The influence of lipopolysaccharide (LPS) on cellular activities in LPS-unresponsive C3H/HeJ mice
by Patricia Anne Nelson Rampy
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in Microbiology
Montana State University
© Copyright by Patricia Anne Nelson Rampy (1976)
Abstract:
It has been previously reported that LPS-unresponsive C3H/HeJ mice are refractory to LPS at the B
lymphocyte level. The current study was undertaken to determine if other LPS-influenced cellular
activities were also defective in these mice. Utilizing adult CBA/J and C3H/HeJ mice as spleen donors,
Graft-versus-Host (GVH) reactions were induced in Balb/c neonates. Spleen weight indices failed to
distinguish between experimental groups so mortality by day 30 was used as the criterion of induction
of GVH disease. Prior LPS treatment of CBA/J adults decreases the ability of their spleen cells to cause
fatal GVH disease in Balb/c neonates whereas no difference was found between injection of normal or
LPS-treated C3H/HeJ spleen cells. Similar observations were found with these cell types using the
mouse spleen mixed leukocyte culture system. There was, however, no suppression of ensuing GVH
disease in Balb/c neonates after treatment of spleen donors of either CBA/J or C3H/HeJ origin with
butanol-extracted LPS, a preparation previously shown to be mitogenic for C3H/HeJ B lymphocytes. In
studies on the effects of serum on GVH disease induction by normal cells, it was found that inoculation
of normal CBA/J cells with normal CBA/J serum results in a GVH reaction similar to inoculation of
the same type of cells in Hank's balanced salt solution while inoculation in LPS-CBA/J serum shows a
significant decrease in resulting mortality. With other combinations of normal or LPS-serum with
CBA/J or C3H/HeJ cells, the critical factor appears to be the strain source of the serum rather than prior
treatment of the serum donors with LPS or not. There was a nonspecific suppressive effect of C3H/HeJ
serum on both C3H/HeJ and CBA/J cells. In another system, layering normal spleen cells on adherent
cells taken from LPS-treated CBA/J animals results in loss of GVH reactivity of the subsequent
nonadherent preparation whereas incubation with adherent cells from LPS-treated C3H/HeJ mice or
normal allogeneic adherent cells produces no such decrease in GVH reactivity. In a carbon clearance
assay for stimulation of the reticuloendothelial system with LPS, it was found that the rate of
phagocytosis is significantly increased in Balb/c and CBA/J mice 72 hours after inoculation of LPS. No
stimulation is seen in rate of carbon uptake in the C3H/HeJ animals after treatment with
phenol-extracted LPS or with butanol-extracted LPS. Finally, pretreatment of CBA/J or Balb/c mice
trypan blue increases susceptibility to the lethal effects of LPS approximately 12 fold. The main
conclusion drawn from this research is that, in addition to the previously reported unresponsiveness of
C3H/HeJ B lymphocytes, C3H/HeJ macrophages are also refractory to stimulation by LPS. STATEMENT OF PERMISSION TO COPY
In presenting th is thesis in p a r tia l f u lfillm e n t o f the
requirements fo r an advanced degree a t Montana State U n iv e rs ity ,
I agree th a t the Library shall.make i t fr e e ly a v a ila b le fo r inspec
tio n .
I fu rth e r agree th a t permission fo r extensive copying of
th is thesis fo r scholarly purposes may be granted by my major
professor, o r , in his absence, by the D ire cto r of L ib ra rie s .
It
is understood th a t any copying or pu blicatio n of th is thesis fo r
fin a n c ia l gain shall not be.allowed w ithout my w ritte n permission.
. Date
THE INFLUENCE OF LIPOPOLYSACCHARIDE (LPS) ON
CELLULAR ACTIVITIES IN LPS-UNRESPONSIVE
C3H/HeJ MICE
by
PATRICIA ANNE NELSON RAMPY
A thesis submitted in p a r tia l fu lfillm e n t
o f the requirements fo r the degree
of
MASTER OF SCIENCE
in
Microbiology
MONTANA STATE UNIVERSITY
Bozeman, Montana,
August, 1976
Ill
ACKNOWLEDGMENTS
I wish to express my deepest g ra titu d e and appreciation
to Dr. John J u tila fo r his invaluable support and guidance through­
out my years a t Montana S tate U n iv e rsity.
I also wish to thank
the fa c u lty , s t a f f , and graduate students of the Department of
Microbiology fo r the p a rt they have played in my education,
e s p e cia lly Dr. Norman Reed and Dr. James C utler fo r taking the
time to answer my many questions.
I wish to acknowledge Dr. James
C utler and Dr. A. Rudbach fo r supplying cultures of Escherichia
c o l l 0113, Dr. Kelsey M ilner fo r analyzing the potency o f the LPS
preparation, and Dr. David Morrison fo r supplying the sample of
butanol-extracted LPS.
