer back to Figure 5B). In mice which received LPS

for a second time on day 3, only a slight decrease in the

number of nucleated cells was 6bserved (from 9.8 to 7.5 x 10

cells/temur at 24 h); however, the already elevated level of

M-CFU/femur was maintained ( Figure 10).

6

58

QiffQrQoti~l-~gynt~_gf_~ztg~eio_~rge~rQtign~_Qf_d~a~itY

P r ;·j d ·L· P r1 t ··· ·::· E• n .,1 t· .,t ·t 1=·· <J· 1 ::~ , ... , ·= )1. ':> (... "' ·1 ·1 .... a ~-~-~£-~---d£~~-~~~---~~~-~-~~~~~

Polymorphonuclear cells

Small mo nonuclear cells

(10- 15 ~ diamete r)

lar ge mon on uclea r cells

(15-21 ~ diameter)

Q9nt t~.Ql

b L~::: .. !5%

~)() ,, ::;/~

.1 - o:~

a Pea k 2 bone marrow cel l s were i solated a nd pooled b y densi ty

g rad i e nt sedi me ntat ion on day 3 after the injection o f saline

(control) or 25 ~ g/ mouse ~- ~gli LPS (tole ri zed) on day 0.

bCytospin preparations of pooled Peak 2 cells were fi xed in

me thanol and stain e d usi ng a modified Wri ght 's stai n .

Differential counts ( 200 cell s per sli de) wer e obta i ned unde r

o i 1. i mm F' t" ~::;ion _

59

60

mice/group) were injected i_p_ on day 0 with eithe r saline or

jl oj mou '=;(:., ). .• :J .• ···- - .. On day :3, th e mi ce injected with

sali ne on day 0 were injected again with either saline

(control) or LPS (non-tolerized) and those injected initially

with LPS were re-injected with LPS (tolerized). At 0, 3 and 24

h after the day J i n jection , the mi ce were sac r i fic ed and the

number of M-CFU determined i n dupli cate as described in

Mate rial s a nd Methods sec ti on_

a: 4 ::» ~ w LL. .......

~ 3 I 0 ,.. )(

::» 2 LL. (.) I

~

1

~ 0 hr post Day 3 injection

D 3 hr post Day 3 injection

IIIIIil 24 hr post Day 3 injection

SALINE CONTROL NON-TOLERJZEO

(Sal/Sal) (Sai/LPS}

TOLERIZED

(LPS/LPS}

61

62

early endotoxi n tolerance was found to be optimal 3 day s after

initial exposure to LPS and the increased number of bone marrow

M-CFU, also observed on day 3, was maintained far at least 2 4 h

following a second injection of LPS (Figure iO), the effect of

multiple LPS injections was investigated next. Groups of mi ce

were injected with either sali ne or LPS one time (on day 0

only), twice (on days 0 and 3), or three t i mes (on days 0, 3,

and 6) .. To assess the effect of repeated injections of LP S on

to lerance, mice were challenged with 25 p 9 ~- CQli K235 LPS 3

post-challenge for meas ur e fu e n t of CSF activity_ The r esults 1n

Figure llA indicate that , with respect to ser um CSF, e ndotoxi n

tolerance was maintain ed at the same leve l , regardless of

I,..; he:.! the r· t h c• rn .i Cl~! t'l·::•ce i ved one, t~..;o, or· th t"eC:! '' to lr:'! r· .i z i. nq ''

in j l::::!c: t.i on-::.-:;_ Control mice i nj ected with saline one, two, or

three times responded normally to a si ngl e challenge i n jection

of LPS to produce high levels of se rum CSF ..

In order to assess the effect of repeated LPS

injections on the number of bone marrow M- CFU, mice injected

with either saline or LPS were sac r ificed 3 days af t e r their

.l<Jst:. ''t.o.lc·r·iz.i.nq'' .injE!ct:.ion and th c!i r bone rn<:tr·roi;.J cel .1.s

cultured in an excess of CSF-1 .. Figure llB demons trates th at

rn .:i.co wh:i.ch r·c'CE!iVPd onE·~ prr.:,v:.i.ous :i.n.:i!:'!Ction of I.F' ~::; r · e~-:;pondecl

with a dram<:ttic i ncrease in M-CFU 3 day s later (compa r e with

F i CJI..tr··e !:::O B) - However, with repeated injections of LPS, the

I of M-CFU was diminished so that after 3 injections, nu.rn ~)er

M-CFU numbers were equival e nt to sa .line-injecte d con tr ol s .

63

Figure llA. ~ffg£1_Qf_rQBQ§1Qd_ioig~tiQO§_Qf_~~-fQli_lf5_QQ

1bg_}gygl_gf_§QLYm_Q§f_fg}}g~iD9_lE§_fb§llQD9Q -- IC R mice (5

mice/group) were injected once, twice, or three times at t hr ee

day i ntervals with sal1ne or ~- ~gli LPS ( 25 pg/mouse) and were

challenged with 25 ~g C- ~gli LPS 3 days after the las t

in.jection. Six hours late r, the mice were bled, th e sera from

each group pooled and assayed for CSF activ ity. rhe data

represent the arithmetic mean ± one s tandard dev iation of

duplicate determinations .

II. (/J (J

:::& :::1 a: 11.1 (/J

2

No prevloua InJection• Injected on day 0

LPS on dey 3

LPS only

aallne only

I 0

D Prevloue Injection• wltll ullne

~ Pravloue Injection• wltll LPS

Injected on day 0,.3

LPS on day 8

2

Injected on day 0,3,8

LPS on dey g

3

NUMBER OF PREVIOUS INJECTIONS

64

65

Figure llB. ~ff~~t_gf_rgR~D1~d_iDJQC1iQO§_gf_E~-~9li_kf~_QQ

1b~_OYill~Qt_Qf~bQOQ_illDLtQW=dgri~~d-~=QEV -- ICR mice (5

mic e /gr6up) were injected once, twice, or three times a t th ree

day intervals wi th saline or f cgli LPS (25 u9/mouse). Mice

we re sacrificed 3 day s after t heir las t injection and bone

marrow c ell s from each group pooled, enumerated, a nd t h e number

of M-CFU de~ermined as described in the Ma terial s and Methods

sec tion . The data ~epresent th e arithmetic mean ± one star)dard

dev iation of duplica te determ i nation s .

66

7

D Previous Injections with saline

6

~ Previous Injections with LPS

5

a: ;:::)

:::E w II. 4 ' ~

I

0 .... )( 3

:I u. (J I ~ 2

0 1 2 3

NUMBER OF PREVIOUS INJECTIONS

67

Cell sizi ng profiles o f the various bone marrow

suspensions were also compared using a Cou l t e r Chann elv ze r .. As

can be seen in Figure llC, one injection o f LPS induced a

marked reduct ion in th e relative numbe r of cel l s in Peak 1

(saline- inj ected - 40% of to tal ce lls; LPS-i n jected = 20% of

to tal cells), and, a compensa t ory increas e in the numbe r of

cells within Peak 2 (saline- injec t ed = 60% of to t al cells:

LPS-i nj ected = 80% of total cells). lh 1s was accompanied by a n

i ncrease in s i ze in Pea k 2. Repeated i nj ections of LPS

Peak 2 (see Figure 6) .

earlv phase endotoxin tolerance is not O- antigen s pecific

(Grei s man and Hor n i.ck, 1976 ; s ee Tab le II), the previous s e r ies

of expe riment s was modified s uch that groups of mice were

injec t ed once, twice, or three t i mes with sa line or ~~ -

QQCU9iOQS~ LPS (25 pgjmouse). Three days after t he las t

· · t' Jr c.·_:Jr ouns o f mi ce we re ei ther challenged with ~- £9li lnJC~c · · .. l.L I , .-

K235 LPS (fo r serum CSF) or sacrificed and t hei r bone marrow

· · ·1 - · .. 1 ·f· - - ... 'L ·t--· · - ·t· . .. · -- : .. , ..... -· .. .. - · .. -. ·f'' ·1· ·1 .. ,, .... C C:.:• J J ~c;.; a n <::t .. . '/ l. 1:·::• <.. U t d .. .. U t' d .1 U f I~::' .. l I I "'·' .l L .lit \:,) j-1 t C.o . . .. L . . ::·· •

illustra t es that hete ro logous tolerance for [. C9l i LPS was

induced by mu l tipl e injections of Ps- QQ[Y9iOQS0 LPS , but on

th e basis of percent s uppression of the con tr o l response,

I I l 'l f .. f"' . .- .. 1 .. I ., ' 1 .. 1. '}· .. l .. ,' .. , c ... .-.·'. '. ~· .. c·.·.-,1-tc··.·.·' . . , 1 .. >

1 .. , ,:.:, ·:1 r· c·?. d t o ) c::..~ ~:-:;om c:;,. ~.-<J •. , a · ·:. . . c· s ~:::; c:: .. ~ · .. · · .1 c a c 1 o u ~-::; \. a :' ·'· !·'::' ·., ... , .. ..

' ~ l .. ·· ' .... f ,,

rn ar t"O\•J p t"ofi.t c~s in F.iqut"E' 1 ~-;~n .i nd .i..catF! t h <::tt :: (1) t h e~ ' ' s h if t'' in

68

Figure llC. ~ffg~!_gf_LQPR~!Qd_ioJ~~tiQDS_Qf_E~_£Qli_Lf~_QQ

~QD~_ill4CLQ~-~gll_§i~iog_ergfi1g~ -- ICR mice (5 mice/group)

were repeatedly injected once, twice, or three times at 3 day

intervals with saline or~ - ~Qli LPS (25 ~g/mouse). Three days

followi ng the last i n jection, the mice were sac rif iced, bone

marrow cells obtained, and cell s izing profiles dete rmin e d

using a Coulter Chan nelyzer. The arrow ind ica t es th e relative

posi tion of Peak 2 _ bone marr ow ce l l s fr om control I CR mic e .

100

80

80

40

20

0 8

..J.

..J. Ill (J

~ 0 a: Ill a:l ::E ;:, z Ill > j: < ..J. Ill a:

Number of previous Injection• with .!:...!2!! L P S :

0

20 40 60 80 100

RELATIVE SIZE OF CELLS

69

70

three day intervals and were c hal l e nged with f- ~Q1l LPS

(25 ~g/mouse) 3 days after the las t i n jection. Six hours

' later, the mic e were bled and the se ra assayed for CSF

activity . Each point represents the arithmet ic mean ± on e

standard deviation of duplicate determinations.

Prsvloua Injection• with :

D Saline

~ Paeudornonaa aeruglnoea LPS

No previous InJections InJected on daJ o InJected on day 0,3 lnJaotad on daJ 0,3 , 8

LPS only .!.:.....!!!! LPS on day 3 ~ LPS onday8 ~ LPSon day 9

aallne only

" 0 2

NUMBER OF PREVIOUS INJECTIONS

3

71

1'-ln n f.. I F'c' In ·j E'C' t j n n ,-·· P r· i C) r· ·t· rJ· c·,·-· ·= ·1 I n , .. c1 .. , -~~-~--=-~----~~~~~-2---~~--~--~~~~~~~~t

Homologous tol e ranc e system

Heterologous tolerance

s yst(·.:.'m

c :~:(]

~.i. .tl.~! ..... E .. ,_(:Q1.1 .... LJ:!~)

60 (, [)

12A (homo logous e ndotoxin toleranc e ) a nd 13A (hetero logous

endotoxin tolerance) .

b Mice were injected once, twic e, o r three ti mes at 3 day

i n te~vals wit h saline, ~- ~gli LPS, or P~ - QQ[YBiQQ~Q LPS.

