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UNIFORMED SERVICES UNIVERSITY OF THE HEALTH SCIENCES F. EDWARD HEBERT SCHOOL OF MEDICINE
4301 JONES BRIDGE ROAD BETHESDA, MARYLAND 20814
SCHOOL OF MEDICINE APPROVAL SHEET TEACHING HOSPITALS
WALTER REED ARMY MEDICAL CENTER NAVAL HOSPITAL, BETHESDA
MALCOLM GROW AIR FORCE MEDICAL CENTER WILFORD HALL AIR FORCE MEDICAL CENTER
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
Thesis and Abstract Approved:
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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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