Last, but c e rta in ly not le a s t, I want to
thank my fam ily and friends fo r having f a it h in me when I most
needed i t .
TABLE OF CONTENTS
Page
VITA .......................................................................................................
ACKNOWLEDGMENTS
ii
.......................................... .................... .... . ..................... i i i
LIST OF TABLES......................................................................
ABSTRACT
V
.....................................
vi
INTRODUCTION. . ,....................................................
I
MATERIALS AND METHODS
4
RESULTS
. . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . .
..................................................
. . .
DISCUSSION.......................................................................................................
REFERENCES
12
.
26
38
V
LIST OF TABLES
Table
I.
II.
III.
IV .
V.
V I.
V II.
V III.
Page
Graft-versus-Host disease in Balb/c
neonates assayed using spleen weight indices . . . . . .
13
G raft-versus-Host disease in Ba!b/c mice
given spleen c e lls from normal and
LPS-treated CBA/J and C3H/HeJ mice .....................................
15
Mouse spleen mixed leukocyte cultures assaying
r e a c tiv ity o f normal or LPS-treated CBA/J
and C3H/HeJ leukocytes against mitomycin
C-treated (Me) Balb/c c e lls
..................................................
16
G raft-versus-Host r e a c tiv ity o f spleens from
mice treated w ith butanol-extracted LPS ......................... 18
Effects o f serum on G raft-versus-Host
r e a c tiv ity of WN c e lls when in je c te d
together in to Balb/c neonates ..............................................
19
G raft-versus-Host r e a c tiv ity a f t e r layering
WN c e lls on normal or LPS-adherent c e lls ......................... 21
Rate o f phagocytosis in normal or LPS-treated
mice measured by a carbon clearance assay .....................
23
A comparison o f LPS LDt5n in normal or trypan
blue pretreated mice . ................................................................... 25
vi
ABSTRACT
I t has been previously reported th a t LPS-unresponsive C3H/HeJ
mice are re fra c to ry to LPS a t the B lymphocyte le v e l. The current
study was undertaken to determine i f other LPS-influenced c e llu la r
a c t iv it ie s were also defective in these mice. U t iliz in g adult
CBA/J and C3H/HeJ mice as spleen donors, G raft-versus-Host (GVH)
reactions were induced in Balb/c neonates. Spleen weight indices
fa ile d to distingu ish between experimental groups so m o rta lity by
day 30 was used as the c r ite r io n o f induction of GVH disease. P rior
LPS treatment o f CBA/J adults decreases the a b il it y o f th e ir spleen
c e lls to cause f a ta l GVH disease in Balb/c neonates whereas no d i f ­
ference was found between in je c tio n o f normal or LPS-tre a te d C3H/HeJ
spleen c e lls . S im ilar observations were found with these c e ll types
using the mouse spleen mixed leukocyte c u ltu re system. There was,
however, no suppression o f ensuing GVH disease in Balb/c neonates
a fte r treatm ent of spleen donors o f e ith e r CBA/J or C3H/HeJ o rig in
with butanol-extracted LPS, a preparation previously shown to be
mitogenic, fo r C3H/HeJ B lymphocytes. In studies on the e ffe c ts of
serum on GVH disease induction by normal c e lls , i t was found th a t
in oculation o f normal CBA/J c e lls w ith normal CBA/J serum resu lts
in a GVH reaction s im ila r to in oculatio n o f the same type of c e lls
in Hank's balanced s a lt solu tion w hile inoculation in LPS-CBA/J
serum shows a s ig n ific a n t decrease in re s u ltin g m o rta lity . With
other combinations o f normal or LPS-serum with CBA/J or C3H/HeJ
c e lls , the c r it ic a l fa c to r appears to be the s tra in source of the
serum ra th e r than p rio r treatment o f the serum donors w ith LPS or
not. There was a nonspecific suppressive e ffe c t o f C3H/HeJ serum
on both C3H/HeJ and CBA/J c e lls .
In another system, layering normal
spleen c e lls on adherent c e lls taken from LPS-treated CBA/J animals
re su lts in loss o f GVH r e a c tiv ity o f the subsequent nonadherent
preparation whereas incubation with adherent c e lls from LPS-treated
C3H/HeJ mice or normal allogeneic adherent c e lls produces no such
decrease in GVH r e a c t iv ity .
In a carbon clearance assay fo r stimu­
la tio n o f the re tic u lo e n d o th e lia l system with LPS, i t was found th a t
the ra te o f phagocytosis is s ig n ific a n tly increased in Balb/c and
CBA/J mice 72 hours a f t e r in oculatio n o f LPS. No stim ulatio n is
seen in ra te of carbon uptake in the C3H/HeJ animals a f t e r treatment
w ith phenol-extracted LPS or w ith butanol-extracted LPS. F in a lly ,
pretreatm ent of CBA/J or Balb/c mice trypan blue increases suscepti­
b i l i t y to the le th a l e ffe c ts of LPS approximately 12 fo ld . The
main conclusion drawn from th is research is th a t, in addition to the
previously reported unresponsiveness o f C3H/HeJ B lymphocytes,
C3H/HeJ macrophages are also re fra c to ry to stim ulation by LPS.