Thr ee days afte r the last injection , all mice we r e c ha llenged

with F ~glJ LPS.

c The data represent t he percent suppr ess ion of the cont rol CSF

72

73

~ espon se ( sa l i n e - inj ec t e d 1 to 3 times f o l lowe d by c ha ll e nge

wi th E - ~gli LPS).

74

Figure 128. ~ff§~1_gf_r§eQ0lRd_ioig£liQO§_gf_2$Q~dQIDQ00$

0QL~9iGQ§Q_kP~_QO_bQQQ_ffiQCLQ~-~gll_§i?iG9_ecgfilQ$ - - ICR mice

(5 mice/group) were injected with saline or P$- ~~LY9iOQ~Q LPS

once, twice, or three times at three day 1ntervals. Three day s

following the last injection, the mice were sacrificed, the

bone marrow cells obtained, and cell sizing profiles determined

using a Coulter Channelyzer. The arrow indicates the relat i ve

position of Peak 2 bone marrow cells from control

( s aline-injected) ICR mice_

75

Number of previous

Injections with 100 Ps. aeruglnosa LPS:

80 0

80

40

20

fiJ ..J ..J 10 w (.J

LL. 80 0 a: w 80 a:a ::& :;:)

40 z w 2: 20 1-cc ..J w a:

100

80

60

40

20

20 40 60 80 100

RELATIVE SIZE OF CELLS

Peak 2 seen three days afte r one injection of E- f9li LPS was

also s een after a single injection of P~- 0~CY9iQQ50 LPS, a nd

(2) the reduct .L·o,-, ·L· ,- ·f· ~~ . . I .. Ill"'. of cr!.l.ls :tn

observed after repeated injections of r f91i LPS was al so

(compare Figure 12B with Figure llC).

In order to gain a better understanding o f the cell ular

mechan isms which underlie early phase endotoxin tolerance,

several additional murin e models were studied with respect to

se rum CSF induction, bone marrow M-CFU, or bone marrow cell

sizing profiles after prior exposure to LPS.

diffgr_io_tbQir_inbQri!Qd_5QO~i1i~il~_lQ_kP~ - - C3H/He J mi ce

bE~at· a sing le <:tuto~-:; omal mutation, toE::~~ d (I,Jat.son t~1 ~:;,_1. ., 1')?:3) .,

whi c h renders thi s s train high ly refracto ry to the e ffe c ts of

LPS both io ~iyg and io ~i1rg (reviewed by Morri s on and Ryan,

19? 9). Use of this naturally LPS-hypores ponsive strain in

conjunction with syngeneic, fully LPS-responsive strain s

(~p~ n), has provided evidence that macrophages figure centrally

i n the i nduction of LPS- induced pathophysiologic changes

assoc iated with endotoxi c it.y (r e viewed by Rosenstr eich and

Vogel, 1980) . We next sought to dete rmine if any of these

76

~~~ I.

changes wer e present in t he LPS-hypores ponsive C3H/HeJ strain.

c:·'S H/HG.J ( L p s ··-h vi:> 0 t"" p c; F~> 0 n :·:~ 1 v p )' "'lf""J cl ·-=; \1 , .. , CJ F·' , .. , f ·' .,· r: . I 1::> <:: ~ ..... t" F' C" I'") n , .. -~: · 'j. ·.; G' I .• ... . ·-. . - c.: .• ··- I ... ... . ..... - • .... ._} ... ··-' ..... I ··-' . . . ..

C3H/OuJ mice we re sub j ected to t he tolerizing r egimen wh ich had

been established for ICR mice. Each mouse st ra in r eceived

e i t .h c::! t" :·::;a l1' r1 c• ( t J ' E- J · I<,-,-· ,- 1 F' ... , ( ,_, .. ' ' · ~ con :ro.J or~- ~Q=l ,~~ --~ -'~ p9;mouseJ 1-P-

on day 0. On day 3, mice were challenged with C- ~Qli LPS

( ') o:.-, 1 I CJ/ fn(")J [ "- :l ') -~·- \_. .... .:: " ·- ~-=' (:: ' b l c~~ d 6 h .l a tc' r· , and sera assayed fo r CSF

activity. The res ult s in Table VII s how that C3H/OuJ mic e

r esponded to LPS-challenge similarly to outbred ICR mice, i.e.,

a dramatic increase in ser um CSF from control

(saline-pretreated ) mi ce and a mitigate d CSF r esponse in

tolerized mice (i .e., those pretreated with LPS) .. In contrast,

C3H/HeJ mice did not res pond with de tectable levels of se rum

CSF after ei ther treatment.

Bon e marrow cell sizing prof i l es of LPS-responsive

(( .. ,.,,.1 1/ .-l "f ' ·· • . .j L l·, c- ..... ,. v · ·· -,.- ) .. - .... . ,, -, (t""""'' l'/1·1 .. , T' 1· ... .. , ., ... , ..... rill"" ., J·'r> "·l ,., ,,:,· I .. U •. ) <:til!.~ .. • •. .> I,POtC·: .. .. :.•r-UJI:.,J.,(·: .... , .. )"1 ·(·:; . •. ) rn ... C...C·: .. ~'J!·.:·. t f: C...U .. I<J ::.1 .. :-:;

days af t e r injection of saline o r LPS ( Fi gure 13 ) . Cell

profiles from LPS- res pon s ive C3H/OuJ mice, i nj ec ted on day 0

with 25 ~g LPS, s howe d a cel l sizi ng p rof i le simi lar to tha t

seen in LPS-injected, outbred ICR mice, i. e., a dramati c

i ncr·ease i n the average s i ze and numbe r of cells in Peak 2 . In

contrast, the cell sizi ng prof i l e from LPS-hyporesponsi ve

C3H/HeJ mice was not altered. One mg LPS per mouse wa s

requ ired to cause any significant alte rat i on in the cell

profile.. Speci fical l y , i n r esponse to sali ne, 25 p 9 o r 100 ug

LPS, Peak 1 compri sed approximately 4 0% of the total cel l

1 t . -~- 1·1, r es1~ on se to 1 m(_:.J LPS, Peak 1 comprised 33% o f popu. ;:, ··.lu ' -

77

~ .~

:·:·~ .I

~ \ .. . ' ~ . ;: ' f •,

~ "tM '.il

1i

'.11·.·.

I' 1!

'

! ., -: l :t

C' '"'' U 'LJ ")' ( I d . .. ,.) r J/ r E·!.. ·" l:?~i )

!~)(:LJ inE:·

~::al inF:.'

L PS

::::al inf:::~

Sa l i ne

LP!3

::>al.inr:~

L F'~3

LP!3

~:; al int::'

[_p::;

L P!:::

("'C'F ( C'FII '''tl I ) b .. ~! ~.~ .... ..... :-.. ·""· .... ·"" L ! .......... ... .

0

7 ' 000 ± 2SO

.l ' 3UU ± 1.10

0

0

Cl

a Groups of S C3H/OuJ or C3H/HeJ mice were injecte d i.p . with

day s la t er, mi ce were injected with s aline or c hall enged with

E- ~Qli LPS (25 ~g/mouse) .

.l. c've l :::.~ t•.JF·rc·:: determ.inF:!d on pooJc:)d :::; c) t"U.tn :::.; amp .l e~-::_; cc::.JJ.cc!Ctc0)d ( ,

h after the i n j ec tion s on day 3. Resul ts represent the

arithmetic mean ± one s tan dard deviation of duplicate

78

i J .

l ! ' ;

(:. I' I I,

11 I

79

F .i qu. t· P 1 :s . E.t.ff:~~:.:t_Q.f ... .:tt! t~_ . .! tJ1g.cizto.8_t!R.9i m~zrJ .... t.:Jn ... tt.I.S:! ... !::?9Dl~~-~JJ~L.t.t:.G!~J

LES=biPQ[Q§PQDSiY@_ffii~g -- LPS-responsive (C3H/OuJ) and

LPS-hypores pons i ve (C3H/HeJ) mi ce (5 mi c e/gr oup ) were injec t ed

1-P - on day 0 with either saline or E - cgli LPS (25 pg / mouse).

At 3 days pos t-injection, bone marrow cells were pooled and

slzing profiles obtained usi n g a Coulter Channely zer. Bone

marrow cells obtained 3 days pos t - in jection were also p lated in

an exc ess of CSF- 1 for determination of the number s of M-CFU as

' . described in the Ma terials and Methods section. Based on two 5

sepa rate expe r i me nts, the number of M-CFU/10 nuc leated bone

ma rrow cells we r e as follows: s aline-treated, C3H/OuJ = 101 ±

11. 9; LPS-treated, C3H/OuJ = 2 33 ± 9.8; saline-tr ea t ed, C3H/HeJ

= 92.6 ± 14; LPS-t reated, C3H/ HeJ = 102 ± 2.8.

80

100 C3H/ OuJ

80

60 LPS on Day 0

40

(I) .J

20 .J w u u. 0 a: w m :i 100 = z C3H/HeJ w > }: 80 ~ ....

Saline on Day 0 w a: 60

40

20

20 40 60 80 100

RELATIVE SIZE OF CELLS

the total cell population with a compensatory increase in t he

increased in bone marrow cells derived from C3H/OuJ mi ce

exposed on day 0 to LPS. No s uch increase was observed in bone

marrow cells derived from endotoxin-treated C3H/HeJ mLce

(indicated in . the figure legend, Figure 13).

tho initial description of a hait"1.(:0SS , mut.ant (nucl(:;~) mous e~ b\:

Fl anagan (1966) and further observations by Pantelouris (1968 )

which showed that such mice were athymic, T cell-deficient,

nude mice have been used to study the rol e of T cells in

antibody- and cell-mediated immune responses (reviewed by

K indr·ed, .1. 90.1.) . Homozy~Jous cxpn;~ssion of thr::.• "nuch.:·~" a.llc•lr.:·!

(QY/Qy) results in congenitally athymic mice that possess

extremely low levels· of functional T cells. Several recent

. reports suggest the existence of LPS-responsive T cells (Vogel

Ql §l .• 1983 ; Mita 01 §l., .1.982; McGhee ~1 ~l-, 1979;

Koenig ~1 Dl-, 1977; Scheid ~1 §l., 1973 ) . There for e, to

determine if T cells were involved in early e ndotox in to l e ranc e

induction, nude mice were studied.

BALB/c athymic nude (oy/oy) mice and their e uthym i c

( nu J·+· ) l:ittermates L'-!erP .inject(:;·cl with c~.ith(~r ~::;alinlo! o~· C- .LQ1J . ......•. /

1;·::, ·:.· ': 1 1::.c· (. ·:~ · ·· C'jmol.t~::::E~ ) i. p. on da v 0 and cha11 enqnd ::s da '/ s: ,_JJ - 0 ~J ~~ .

later· with~- cgli LPS (25 ~gjmouse) . Mice were bled 6 h

followinq challenge and sera tested for CSF activi t y. When

81

~~

I{

r.

82

athymic and euthymic mice were injected with sa line on day 0,

both responded to an LPS c hall enge on day 3 with the production

of se rum CSF (Figure 14) . Exposure to LPS on day 0 tolerized

both athymic and euthymic mice as evidenced by a greatly

reduced production of se rum CSF wh e n challenged on day 3 with

LPS.

Recent studi es have suggested that certain athymic

mouse strains contain substantial numbers of T cells (Ikehras

gt 01., 1984; Habu and Okumura, 1984). The spleens of the nude

mice used in this study wer e analyzed for the presence of a

T-cell mar ke r, Thy 1.2, by analysis with a fluorescence

activated cell sorte r (FACS). Single cell spleen cell

suspensions from individual BALB/c (oyfoy) and (oy/+) mi ce were

prepared. Aliquots of each suspension were treated with

fluoresceinated anti-mouse IgM (~heavy c hain specific) to

detect B cells, anti-Thy 1 .2 (to detect T cells), or control

antibody, as described in the Materials and Methods section,

and fluorescence a nalysis was car ried out. Th e BALB/c Coy/ny)

mice used in this study were very deficient in the number of

cells whi ch stained specifically for Thy 1 .2 (3%) when compared

with spleen cells derived from the euthymic con trols (24.4%)

(Table VIII). The percentage of Thy 1.2 positive cells a l so

differed with respect to median fluorescence intens ity (MFI).

Thus, the few spleen cells f rom athymic mice wh ic h stained

positively for Thy 1.2 exp ressed far less of t h is su r face . '

antigen than spleen cells from euthymic mice. The percentage I .

of spleen cells exp ressi ng ~ was as expected higher in the

83

I L. ' • lOY .DYl_ ffil~Q BALB/c euthym ic and athymic mi c e (5

mice/group), were injected on day 0 with either s aline or E-

~gli LPS (25 p gjmouse) a nd c ha llenged 3 days later with 25 ~g

C- ~Qli LPS. S ix hours later, serum was coll e cted and assayed

for CSF acti v ity a s de scribed in the Materials and Methods

s ection. Th e resul t s represent the arithmetic mean ± one

standard deviation of duplicate determinati o ns.

10000

-e 8000 ...... ;:) Ll. (.) -Ll. 6000 V) (,)

:E ;:) a: LIJ V)

4000

2000

BALB/c ( nu/ +)

Euthymlc

BALB/c ( nu/nu )

A thymic

D ~

84

Saline on day 0

LPS on day 3

LPS on day 0 LPS on day 3

sou;·· c (:' a

13 AL.I3 / c £-:·)1..1. t:hy rn :i. c

r / ' ~.r.J .U +;

I:!.ALB/c athym.ic

(!:J.V/f."J.1A)

I tJ Y.. .•. .1 .,,. _;? J.g tj

24.4 ±1.5 (473±28) 58 . 2 ±2~8 (352±36)

3. 0±0.7 ( 290±3 4) 7 1.0±4.6 (392 ±5.4)

------------------------------- --- ------------------------ ----a ·r , .. , (J ·L· ' , J. (J' t ""t ·1 '=; 1:> l '·" <=-' ,-, r: <·" ·1 l <.:-; ' t ,,~ 1::> '"·' , •. , •-:~ 1. <-> , •. , •::- ·f·' r·· <") r11 1::- .,t ·'· 1-, " rn J. (··. ., , •• ····1 ":-. ~ .• 1 . " . _ .. I ... (, . 8 · - \ _. - · .• ~· . - . • .••.• ··~ '·· ··- . - • __ \ • , _.) <- L· 1 . - - · < . .l I •., ,_)

e uthymic BALB/c mi ce were treated with fluoresceinat e d antibody

us .i.nd:i. c at.<:? d i n thP t·1atc't·.i. al:::; and ~1<:: thod~::; :::>Hct..i.on .

b Fluorescence analysis was carri e d out on eac h sampl e us1 ng a

fluore s cence-ac tivated cell sor ter, and t he percent age o f cel ls

whi c h sta ined with the indicated anti body and median

fl uo rescence intensi ty (MFI) determined. Resul t s, exp ressed as

percent positive cells, repr esent t he a rithmetic mean ± one

standard deViation of meas urements made on ind ividua l s pl een

!! ss a

., . ~~

! 1."

86

cell suspe ns i o ns.

87

athymic mouse strai n due to the anticipated T-cell deficiency.

i~idl_ffiQY§Q_§tr§in -- CBA/N mice possess a defective X-li nked

gene, ~id, which interferes with th e development of cells that

exp ress the Lyb·s s urfac e antigen (Ahme d Q1 Dl-, 1977 ). These

mi ce are unable to respond to a number of B cell mitogens,

including purifi e d poly sacc h a rides and LPS (Amsbaugh Qi Dl-•

1974; Sc h e r, 1982). Although s p leen cell s from CBA/N mice are

de f icient with respect to LPS- induced mi togenesis (Glade and

Rosenstreich, 1976), these mi ce are fully responsive to th e

toxic effects of LPS a nd possess ma c roph ages wh ic h r espond

n o rmal ly to LPS in yitrg to pr oduce l nterleuk i n 1 (Rosen s t reich

Qt §l -, 1978). Za ldivar a nd Sc h e r (1 979) have compared

immune-de f ective (~i~ ) F 1 mal e mice with immunologically normal

F1 f e male mi ce de rive d fro m the c r osses of the CBA/ N s train

wi t h DBA/2 mice. Their resu lts i ndi ca t e that al th ough th e