INTRODUCTION
The lipopolysaccharide (LPS) fra c tio n o f gram-negative
b a c te ria l c e ll w a lls , also re fe rre d to as endotoxin, has been shown
to possess a wide v a rie ty of b io lo g ic a l a c t iv it ie s when in jected in to
higher animals.
These properties include le t h a li t y , t o x ic it y ,
pyrog en icity, immunogenicity, a d ju v a n tic ity , and Schwartzman
r e a c tiv ity ( I ) .
LPS is also a B c e ll (bone marrow-derived lymphocyte)
mitogen (2) and a s tim u lato r o f polyclonal responses (3 ).
In 1940, H ill f i r s t reported the development o f an endotoxinre s is ta n t s tra in o f mice by successive breeding o f challengedre s is ta n t generations (4 ) .
More re c e n tly , S u ltzer found the C3H/HeJ
mouse s tra in to be highly re s is ta n t to the le th a l e ffe c ts o f LPS and
to e x h ib it a d iffe r e n t pattern o f leukocyte accumulation than is
usually seen a f t e r in tra p e rito n e a I ( i . p . ) in je c tio n o f LPS (5 ).
In a d d itio n , LPS does not function e ith e r as a mitogen (6 ,7 ) or
an adjuvant in these mice, and the immune response induced is
tra n s ie n t ( 7 ) .
Although the genetics of the LPS-nonresponsiveness
has not been resolved to date, S u ltze r indicated th a t the le th a l
e ffe c ts o f LPS and the d if fe r e n t ia l leukocyte accumulation are under
polygenic control ( 8 ) , whereas Watson and R ib le t have demonstrated
th a t a sin g le dominant autosomal gene governs mitogenic and immune
responsiveness (6 ) .
Using c e ll mixing experiments, i t was shown
2
th a t the f a ilu r e o f C3H/HeJ spleen c e lls to e x h ib it normal responses
to LPS was due to nonactivation o f B lymphocytes, even though the
c e lls bound LPS as w ell as B c e lls from LPS-responsive mouse strains
(9 ).
I t was suggested th a t a membrane defect was involved and i t
has since been reported by Glode e t aI (10) th a t the binding of LPS
to C3H/HeJ B lymphocyte membranes leads to th e ir in a c tiv a tio n ,
causing a dose- and time-dependent suppression o f B c e ll stim ulation
by Poly I , another B c e ll mitogen.
The e lu c id a tio n o f a B c e ll membrane defect to account fo r
some o f the observed nonresponsiveness to LPS s t i l l does not account
fo r the high resistance o f the C3H/HeJ to the le th a l e ffe c ts of LPS.
A c tu a lly , the p o s s ib ility is raised th a t s im ila r membrane components
may be involved in other LPS in te ra c tio n s in vivo w ith c e ll systems
not y e t explored.
A c la s s ic system th a t has been used to study immunological
c e llu la r in te ra c tio n s is the G raft-versus-H ost (GVH) re ac tio n .
This
phenomenon was f i r s t described in 1916 by Murphy (11) a f t e r he
inoculated a d u lt chicken spleen c e lls onto the c h o rio a lla n to ic
membranes o f young chick embryos.
The sig n ifican ce o f th is reaction
was not re a liz e d u n til 1953 when Dempster and Simonsen (12) inde­
pendently expressed the idea th a t the g r a ft may be capable of
mounting an immunological reaction against the host under c e rta in
circumstances.
A considerable amount o f lit e r a t u r e has been
3
published in th is area since then, and an extensive review has
re cen tly appeared by Grebe and S t r e ile in (1 3 ).
Since GVH disease
is a real problem to immuno-deficient patients who must be reco nsti­
tuted w ith lymphoid or lymphoid precursoral tis s u e , the abrogation
o f GVH disease has been the s p e c ific subject of in ten sive research.
In th is regard, Chedid (14) has shown th a t pretreatm ent o f donor
splenocytes in vivo and in v itr o with LPS w ill suppress the GVH
reactio n .
Since then, Thomson and J u tila (15) have reported th a t
i t is the adherent population o f LPS-treated spleen c e lls responsible
fo r th is suppression, and th a t these c e lls in te r fe r e in some way
w ith a llo a n t igen-reactive c e lls in the nonadherent population which
cause the GVH disease.