immun e-defect ive male mi ce were unable to make a specfic immune

r esponse to ~- ~Qli LPS, th is did not influence ei ther thei r

n a tur a l resistance to LPS lethal cha l lenge or th e development

ot late phase t oleranc e ( a n tibody-me d iated) to LPS after

pretrea tme nt with a subletha l a moun t of LPS and a dm inist r ation

o f LPS 10 day s later.

In 1983, Mond gt 0l- s howe d th a t sp leen cel l s from th e

C3 . CBA/ N mi ce (which possess the xid gene defect on a fully

LPS-responsiv e C3H/HeN background) fail to respond iD Yi1r9 to

B-cell mitogens, including LPS. I n the following se ries of

88

experiments. C3 .CBA/N mice were utilized to determine whether

the population of LPS- respansive B cells whi c h i s defici e n t in

these mice were involved in the induction of ear ly endotoxin

to.l.er·ancG. e .. ,,. r···J::l A/I' I ·f""' ·- J -~ ...... .> • ·' -' ' b m.:t .. L mice,

the xid defec t Cxi d/xid), were age-matched with C3 H/HeN female

mice and were subjected to th e tolerizing regimen. Mice from

eac h strain were injected with either saline or ~ - ~gli K235

LPS on day 0 and· either challenged 3 days later with LPS and

bled to obtain s erum for assessment of CSF activity, or

sacrificed an day 3 to obtain bone marrow cells for c ulture of

M-CFU and cell sizing profiles. The xid defect o f C3.CBA/N

mice had no effect on the induction of serum CSF after LPS

challenge of mice injected on day 0 with saline (Figure 15)~

Moreover, pre-exposu re on day 0 to LPS resulted in early

e ndoto xin toleranc e as assessed by a r e duct ion i n serum CSF ~ following LPS challenge.

't. ., •i: .,

Bone marrow cell s i z ing profil es fro m control I

t.

' (saline- injected) C3.CBA/N and C3H/HeN mi ce revealed a

characteristic bimodal distribution of cells. The pattern was

altered in both the normal and defec tive mi ce by exposure to

LPS 3 days ear lier. Both strains also ~esponded to p~ior LPS

ex posure with an increase (approximately 3-fold) in the number

of bone marrow- derive d macrophage p~ogeni to r cells (Tab le IX) .

~1 1'..: I'

mice was recen tl y established by Williams 91 Dl- (1 9 8 3 ) to

89

Figure 15. Eff~~t_gf_lbQ_1Ql~ci~iQ9_LQ9iffiQQ_QQ_~E~=irrdYC9~

50LYffi_Q$E_LQ§RQO§Q_iu_Q~~L~QN_~nd_Q~~Q~AL~_ixi~l_mi~Q -­

C3H/HeN and C3.CBA/N mice (5 mice/group) were injected on day 0

with either sa line or E- ~g1i LPS (25 ~g/mouse) and challenged

3 days later with 25 ~g E- ggli LPS. Six hours later, se rum

was collected and ~ssayed for CSF activity as described i n the

Material s and Methods section. The results represent the

arithmetic me an ± one s tandard deviation of duplicate

det0rminations .

90

3000

D Saline on day 0

LPS on day 3

~ LPS on day 0

LPS on day 3

-E 2000 .......

:;) LL. 0

LL. (/) ·,

0

:::t ~ ;:) ,, a: ~~ ' w F (/) 1000 w

!

\ •'

t':

C3H / HeN C3.CBA / N (xid)

I

91

c 2_£gJl5 (~)

C3 H/ HeN Sa l ine 13 ' 260 ± 1 ' 441 59 10

LPS 26 345 ± ~77 77 11 4 , L ~~ .

C3.CBA/N Salin e 1 1 , 0 50 ± 9 19 6 4 10

LPS 29 70 ~ ± 487 84 1 1 4 , J,~ .

-------- --- -------- ---- ------ --- ------------- - - - - -------------· a

M1 ce (5 mi ce/g roup ) were i n jec t ed i.p. with sa l i ne or E-

~gli LPS (25 pgjmouse) on d ay 0 a nd sacrificed on day 3. Bone

marrow cells f rom eac h gr oup were then pooled and sizi ng

p r ofiles obtained u s in g a Coulter Channelvze r .

b 4 Five X 10 pooled bone marrow cells f r o m eac h g r oup were

plated in a n excess of CSF-1 a nd t he nu mber of M-CFU per femur

deter mi n e d as described i n t h e Materials and Meth ods s e c tion .

c calc u lated from i n teg rat ion data wh ic h wa s obtained us ing t he

Coulter Channelvzer which was calibrated according to the

man u fact u re r' s instructions. ,j ~~:

identify those ce lls which p~oduce CSF in ~espon se to LPS. In

that study, it was demonstrated that ea~ly endotoxin tole ~ ance

could be ~eve~sed by injection of tole~ized mice with spleen

cells o~ a mi x ture of splenic lymphocytes (macrophage depleted)

plus thioglycollate-elicite d peritoneal macrophages from

control mi c e 24 h p~ior to LPS challenge. The authors,

therefore, suggested t hat spleen cell s contribute significantly

in the production of ci~culating CSF . To assess further the

importance of the s pleen and its resident cells in the

induction of early phase endotoxi n tole ranc e, a different

app~oach was employed. ICR mice were divided into three main

experimental groups, i.e., s plenectomiz e d , sham-sp l e nectomi zed,

and untreated ( no su rgery). Mice whi c h had surgery we~e

allowed to r ecover completely and were then i nj ected on day 0

with either sal ine or ~- CPli K235 LPS. Three days later, th e

respective groups were either c ha llenged with LPS and bled 6 h

later ( for measurement of s erum CSF activity), a ~ sac rifi ced

without challenge for the c ollection of bone marrow cells (fo~

de ie~mination of the numbers of M-CFU colonies and cell sizing

profiles ) . Res ults in Figure 16A illustrate that: (1) high

l eve l s of LPS-induced serum CSF were found i n all th~ee

experimental groups, regardless of treatment and, (2)

splenec tom ized mice had a reduced capacity to gene ~ ate CSF in

~ esponse to LPS challenge ( i. e., th ey could be tolerized to th e

same extent as control s ) .

The ef fect of the tolerizing ~egimen on bone marrow

cell sizing profiles and M-CFU n umbers was also assessed. Bone

92

' . ~ :I. ''

93

Groups of S

ICR mice from each ex perimental group, 1.e., s pl e nectomized,

sham-splenectomi zed, a nd untreated (no surge ry), wer e injected

on day 0 wi th either sal ine o r E. ggli LPS (25 p9/mo use) a nd

cha ll e nged 3 days later with 25 pg E- CQli LPS. Six h o ur s

la t er, se rum was collec ted a nd assayed for CSF activity a s

described in th e Materials an d Methods sec ti o n _ Th e resul t s

repr esent the arithmetic mean ± o ne standa rd deviation of

dupl ica t e determinations.

94

5000

D Saline on day 0

LPS on day 3 -E ' ~

LPS on day 0 ~ u. LPS on day 3 (J

u. en (J

:e ~ a: w en

\ •'

1000 n· t·

SPLENECTOMIZED SHAM- NO SURGERY

SPLENECTOMIZED

'·' I, 11 1

w I ~ i

,H k

marrow cell s i z ing profiles from splenectomized and

s ham-splenec tomi zed mic e were obtai ne d 3 days after injec tion

of sal ine o r lPS. The bone marrow profiles shown i n Figure 168

illustrate t hat r dmoval o f sp l eens has little or no effect on

the LPS- induced bone ma rrow cell sizin g profile, · i.e., bo th

s plenectomized a nd sham-splenectomized bone marrow cells

exhibited a reduc t ion in Peak 1 and i ncr ease in cell size and

number in Peak 2 following inject i on with LPS wh en compared to

th e profiles obtained from s aline-treated mi ce. The .number of

bon e marrow M-CFU was al s o shown t o be increased in both g roups

three day s following administ rati on of LPS (indicate d i n fi gu r e

l egen d, Figur e 16B).

Bacterial e ndotoxins a f fect th e pathogen icity of a wide

variety of organis ms including bacteria, fungi, parasi tes, and

v ir· uses (reviewed by Cluff , 1971). Endo toxi ns induce

res i s t ance t o infection over appro xi mat e ly t h e same time

interval as th e y induce LPS-hyporesponsi veness, (Beeson. 194 / a;

Bees on, 194/ b) . Endotoxin-induced resistance to infect ion

occu r s i ndependentl y of t he ge n e ration of a-specific antibodies

( "I "I · ·r ' ')"? ... ,. ·, How (·~' \/E·' r • t hE' co t" t" E' .Lat. .1.. on of on do to>< .i..n ,· :t .. , n (·~r, .. ;·, .. ) )-

tol e t" an c (:;~ t•J :i. th r E!~::::: .i ~:::.:ta n c ~:::• to in fr:~c t j , on Vdt" .i (:;~~::: r:.~ n or rn c:t~..ts.l v .. This

vari<Jtion has been <J ttributed to dif f e r ences i n the dos e of

95 I ,! !:· ··' H I;' i

·~ ~. \

~

I ~~

.• l ti-":i i!!i

•' jo,f

''I

•.1

96

0dmiai§tr0tiQU -- On day O, splenectomized or

sham- splenectomized ICR mice (5 mic e /group) were injected with

saline or~- ~Qli LPS (25 ~g/mouse ) . Three days later the mice

--· were sac rificed, the bone marrow cells obtained, and th e cell

s i z i ng profiles determined using a Coultet· Channelyzer. The

arrow indicates the relative pos ition of Peak 2 bon e marr ow

ce lls from control (saline-injec ted) ICR mice . The numbe r o f

5 . M-C FU/ 10 nucleated bone ma t·row cel l s were as follows:

sa l ine-t r eated, splenectomi zed = 110 ± 3; LPS- treated,

splenectomi zed = 192 ± 9; saline-t r eated , s ham-splenec tom ized=

'):::: ± l !.; L.P ~:: -·- t:ru <Jtt::'d, sham -··::; p}.\-?}n b' c::tomizt·:·~d :: ;-:,> :.Sl.i. ± :;:~1 -

97

Splenectomized

100

80

80

40

20

100 Splenectomized

80 LPS

(/) 80 ..,j

..,j

w 40 (.J

II. 20 0

a:: w ID :::E :::::1 100 z Sham-splenec tomlzed

w 80 > Saline

i= < 80 ..,j

w a:: 40

20

100 Sham-splenectomized

80 LPS

60

40

20

20 40 60 80 100

RELATIVE SIZE OF CELLS

98

endotoxin, the route of administration, the time interval p r io r

to infectious challenge, and the pathogenic course of the

To assess the potential of t he early endotoxin

tolerance regimen established in thi s study to protect again s t

a Gram negative infection, it was deemed important to choose an

infectious model whi c h coincided temporally with t he tran s iency

of the tolerance s ystem (see Figure 1). A well - defined model

for E§ . §~C~9iQQ§§ infection was e s tablished by Stieritz and

Holder (1975) and modified by Pavlovs kis Qi 01. (1977 ). With

this system, mice are first administered a third- degree burn

and then are immediately infected subcutaneously with Ps.

§§r~SiQQ§§ at the burn site. Death s are recorded for a seven

Groups of 5 or 10 m1ce wer e injected on day 0 with

either sa line or 25 pg r ~Qli K235 LPS i.p. Three day s later,

the mice were burned and injecte d subc u taneo0sly at the burn

si te with varying concentrations of f§. ~~rY9iOQ§Q . The

results s hown in Table X indica te that mi ce tol e rized with ·~-

~gli LPS exhibit a higher degr ee of resi s t a nce to infection

(compared with control s aline-inj e cte d mice), particularly at 7 8

the high e r doses (J x 10 - 3 x 10 bacteria/mouse ) . Thi s

r es i stance, as assessed by mean time to death ( MTD ) , howeve r ,

was transient, wan e d with i n 2 day s pos t -challenge (day 5

pos t - LPS ). and was not obs erved with lower challenge inocula.

Thc:.::> rE! was no st.a t:i. s t :i.cal d.i..f fe t"c·~ncc) in t hE·) l_Dso ' :~; ca l cu.l<Jt.Gd

·fr orn con t.t"ol an·cl tcd.e r i ze d C' X P(·)r· i rn r;,:) n ta 1 q rou.ps.

ll!

C't .. Oi l :la .. ·:! ....... , •. :.,.!...

Contr··c.l ::) X

· - :~· X '-' Tol E~ t· · i z~::'d

~:~·

>< ~ .. J Cont r·· o .l

3 X

Cont.rol :·_:s v ·"·

·roli:·)t .. i ZfZ~d 5 v "

Con t.ro.l :s " ·"·

ToJ.. (~~ t- j_ l.C'd :s X

Con t: t· o .1. ··::· x: '·'

roJ..et .. .i. zed ··::.· v '·' .'\

Cunt. t'" O.l :s X

·rolet--:L z (O>d :s ><

8 10

8 10

7 .1.0

7 10

6 .1. 0

6 l U

5 .10

5 .10

4 10

4 10

3 J U

3 10

__ 1 __ ________ 2 ______ __ ~ _____ ___ 4_

60 1.00

c ~~-~ "? ::::·?

~:> 5 .1. 00

c 0 ::::::?

D 47

0 60

0 ~?0

0 :;:~ u

CJ u

0 0

Cl u

0 0

.I. DO

c · ~j ::s

.1. 00

c J.C) O

~~:,;:·

-{:, ;.:~

::;:s

40

0

0

lOU

·)~5

100

lDU

-?~:s

t~: . ~~~

40

.::j.()

0

Cl

-, /

- ------------- --------- - --------------------- -------------

99

,. J,

a ICR mice were treated on day 0 with either saline or ~- C9li

with ESQY0QillQ00S 0~LY9iD9S~ at a burn site with the i ndi cated

number of bacteria (inoculum).

b The resu lts are presented as the cumulative percentage of

deaths for each day after bacterial injection and represent th e

combi ned data from two separate experiments in which a total of

15 mice per experimental group were treated. The calculated LD50

(based on two separate experiments) for control and toleri z ed 5 5

mic e were 8.8 X 10 and 7.6 X 10 bacteria, respectively.

c p < .05 by a one-tailed, Kolmogorov-Smirnov two-sample test

100

~. ;

I!

"

I~PUQIIQN_QE_E~B~Y_E~~~E_E~QQIQ~lN_IQ~~B~~QE_~y_A_QEIQXIEIEQ

~IPIR_A_QEBI~~II~E~-~Q~QPUQ$PUQBYL_LIPlP_A_iUP~l

Although endotoxin has been shown to induce many of th e

harmful manifes tations seen in patients with Gram nega ti ve

sepsis, some of the effects of endotoxin are bene f i cial

(reviewed by Nowotny, 1983). However, therapeutic approaches

whi c h use endotoxin to induce beneficial effects have been

l1m1ted because man is very sensitive to the toxic effects of

e ndotoxin. Investigators have explored the possibility of

usi ng chemical modification techniques to reduce th e toxicity

and pyrogenicity of endotoxin while retaining beneficial

effects such as adjuvanticity (Nowotny, 1963; Johnson §1 ~l-,

1964; Ribi gt §l., 1979 ). Takayama Qi ql. (1 984) have rece~tly

described a detoxifie d Lipid A derivative, monophosphor yl Lipid

A (MPL). Therefore , in this fin al series of experiments, we

have studied the capacity of the nontoxic, monophosphorvl Lipid

A derivative to induce early endotoxin tolerance.

K235 LPS into mi ce was followed by a rapid rise of serum CSF

which r eac hed a maximum level at approximately 6 h

post-injection. To test whether the non-to xic Lipid A

derivative, MPL, was also able to induce thi s manifestation of

endotoxin ~esponsiveness, groups of 5 mice were injected

101

102

intravenously with varying doses of MPL. At 6 h following

injection, the mice were bled and the serum subsequently tested

for the presence of CSF. The results in Figure 17A s how that

serum CSF was induced over a wide dose range (1.0 pg -

100 pg/mouse) and was induced optimally with 50 p9 MPL/mouse.

Additionally, MPL induced a similar (although somewhat reduced)

dose-dependent response in mice injected intraperitoneally.

These data are s hown in Table XI.

Williams §1 ~1- (1983) and those presented in Figure 1

indicated that early e ndotoxin tolerance was optimal 3-4 days

after initial exposure to either a homologous or heterologous

preparation of LPS. To test whether an initial exposure to MPL

would also render animals refractory to LPS, we injected groups

of mice with varying concentrations of MPL , and 3 days later

challenged these same mice with C ~gli LPS (25 pg/mouse). Six

hours after challenge, blood was collected and the se rum

assayed for CSF activity. As can be s0en in Figure 17B,

increasing the dose of MPL given on day 0 from 1 pg to 100 pg

reduced significantly the LPS-induced se rum CSF response upon

r··l''t'''tl lc::-· r·tcj(·:• I•)J. 'I"J'''I ··;.•:; ('"I F' c:n'J j I p<; on d<::t\1 5 TlliS d(·:-•qr"(!(> of .... <. ... . ... .:: -· ,, .• .• '-·'· p .:0 ·"' • -"-"~·'-"-'·=· .... ,,. .. - I •

LPS-hyporesponsiveness was equivalent to that seen when 25 p 9

~- fQ!i LPS was used as the tolerogen (Figure 178)_

Another indicator of LPS responsiveness io YiYQ lS the

induction of serum i nterferon (IFN) which usuall y peaks 2-3 h

following intravenous administration of intact LPS or Lipid A

103

ICR mice (5

mi ce /gro up) were injec t e d with either saline, E- ~Qli LPS

(25 ~ g/ mouse), or MPL (1 - 100 ~g/mouse) i.v. Six hour s

late r, th e mi ce were bled, se ra collected, pooled, and assayed

for CSF ac ti v i ty as described in the Ma terial s and Me th ods

sec t ion. The re s ults r e present the a r ith me tic mean ± one

s t a nda rd devi ation of duplicate samples of se r u m poo l s from

three sepa rate ex perimen ts i n which 5 ml ce pe r t r eatment per

ex pe ri ment we re pooled.

5000

- 4000 .J ::e ' :J ~ 0 .... 3000 ~ tn (.)

~!: ::e :J 2000 tc UJ tn

1000

'f

0

A

E. coli LPS ( 2 5 }l9)

/"\ /! i ~

Saline only

I ;------2 1 10 25 50 1 00

CONCENTRATION OF MPL INJECTED (f4g / MOUSE)

104

II. .. f

105

Induction of serum ~SF in rrp mirP in·I·Pr·t· p-1' ----------------~--~~--=---~~--~~~-U--~~~:~

Concentration

of MPL injected

_________ §QrYm_GBf_iGEWLmll ________ _

0 0 0

1 560 ± 37 NT

10 0

' , 170 ± 186 640 ± 130

0C LJ

0

' , 900 ± 365 1 ' 900 ± 254

50 ~ 500 ± 1 ~ 4 2 560 ± 488 w ' / '

100 0 L ' 400 ± 218 1 ' 2 10 ± 285

a ICR mic e (S mice/group) were injected with either saline or

MPL (1-100 pg) i.v. or i_p _ Six hou rs later, the mice were

bled, sera collected, pool ed, and assayed for CSF activity as

described in the Materials and Methods section. The results

represent the arithmetic mean ± one standard deviatio~ of

duplicate samples of serum pool s from three separate

experiments in which 5 mice per treatment per experiment were

pooled.

. , ·,

106

LPS -- ICR mice (5 mice/ group ) wer e i n jected on day 0 wi t h

e1t h e r sa l i ne , ~- ~gli LPS (25 p g/ mouse ) , or th e indicated

concent ration of MPL (1- 100 pg/mouse). On day 3 , mice .we r e

I .J J I 1," ]·_ ·t·i·l 1:·· ·· 1 · 11 , .. , .,,. o:·· I I"' c· ( , .. , oc· c· / ·· I ····· -, ' c 1a .. e ngec v ~- ~Q_l .~JJ _-0 ~J ~ J mu ~ ~~J .

later se r a were co l lected a nd assay e d fo r CSF activi t y as

desc r ibed i n t he Mate ri als a nd Me th ods section. Th e resu l t s

represent th e arithmet i c mean ± one s t anda r d d eviation o f

dup l i cate samples of se r um poo l s f r om t h ree sepa r ate

expe r i me nts in wh ic h 5 mice pe r t r ea tm e n t pe r expe r i me nt were

pooled . The treatmen t design a ti o n 1n Figu re 178 i ndicates the

trea tments whi c h were a dm inis t ered o n d ay 0/day 3,

5000

..J 4000 ::: ' ~ Ll.. u

3000 LL V)

u ::: ~ a: 2000 w V)

1000

B

Saline / E . coli LPS T .J..:

_?_--, I-o MPL / E. coli LPS

E. coli LPS I E. coli LPS

Saline I Saline

10 25

CONCENTRATION OF MPL INJECTED

ON DAY 0 (n / MOUSE)

50 100

107

(Youngner and Stinebring, 1965). Figure 18 i l l ust r ates that

t he level o f se r um IFN induced by MPL was comparable to that

s timulat ed by ~ - ~9li LPS; however, th e tolerance i nduced by

MPL for IFN was less than the to lerance induced by the i n tac t

LPS for IFN (30% vs . 99% s uppression of the IFN r esponse) or

the MPL- induced tolerance f or CSF (see Figu r e 17B).

I t was also found that prior ex pos ure to a n initi a l

do~·:: P n f ~1 P L ( 1 (] (] 11l:t / "l n 1 1 .:::; P ) ·L· 1-1 r- , .. f" ·::t ,._.. "'·' cl ··:·· 1. r" I"J ·1' ·f' ·1· c·· '''ti"J ·t· l '/ ·t· l"1 t''·' L r ·1 5o ·• ~ •• .. " - . - .. to-' .;:1 It .. •• • '··· ..• , ... f , •• <., .... ' \';:, ., .,,";; _ . .:.:J . , , ... • c.. . , ... _ .•

(approxi ma tel y 5- f old) a nd essenti a lly elimi nated overt

s ymptoms of e ndotoxici tv, i.e., ruff led f ur, c on junc t ival

disch a rge, and diarrhea in respons e to a suble thal chal lenge of

LPS. Th ese data are s hown i n Tab l e XII .

ef ficac y of MPL to i ncrease bone ma rrow M-CFU (as s hown earlie r

fo r f ~gli LPS; see Figur e SB), groups o f 5 mice we r e i n jec ted

wi th sal ine, E ~gli LPS (25 f.lg/mouse). o r va rying

c onc e ntr a tions of MPL, and on th e third day fol lowi ng

i n jection, bone marrow ce ll s f r om the femurs o f eac h group were

enumerated a nd cu ltured in an exc ess of CSF- 1 to dete r mi ne t he

number of M-CFU. Th e r esults ln Figure 19 i ndicate that MPL

in creased the number o f M-CFU 1n the bone marrow in a

dose-dependent fa shion. When mice we r e given 100 ~8 MPL per .

mouse , t he level of M-CFU pe r f e mur was equal to that i nduced

by :;:~5 f.! ~J !~~.. c:.L-J.L LP::;.

These fi ndings we r e further suppor t ed by c ha nges in

108

109

Fiqu~e 18. lot~rf§rgo_iodYctigo_b~-~E~-~od_§ffg~t_gf

ME~=tr§~tm§ot_go_~t~=io~ycgd_iot§rfgrgo_ergdyctigo_io_viYQ

ICR mic e (5 mice/g ~ oup) we re injected with sa l i ne, E- CDli LPS

(25 ug/mouse) , o~ MPL (50 or 100 u9/mouse) i.v. on da y 0.

Thr ee day s la ter, the vari o u s groups of mice were injected i.v.

with either sa l i ne, LPS, or MPL, as indicated. Se rum s ampl es

wer e collected 2 h later and a ssayed f o r i n terferon (antiviral)

activity as desc ribed i n t he Materi a l s and Me thods s ection .

Results represent the ari t hme t ic mean ± standard error of the

a~ithmetic mea n of antiviral act ivi ties of individual se rum

samples . Along the absc i ssa are indicated th e tr ea tm e nts on

days 0 a nd 3, r especti vely .

I• 1000

I• en 100 1-z :::1 ..J < !!: > ;::: z < >-!:: ~ 1-u < z 10 0 IX: w u. IX: w 1-~

a

INJECTION ON DAY 0: Saline

DAY 3 : Saline

110

,..-

Saline LPS Sa line MPL(SOJ Saline MPL(100)

LPS LPS MPL(50) LPS MPL( 1 00) LPS

TREATMENT GROUP

b L.Dso

· r·· r·· 1 1 r·· c nu ······ . (·:x ···1.H"·

Con .:iunc tiva l

d :.l.schat-q(;:- c

Diar· c rh ~-:::·a

Non - toleri zed MPL-tol e ri zed LPS-tole ri zed

j l"l/ 1':> . .. (,., U/ .1 !S l/ .1.~=>.

S/12 ,.,/ 1'-f".:~ . ~ ::. 0/ .1 ~:.'

(.j 1 ~? 1./1 !:) .1/ ].;?

a Mice were injected i.p. on day 0 wiih sali ne , MPL

(100 ug/mouse), o r ~ - ~gli K235 LPS (25 u9 / mouse ). Three days

later, the m1c e were challenged with f- ~Qli LPS fo r LD 50

dete rmin a ti on and assessment of s ymptoms as described i n the

Materials and Methods section .

b Cal cu. .I. a tc~d by thf:~ rnf:~ t:h od o f Hc·~Pd a nd 1'1UcJnch ( 19 ::.:;·)) .. [ fh 1:-~ L.D so

for MPL was not ciPte rmin ed in t his s tudy. Th e MPL LD 50 has

been r e po rted to be greatPr than 10 mg for mice (Hibi, 19 84 )] .

111

II

112

a The proportion of mlce clearly exhibiting the indicated

symptom 6 h a f ter i.P. i n jection of ~- ~gli K235 LPS

(25 ~g/mouse) o n day 3. I n jection of MPL (100 ~g/mouse)

i n d u ced n o n e of the i ndica t ed symptoms in any of the 15 mi ce

t es t ed in t h is exper im e n t.

113

OYW~Qt_Qf_m§Ct 9Rb09§_ RtQ£Yt§QL§_io_tb9_bQO§_ ffi0t tQW - - ICR mic e

(5 mice/ g r o up) we r e i njected o n day 0 with eithe r sa l i ne, ~ -

G9li LPS ( 25 pg/ mouse), or MPL ( a t the indica t e d doses)_ Th ree

days l a te r , t he bon e ma rr ow ce l ls were co l lected a nd poo l e d (5

mice pe r t r e atme nt gr o up )_ 5 X 104

ce l ls f r om eac h cell

suspension we r e th e n pl ated i n dupl i c ate in a n excess of CSF - 1

and th e numbe r of M-CFU de t e rm i ned as desc r i be d i n t he

Mate ri als a nd Me th ods sec ti on. The r esults r ep r esen t th e

a t .. it hJn f::tic mra <:~n ± on e• s t anda r d de viation o f dupl ica t i':)

dfO.' tE~ r· rn.i. n<:t t. i on s _

114

7 -

6 -a: ::1 ~ 5 -w &&.

....... "'t

I

0 4 -,... )( ..::c. ::1 &&. 0 3 -I ~

.:z:..

2 -

1 ..

Saline LPS MPL(25) MPL(SO) MPL(100)

INJECTION ON DAY 0

bone marrow cell sizing profiles obtained using a Coulter

Channelyzer. Bone marrow cell suspensions from groups of 5

mice injected 3 days earlier with saline, E- ~Pli LPS, or MPL

were compared. As can be seen in Figure 20, llke LPS,

injection of MPL induced a marked reduction in the relative

number of bone marrow cells in Peak 1 (20% of t otal) and a

compen sato ry increase in the r1umber of cells within Peak 2

(80%). Thi s was accompanied by an increase i n cell s1ze 1n

Peak 2. As was previously observed with E- ~9li LPS , t hi s

c hange in .cell sizing profile induced by MPL coincided

temporally with the acquisition of optimal tolerance.

115

.. 11 6

Figure 20 . Cffg~1_gf_1QlQriziog_dQSQ§_gf_~p~_go_bQDQ_W§rrg~

~gll_siziog_~rgfil§S - - ICR mice (5 mice/g roup ) were injected

i.p. on day o with either saline, r CQll LPS (25 pg/mouse), or

MPL (at th e indicated doses) and sacrificed three days later.

Cell s izing profiles were obtained from suspensions of bon e

ma r row cel ls using a Coulter Channelyzer. Th e· arrow indicates

the initial position of Peak 2 in mice injected with saline on

dav 0 ..

II) .... . .... w 0 I&. 0 a: w

. al · ::e ::J z w > j: <( .... w a:

{'

Peak 100

80

60

40

20

100

80

60

40

20

100

80

60

40

20

100

80

60

40

20

Peak 2

Saline

20 40 60 80 100

RELATIVE SIZE OF CELLS

117

-·

For some time, i nvestigator s have attempted to clarify

the · extremely complex cellular mechanisms which underlie

responsiveness to Gram negative b~cterial endotoxin (LPS) _ By

far, one of the most extensively used animal models has be en

the C3H/HeJ mouse strain, which, due to the presence of a

single autosomal mutation (Watson ~1 §l., 1978), is markedly

hyporesponsive to LPS (reviewed by Morri s on and Ryan, 1979) _

Thi s genetic defect (LR§d ) is reflected in a number o f ce l l

types, e.g . , decreased mitogenic resp6nse of B cell s (Sult ze r

a nd Nilsson, 1972), reduced ability of C3H/HeJ pre- T c el ls to

express Thy-1 a n tigen (Koen ig gt §l., 1977) or to proliferate

(Vogel R1 ~l - . 1983) in response to LPS, reduced a bility ot

C3H/HeJ macrophages to respond to LPS (reviewed in Vogel e t

01- . 1981), and the failure of C3H/HeJ fibrobla s ts to be

altered metabolically by LPS (Ryan and McAdam, 1977) _ Us ing

this model in ~on j unction with syngeneic LPS-responsive mice

(be~) . much information has been obtai ned that points to th e

macrop hage as the cell which figures centrally in me diating

endotoxicity. Michalek ~1 0l- (1980) demonstra t ed that th e

c ells principally responsible for e ndotoxi n respon s iveness are

radiosensitive a n d bone marrow- derived. Further, i t was shown

that m~ny soluble, macrophage-derived f actors whi c h are induced

by LPS iD Yitrg [e.g., endogen o us pyrogen (Atkin s 91 ~1 ..

1967 ), colony stimulating factor (Apte Rl ~l -• 1~/6; Ap t c and

Plu z nik, 1976; Metcalf, 197 1; Quesenberry Ql ql., 1972),

118

interferons (Ho, 1980), and prostaglandins of the E series

(Skarnes and Harper, 1972) ] are associated with io YiYQ

endotoxicity_ In addi tion , an association between mac r ophage

differentiation and macrophage sensitivity to LPS has been

demonstrated (Suter Ql 0l-, 1958; Benacerraf Ql 01-, 1959;

Rose nstreich and Vogel, 1980; Moore Qi gl., 1980) _

A second, less frequently employed model of

L P ~3-·· hy por t?.~:;:;p on~:::: i vc~nc~s:::; i. ~:::; an .i. nduc i bl P st. a t.f?! of '' Gndotox .i.. n

tolerance'', as it was designated originally by Favorite and

Morgan (1942). This condition is characteri zed by a

progressive decrease in LPS respon s ive ness when endotoxin is

re-administered at appropriate intervals (reviewed by Greisman ,

1.'7H5) _ 1\n ''(·::)at .. .l.y phas(·::)' ' of endotoxin ·.t:oli-;)t·ancc) occur:::,; ~J:tth :tn

the first few days after exposure to LPS, i s transi ent

(subsides within a week), and is not 0-antigen-specific

(Greisman and Hornick , 1976) . Previous studies which employed

this model also suggested mac rophage participation in the

·mediation of LPS- induced toxicitY. Specifically, it was s hown

that the l e vels of e ndogenous pyrogen and prostag landins

produced iD yitrg by macrophages derived from tolerized mi ce

were decreased (Dinarello gt ~1-, 1968; Ri etschel 01 01.,

1980). The data presented herein demonstrated that the sterile

inflammatory agent, thioglycollate, elicited markedly reduced

numbers of peritoneal exudate macr ophages from

endotoxin-tolerized mice (see Table IV). Thi s reduction in the

rela tive numbe rs of peritoneal macrophages in mic e pre-treated

with LPS s ugges ted that, in addition to LPS-induced macrophage

119

refractoriness to LPS, fewer macrophages might be available to

respond to LPS in the periphery. Taken together, these

findings prompted us to test the hypothesis that early

endotoxin tolerance is related to the presence of immature

monocytic cells which accumulate in the bone marrow and could,

in turn, limit the potential number of LPS- responsi ve cell

types in the periphery.

Using colony stimulating factor (CSF) as an indi c ator

of in YiYQ LPS-responsiveness, we confirmed th e findings of

Williams §l ~1- (1983), that early endotoxin toleran ce was

optimal at 3 and 4 days after the .initial exposure to LPS and

that this state of hyporesponsiveness waned within a week. We

found that the acquisition of early toleranc e followed a normal

response to LPS. Optimal levels of serum CSF were demonstrated

6 h after the first exposure to LPS, as had been reported

originally by Metcalf (1971) . On days 1 and 2 post-LPS

injection, the number of nucleated cell types in the bone

marrow was markedly reduced . This latter observation was

consistent with the findings of Sultzer (1969) and Moeller et

dl- (1978) who have shown an efflux of granulocytes and

monocytes from the bone marrow into the blood and peritoneum

following intraperitoneal i r1 jection of LPS. Furthermore, we

. . . . I 'l ,. have shown that the acqu1s1tlon, ma1ntenance, anc .. oss or

tolerance coincides temporally with an increase (followed by a

decrease) in the number of macrophage precursors (M-CFU and

HPP) in the bone marrow. The increase in macrophage precursor

frequency correlated well with: (1) an alteration in bone

120

marrow cell sizing profiles, (2) an e nrichme nt in a denser cell

population which contained all of the pr ecursor activity, and

(3) an i n c rease in immature macroph age forms, as shown by

cyto logy_ Taken collectively, these r esults i nd icated that t he

development of ea rl y endotoxin tolerance is ass0ciated with an

abrupt depopulation of th e nucleated cel ls in th e bone mar r ow

which is replaced by a less matur e population o f ce l ls e nrich ed

for macr o ph age p recur sors.

The incr e a se i n the l e vel of macrophage precur s ors was

s us tained in the bone marrow for a t least 24 h afte r a second

(c hallenge) injection, but returned to normal after a se r ies of

repeated in jections . Thus, it is possible that in the initial

p hase of e ndoto x in toleranc e, those mac r ophages which are

available to respond to the second challenge dose of endotoxin

a r e immature and less responsive to the LPS challenge. Th e

hypothesis t hat immature mac r ophages are l ess LPS-responsive

has been s upported by th e independe nt findi ngs of a number of

investigators. Specifically, Ne umann and So r g ( 1980) showed

that bone marrow-derived macrophage precursors grown in the

pr esen ce of CSF-1, acquire LPS-sensitiv ity late i n cul tu re

relative to t he a cqui sition o f other macrophage differentiative

functions. Similarly, Moore Ql 01. (1980) have shown that

CSF-1-pr opagated bone marrow mac r ophages (from e ndotoxi n

responsive mice) coordinately exhibited e nh anced

differen tiation and LPS-sensitivity i n ~itLQ- In addition, 1t

has been shown that lympho ki ne-ric h culture s upernat an t s

induced increased differentiation of C3H/ He J macrophages in

121

Yi!LQ, and rendered them partially responslve to LPS (Vbgel and

Mergenhagen, 1982). It is conceivable that the s equential

expression of certain functions during macrophage development

and differentiation may be a prerequisite for full expression

of endotoxin sensitivity. A blockade in development which

results in the accumulation of macrophage proge nitors in the

bone marrow may limit the availability or, if released, dilute

the numbers of mature lPS-responsive effector cells in the

periphery. This latter possibility was s upported by t he

fi n ding that the number of M-CFU in th e spleens of mice

injected 3 days previously with ~- ~gli LPS was inc reased

fourteen-fold. One approach which could be taken to test th is

hypothesis would be to reproduce the hematopoietic alterations

seen in response to LPS with a non-endotoxi c agent, then

subsequently challenge with endotoxin. If th e mice were found

to be endotoxi n hyporesponsive, it would strengthen the notion

that the induction of early endotoxin tolerance depends upon a

repopulation of the bon e marr ow with immature macrophages.

The studies of van Waarde ut ~l- (1978) provide

possible mechanisms by which increase d number s of macrophage

precursors could accumulate in the bone marrow. These

i n vestigators have s hown t hat the injection of certain

inflammatory agents resu lts in the induction of two soluble

fa cto r s (i.e., factor increasing monocytopoiesis (FIM) and

cell cycle time for promonocytes and results in i n c r eased

numbers of monocytes, while MPI, which is produced after th e

122

in f lammatory s timulus has been r e moved, cur tail s monoblast

proliferation and acts to curtail the action of FIM. Although

endotox in wa s not utili zed as an inflammatory st imulus in t hese

studies, it is possible that the increas e in macrophage

precursor pools which we observed in toleri zed mice res~lted

f rom a n overproduction of FIM or a failure of LPS- tol erized

m1 ce to produce th e inhibitor, MPI .

The finding that tolerance i s s u s tained with repeated

LPS injections (ho mologous or h e terologous), even t h o u gh t~e

number of precursors r eturns to no rmal l eve ls, suggests t hat

additional mechan isms for s us taining t he toler a nt state come

i nto play. One possibility is that repeated LPS injections

res ul t in the appearance of anti-LPS antibodies whi c h ·act to

clear the endotoxin fro m the c irculation. However, since thi s

was seen for both homol ogous and h e ter ologous LPS in jec t i on

sequences, one would need to invoke the presence o f eithe r

cross- reactive anti - Lipid A or anti-core a nt ibodies which could

act to c lear both E- cPli and £§. ~QLY9iDQ§~ LPS fr o m the

circula t ion o r a polvclonal activation e ve nt, in wh ic h B cell

clones with antibody spec if ici ties f or an assor tment of LPS

species are expanded . Although the LPS a nd Li pid A of f§.

0~tU9iQQ$0 s hare same st ructura l simila r it i es with t hose

produced b y f. ~Qli (Wilkinson, 1977 ), nei ther th e Li pid A nor

the c ore moi e ties are ex t reme l y ant igenic in t h e mo use (Galanos

.L .:_·_.{}"" .·1) • ~:;~ .t {~~- J. .. ' Fo r this r e a s on , t he fir s t possibilJ. t y is less

likely; Neverth e l e s s , since it is we l l know n th a t LPS acts as

a p a lvclonal B-cel l activato r, such th a t a s pectrum of a n tibody

123

specificities 1s induced (Andersson ~1 ~1., 1972), repeated

injections of either homologous or heterologous LPS could

possibly expand B-cell clones with specificity for a

heterologous LPS preparaticin.

A second possible mechanism to account for the firidlng

that tolerance is sustained even after M- CFU numbers return to

normal comes primarily from the work of Ulevitch ~i ~1· ( 1979,

1981, 1984). These authors reported that the binding of LPS to

high density lipoprotein (H DL) markedly affected the kin e ti cs

and specificity of LPS-cell interactions and the biological

activity of LPS. Specifically, pyrogenic activity and the

ability of LPS to induce neutrop enia in rabbits were inhibit ed.

Further studies were performed by Ulevitch and co - workers Lo

compare the binding of LPS to HDL in normal rabbit serum and

serum derived from LPS-pretreated rabbits. Their results

suggested that a new protein, which they termed gp60, was

produced as an "acute phase reactant'' (as defined by Sipe and

Hosenstreich, 1981) in response to LPS, and thAt gp60 increased

the binding of LPS to HDL. Perhaps in c reased LPS-HDL binding,

facilitated by an LPS-induced acute phase reactan t, u cc u~ s

following repeated injections with homologous or het erologo us

LPS preparations. This, in turn, could limit the availability

of LPS to induce toxic effects, e ven after the numb er of

immature rnacrophages had returned to normal. Such a mechanism

co uld be operative, even in h e terolo go us injeclion sequences,

without invoking the presence of antibodies to clear the LPS.

The effect of ac ute phase proteins on LPS aclivily and Lheir

124

relationship to the tolerant state remains to be explored.

It is also po~s i ble that the alterations in macrophag e

progenitor numbers is an epiphenomenon; the actual mech a nism

which underlies acquired unresponsiveness to LPS is that

exposure of macrophages to LPS renders them refractory to

subsequent stimulation. This could be due to receptor blockad e

and/or down-modulation of the putative "LPS receptor" from the

surface of the cell. The findings of Sullivan ~i ~1· ( 1983)

are consistent with such an hypothesis. These investigators

found that in response to an initial exposure to endotoxin 1n

Yi1rQ, human monocytes produced CSF; how ever , the cultures

rapidly became refractory to further stimulation by LPS, such

that by the fourth day in culture, CSF secretion was n ot

detectable. The diminished response was shown not to be du e to

degradation or "detoxification" of endotoxin, accumulation of a

soluble inhibitor of monocyte synthesis, or n egative feedback

inhibition by newly-synthesized CSF. To date, theirs is the

only repo rt which suggests that human monocytes can be rendered

tolerant to LPS by i9 yA!rQ exposure to LPS. Pr ecedent for

r eceptor down-regulation following ligand-receptor interaction

has been demonstrated in a numb er of systems. For· exampl e ,

after epidermal growth factor (EGF) binds to specific recept o rs

on target cells, aggregation of the hormone-receptor complexes

occurs a nd occupled EGF receptors in coated pits are

e ndo cytosed. The rapid degradation of receptor molecul es by

lysosomal enzymes results in their down ·- regulaLi.•>n because the

receptor mol ec ul es cannot be recycled to the membrane

125

(Schlessinger ~1 !!·, 1984). Other studies have shown that

down-regulation of insulin receptors occurs follow i ng binding

of insulin to its receptor (Marshall and Olefsky, 1979; Conti

et ~!· 1976; Marshall and Olefsky, 1980). This receptor-ligand

interaction induces clustering of insulin receptors

(Schlessinger ~1 ~l·, 1978), and this clustering leads to

internalization of the ligand- receptor complex and, like EGF, a

failure to recycle the receptor to the cell membrane (Anderson

~1 ~!-' 1976).

In the second section of this study, we sought to

characterize further the cell types which participated in the

induction of early endotoxin tolerance. To this end, o ther

murine models with deficiencies or altered responses in

specific cellular compartments were subjected to the same

tolerizing regimen which had been established for outbred ICR

m1ce. Early endotoxin tolerance was induced in inbred,

LPS-responsive (12§0) C3H/OuJ mice, but not in syngeneic,

. ~

LPS - hyporesponsive C3H/HeJ C1E§ ) mice, as assessed by

increased serum CSF, bone marrow M- CFU numbers, and a mark e d

shift in bone marrow cell sizing profile. These results

indicated that expression of the 12§d gene defe c t resulted in :

( 1) an inability to respond to an initial e x posure of LPS to

produce serum CSF [as had been reported previously ( Apte ~! al.

1976 ) ]; and (2) no alteration in either bone marrow M- CFU

numbers or in the bone marrow c e ll sizing profil e . Th e s e

findings are consistent with th e hypothesis th a t C3H / HeJ mi c e

fail to respond to LPS due t o an 1E§d - encoded membrane defect

126

(Coutin ho Ql Ql- 1977; Jakobovitz Ql gl. 1982). If th8

mechanism(s) by which C3H/HeJ and endotoxin-tolerized mice fail

to r espond to LPS were the same. one would expect to obse r ve

both a s hift ed bone marrow cell sizing profile and increased

M-CFU numbers in untreated C3H/HeJ mice. Nei th er wer e observed

in thi s study_ However . one repo rt in the literature has

suggested that M-CFU numbers a r e inc r eased in C3H/HeJ mic e whe n

compared with C3HeB /FeJ ( I !::> ·::; n... rn i c: P ( tvl ·::, c: V ; t ·t· ·1· P .,,,-, cJ· I>JP 1. , •. ,, .. , ,., ,. c· , .::.~ ,,_::::. ) . , _ -· • (. .. .l. , • . - · <.. .• • .•• . . t ... •\_. I .:J 'I

1980). Further studies will be required to confir m t hese

observations in light of recent f indi ngs that C3HeB/FeJ mice

(o) xpr .. r:::'=-~~:; c thE! r· q('! ne ck·' f('C t s ( dj_ s t. i nc t f r"Ofll LE!. ~i d) ,,.Jh 1 ch fll <'.1Y

compromis e;~ thr:? functj.onin9 of th1?ir- mac t .. oph a<;i!'~S (O'B r .i.c•n .:Jnd

Rose nst r eich, 1983).

Wh e r eas normal responsiveness to endotoxin was not seen 1n

mi ce, · BALB/c euthymic (oy/+) and athy rn i.c ( I} IJ./ [! 1.~\ )

mice responded comparably to LPS t o produce CSF and to become

extremely T-cell deficient by FA CS analysi s of their

s plenocvtes (Table VIII). Th ese findings sugges t e d that T

cell s do not contribute s ign ificant ly to the generation of

LPS-induced serum CSF o r t o t he induction of early e ndotoxin

t. o.l.c-:;t-ancH. Previous s tudies in dicated that the presence of th e

nude ge ne on either an LPSn or Lesd backg round fai l ed to alter

LPS-responsiveness o r hypo r esponsiveness, respectively (Vogel

CBA/ N mice express an X-li nked genetic lesion Cxid)

whi ch r esults i n a f ail ur e to respond to certain B ce l l

127

mi tog e ns, including LPS "( Amsbaugh ~1 !!·, 1974 ; Sch er , 1982 ) .

This genetic defect manifests itself as an abs e nce of th e Lyb 5

positive B cell subset in these mice (Ahmed§! !l·, 1977 ) . In

the present study, a third murine model, the C3.CBA / N mous e

strain was utilized because it carries this X- linked gen e ti c

defect (Mond ~! !l·, 1983). This strain was developed by

introducing the ~!~ gene into the C3H/HeN inbred mouse strain

by r e peated cycles of backcrossing. Th e initial cross

consisted of mating a female CBA / N (homozygous for the xi d

g e ne ) with a male C3H / HeN. One of the mal e progeny ( h e mi zygo u s

for the ~!~ gene) was then backcross e d to h is moth e r. Las t ly ,

females from this cross, now termed C3.CBA/N and homo z y g ou s f o r

the xid gene, were crossed with male C3H / HeN mi ce . C3.CBA / N

mic e us e d in this study were at the 13th c y c l e of t his

backcrossing procedure. The probability of homo z ygosi t y a t a

specifi c locus after 12 generations of backc rossing to a n

inbr e d strain has b ee n calculated to be 99.9% ( Klein, 1 9 75 ) .

Mond ~ 1 al. ( 1983) v e rif i ed that, lik e CBA/N mice ,

C3 . CBA / N sple e n c e lls have a grea tly r e du ce d res pons e to LP S

!~ Y!!£2 and the B cell defi c it in this mo~se stra i n could b e

d e monstra te d b y FACS analys i s of ce lls fr om t h e sp l ee n, l y mph

nod e s, and Peyer's patch e s. It wa s shown t ha t th ere a re 2 1 .9% ,

2.7% a nd 18.0% s i gM p os i tiv e ce ll s , r es p ect i ve l y , in these

organs from the C3. CBA/ N mous e , wh ere a s cel l s fro m C~H / H e N

s pl ee n, l y mph nod e s, and P eyer's p a t ch e s yie l ded 51 . 9% , 20.9%

a n d 30. 4% s igM positi ve c el ls, re spe c ti vel y . I n t he cu r r·e n t

t d Lh esc mi ce r e spond e d n orma l ly to a n i n iti~l i n jec t i on o f s . u y ,

128

LPS with a characteristic i nc r ease in the level of serum CSF.

Thi s increase was s hown to be s uppressed after a second

( c hallenge) injection of LPS on day 3. C3.CBA/N mice also

displayed th 0 toleriz e d bone marrow profil e a nd an increase in

bone ma rrow M-CFU number s 3 days post-LPS injecti on . Mice

which express the ~id defect have been s hown to r espond

normal l y to the toxic effects of LPS io YiYQ , and t heir

mac rophages produce norma l leve ls of IL 1 in response to LPS

io ~itLQ (Rosenstreich ~1 ~1-. 1978 ). Thus , the population of

LPS-respons ive B cells which is de f icient in t h is st ra in does

not appear to play an essential role in either LPS

re s pon siveness o r th e induction of e ndotoxi n tolerance.

The recent findin gs of Williams Qt gl. ( 1983) i ndicated

that i n jection of no rmal s plee n ce l ls into LPS-tolerized I CR

mice restored the capacity to respond t o LPS, as asse ssed by

serum CSF induction. These i nves t igator s , howeve r, made no

attempt t o tolerize n o rmal I CR mic e by tr a nsfe r of spleen cells

from tole ri zed mi ce to normal mice because th e numbe r of ce ll s

necessary to ove rride th e n o rmal leve l of responsiveness was

deeme d by th e investigato r s to be experimentally prohibitive

( D. Pluz n ik , personal communi cation ). I n o rder to evaluate the

cont r ibu tion of th e spleen in CSF production and in the

induction of tolerance, sp l en e~tomi zed ICR mi ce were subjected

t o t h e standard tolerizing protocol which was es t a bli shed fo r

normal mice. Th e data indicate that the presence or absence of

a spleen had no influence on the ability of mice to respond t o

LPS t o produce ser um CSF and to be toleri z e d . Thus, sp l een

129

130

cells cannot be considered as a major s ourc e of LPS-induced

CSF, a s s ugges t e d by Williams gt 0l- (1 983 ). Thi s supports t he

a 1 t H r· n a t i v p c; I l CJ CJ p .-::; ·t·· ]. n 1"1 t·-, ''I I.J 1. 1 "J 1. .,, '0 '"' e ·t ''-( 'I (" "I () () .. ,,. ) ·t·.J··, ;_,, ·t. ("_) ·t·.·. L., ~' .... ·'. '•-,,. ' ' " •• ._- • .-•,,, .. , • • w ' V , .. , ··~ , I .. II ._\ ,,::::,, .. : ~ .. ...:.. 0 , ,• 1 .. ,1.._) ~ I '

organs, such as lungs or resident peritoneal macroph ages, may

contribute s ignificantly to the production o f sol ub le se rum

factors after admini s tration of LPS. Changes i n M-CFU numbers

and alterations in bone marrow cell siz ing profi les i n

tol e ri zed, s pl e nec t o mi zed mic e we re also consistent with the

notion that th e s pleen plays little r ole , if any, in th ese

manifes tation s of LPS- induced tolerance.

Th e nex t phase of this proj ect sought to establi s h

whether acquired res i stance to LPS could extend to protection

agai nst bacterial infec tion. The responses observed in t he

early phase of endotoxin tolerance and endotoxin-induced

s t i mulation of resis t a nc e to infections are antibody

independent (Milner, 1971 ). S tudi es by Landy (1956) s howe d

that various bac teri a l lipopoly sacchar ides evoked i n mic e a

rapidly developing, non -specif i c increase in r esistance to

different Gr a m negat i ve bacterial infections. Th e development

of increased resis tance to infection by ~- CQli, e rg t gy§

injection wa s sh own by Landy a nd Pillemer (1956) to be

associated with elevated properdin levels , whi c h, in control

a n i mals was found to fall progressively as th ey became

infected. Th ese i nvestigato r s al s o s howed that r e s istance t o

Cs- §Q[Y9iQQ5Q infection was i ncr eased optimally 24 h after

injection of 1 p 9 §01WQOD110 tYRbQSU LPS i.P- (65% survivo r s),

131

but was r e duced only sligh t l y (SO% survivors) if mic e were

injected with 25- §~[Y9iQQ§0 72 h or 120 h after LPS

administration.

Increased r esis t a nc e to infection s timulated by LPS may

also be related to th e production in vivo of a numhPr nf -- ---- -- -

biolog ically ac tive material s wh ich h ave been fo und to

s timul ate macrophages to become activated. Production of CSF

during infections is a ssociated with enhanced fu nctional

activities o f mac rophages. CSF has been s hown t o sti mu la t e

macrophages to produce prostaglandins (Kurland et 01., 1979) ,

plas minogen activator (Lin a nd Gordon , 1979), and Interl e ukin 1

(Moore ~t 01., 1980 ). I n one report, Handma n a nd Burges s

(1979) demonstrate d t hat partially pur ified

granulocyte-macrophage col o ny stimulating f a ctor caused a

d r amatic increase in the uptake and killing of t he

intracellu lar par a site, L~i5hW0Qi0 !CQPiC0 by macrop hages .

Trudge t t ~t 0l- (1973) s tudied th e e ff ec t o f CSF o n the io

vitLQ phagocytic and mi c r obicidal activity of mouse per itonea l

macrophages in c uba t ed with ~BliDQD§llB t~~bimYriYm- They found

that CSF induced a signif icant e nhan c ement of ~- iY~himYriYm

killing by mac r o ph ages, although there was no e ffect on th e

number of bac teri a phagocytosed . Ano ther macrophag e product

th a t is produced in res pon se t o LPS is interferon (Youngner and

Sti n ebring, 1965). Infection of experimental ani mals withE-

(Remington a nd Merrigan, 1970) has bee n s hown to be s uppr e ssed

bY prior injection o f interferon or inter f eron inducer s .

Lastly, the survival rate of mice injected with KlQ~Si?ll~

eQQYffiQDi0 one day after injection with endotoxin or other known

interferon Inducers was shown to be increased (Pindak, 1970).

I n order to determine the temporal relationship of

early phase endotoxin tolerance to LPS-induced non -specif ic

resistance to infection, control and tolerized ICR mice wer e

challenged with E§QY~QffiQDDS DgrY9iDQS0 using the burn mode l of

Pav lovskis ~1 0l- (1977). The rapid progression of f atal Gram

negative infection associated with this model was necessary if

resistance to infectioh were to be detected during the

transient early ph ase tolerance period (refer back to Figure

1). The two bacterial products most likely implicated in the

systemic infection caused by Es- QQLY9iDDSD are

lipopolysaccharide and exotoxin A. Evidence associating Ps.

QQ[~Si09§0 endotoxin with sepsis is extensive (reviewed by

Young, 1979). Exotoxin A (Liu, 1966) in its pu rifi ed for m 15

highly lethal for animals and produces shock in dogs (Ati k et

01 .• 1968) and rhesus monkeys (Pavlovskis et 01., 1975).

Tolerized mice initially displayed a high degree of resistance

when compared to similarly infected control mice , but such

resistance was shown to wane rapidly (2 days post-bacterial

challenge; 5 days post-LPS admini s tration), at a time when

early endotoxin tolerance would normally be less th a n optimal

(see Figure 1). It i s interesting to not e that a s tatistically

sign ificant delay in death was observed only at the h ighest two

doses of bacteria. This may reflect a contribution of LPS frpm

the bacterial challenge, which acts to serve as an additional

132

''tol:::)r·iz.inq'' inj,~ction. t1ort· i.son <:1 nd Curt·v (.19/9) hd\/ t'.·

es timated that approximately 40 ~g of LPS could be expected 1 0

from 10 Gram negative organisms. In consideration of these

results, repeated tolerizing injections prior to bacterial

infection would have, perhaps, proven to be more efficacious in

the protection of mice against E§~YdQffiQQQ§ infection . A

reduced time interval between LPS administration and bac te ri~l

challenge also could have yielded a greater degr ee of

re s i stance to infection.

The major obstacle which has limited the cl in ical

application of endotoxin for its beneficial effects has bee n

its toxic side effects. Chemical modification of the e ndotoxi n

molecule has resulted in a non-to xic, Lipid A derivative,

monophosphorvl Lipid A (MPL) (Qureshi§! ~1 . , 1982) which is

greatlv reduced in its toxic activity . In the final section of

this study , the efficacy of MPL to r epl ace intact LPS in the

early e ndotoxi n tolerance protocol was exafuined. Unlike

:i.nit:ia.l expcsure to wild typF::· I_PS, t1PL did not indu.cf::! sy rHpt orn:::;

of endotoxicity, i .e., ruffled fur, conjunctival discharge, or

diarrhea, even at a dose of 100 pg per mouse. Des pite the

absence of these overt manifestations of e ndotoxicity, MPL wa s

s hown to induce both s erum colony s timulating fact o r a nd

interferon to l evels induced by wild type LPS . It wa s also

found that injection of MPL on day 0 reduced th e LPS-i nduced

serum CSF upon c hall enge with wild-type ~- ~Qli LPS 3 days

late r. MPL was also s hown to increas e greatly the LD 50 to

challenge i n jection with E. ggli LPS and ef fectively r educe

133

134

overt s ymptoms of endotoxicity upon challenge with intact LPS.

As seen in the LPS-induced tolerance system , which was

established in th e fir s t phase of thi s s tudy, MPL

admini s tration greatly increased the number of macrophage

progenitor cells in the bone marrow a nd this incr ease in M-C~U

co i ncided with an increase in cell size and cell number i n

Coulter Channelyzer pr o f i le ''Peak 2''. Taken collective ly,

these results indi ca te that MP L possesses the necessa ry

st ructur al compon e nt( s) required to induce a state of early

endotoxin t ole rance. Most importantly, MPL failed t o induce

the initial toxic react i ons typically observed with wild-type

e ndoto xin or diphosphoryl Lipid A preparations. One int rigu ing

aspect of th is study was that tolerance, a s ass e ssed by IFN

pr oduc ti on, was muc h l ess affected by MPL than were th e other

.man ifestations of LPS respon si ven ess . One possib l e implication

is tha t IFN may be less i nvolved in the in du c ti on o f th e to x i c

effects o f LPS than other se rum fa c t o r s , suc h as CSF. I n thi s

r ega rd, previous wo rk by Moore Q1 Ql - (1980) s upports a role

for CSF as a n immunoregulatory agent capable of r e nde ri ng

macrophages more sensitive to LPS i o ~i1rg . as assessed by

increased product ion of IFN and Interleukin 1.

In s ummary, an early ph ase endotoxin tolerance model

was established in mi ce based on a syste m deve loped by Wi lliams

t ·1 ('l~n~) ·1·1- 1 ('-~· t " f.'.'.'.--".: l)_.·l. 1:c.; 1~resen~ed h e r ei n indicate that the Q~ ~~- .. ;o~ • ·- _ L

s tate of early tol er ance is a ssoc i ated with marked alteration s

in bone marrow progenitor pools. The cellula r mechanisms which

underlie thi s sta t e of induced h yp o res pon siveness we r e pr obed

further using various murine models to assess the relative

cont ribution of certain lymphoid cell subsets. The temporal

association of early phase tolerance and non-specifi c

resistance to Gram negative infection wa s examined using

P~QY0QffiQQ~~ QQLY9iDQ~0 as the infecting agent. Lastly, a

non-toxi c Lipid A derivative , monophosph ory l Lipid A, was show n

to be efficacious as an inducer of early phase to leranc e_

These findings, taken collectively, ex t end our present

understanding of the cellular mechan isms which contribute to

the acquisition of hyporesponsiveness t o the toxic

man ifes tation s of endotoxin. An understanding of acquired

LPS-hyporesponsiveness in experimental animals should furt her

our under~tanding of LPS responsiveness. Moreover , the use of

non -toxic derivatives of LPS to induce a hypor esponsive state

to the toxic effects of endotoxin holds great potential for

their use in therapeutic inte rvention.

135

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..... I '•.d:~ •·:; ...

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