I t was established by Bona (16) th a t the
macrophage is the c e ll responsible fo r processing LPS so i t is
p e rtin e n t to ask i f LPS modifies th is c e ll or i f i t promotes pro­
duction of a product which m obilizes other c e ll types.
The o b jective o f the current study was to bring research
on GVH disease together w ith the natural LPS-resistance o f the
C3H/HeJ mouse s tra in .
I t was hoped th a t such an in ve s tig atio n
would reveal new inform ation on the suppression o f GVH disease and
possibly bring to lig h t ad d itio n al in vivo mechanisms influenced
by LPS-nonresponsiveness in C3H/HeJ mice.
.
MATERIALS AND METHODS
Mice.
Normal fiv e week-bid C3H/HeJ and CBA/J male and female
mice were purchased from the Jackson'Laboratories, Bar Harbor, Maine,
and maintained in our laboratory in a conventional environment u n til
use.
Conventionally reared Balb/c mice were obtained from our own
stock.
A ll mice were given s t e r iliz e d Purina 5010C and a c id ifie d -
chlorinated water.
G raft-versus-H ost Disease.
Spleens were removed from adult
C3H/HeJ or CBA/J mice 8-12 weeks o f age and placed in 5 .0 ml of
Hank's balanced s a lt solution (BSS) w ith 5 percent fe ta l c a lf serum
(PCS, Grand Island B io lo g ic a l) in Falcon 60.x I S p e t r i dishes
held on ic e .
Spleens were gently teased apart in to s in g le c e ll
suspensions, pipetted and passed through an 80 mesh stain less steel
screen.
in BSS.
The c e lls were then counted, centrifuged, and resuspended
7
A to ta l o f 2 x 10 spleens c e lls contained in 0.1 ml were
in je c te d i . p . in to Balb/c neonates less than 24 hours o ld .
Cell .
preparations o f th is type are re fe rre d to as whole normal (WN)
c e lls .
Induction o f G raft-versus-Host (GVH) disease was measured
i n i t i a l l y with a spleen weight assay (1 7 ).
on day 10 a f t e r in je c tio n o f a d u lt c e lls .
Mice were s a c rific e d
The spleen index was
taken to equal the spleen weight/body weight o f the experimental
5
neonates divided by the spleen weight/body weight o f control neonates
in je c te d with BSS.
Death between days 7 to 10 or an index of 1.50
or greater was regarded as in d ic a tiv e o f an ongoing GVH process.
The mice th a t died before s a c r ific e were given an a r b itr a ry index
o f 2.0 fo r the purpose o f c a lc u la tio n o f the average in dices.
Because spleen indices did not discrim inate between experi­
mental groups, death in the in te rv a l day 7 to 30 was used as the
c r ite r io n o f GVH disease in a ll the la t e r work.
Lipopolysaccharide (LPS).
LPS was extracted from
Escherichia c o li 0113, cultures kindly provided by Dr. J. C u tle r,
Montana State U n iv e rs ity , and Dr. A. Rudbach, U niversity o f Montana,
using the hot phenol-water technique o f Westphal and Jann (18);
The only m odification was to incubate the phenol-water mixture a t
65C fo r 30 minutes ra th e r than 15 minutes.
The f i r s t water extracts
from two separate extractions were pooled and dialyzed fo r four days
in double d i s t i lle d water.
Sodium acetate was added to 0.15 M and
the LPS was p re c ip ita te d w ith 68 percent cold alcohol (f in a l
concentration).
The p re c ip ita te was dissolved in double d is t ille d
w ater, dialyzed fo r an addition al four days and then lyo p h iI i zed.
The potency of the preparation was established by Dr. Kelsey M iln e r,
Rocky Mountain Laboratories, Hamilton, Montana.
The re su lts of
these bioassays showed th a t the LPS had a chick embryo LDg0 of
0.0073 yg and FI^0 value in rabbits of 0.56 yg (1 9 ).
6
The butanol-extracted LPS (B-LPS) used in two experiments
was a generous g i f t from Dr. David Morrison, Scripps C lin ic and
Research Foundation, La J o lla , C a lifo rn ia (2 0 ).
M odification o f the GVH response by treatment o f donor c e lls .
A.
Adult spleen c e ll donors 8-12
i
weeks o f age were given seven or e ig h t d a ily in je c tio n s o f 60 pg
LPS i . p .
LPS in je c tio n o f donors.
W ithin 24 hours a f t e r the la s t in je c tio n , mice were bled
fo r "anti-LPS" serum (LPS-serum) and spleens were removed.
Cells
(LP S -strain) were harvested in the usual manner and 2 x 10
c e lls
per
0.1 ml were in je c te d in to Balb/c neonates less than 24 hours
old.
B.
Normal, or LPS serum given w ith normal donor c e lls .
Spleen c e lls were harvested from normal a d u lt mice, counted,
7
pipetted in to small tubes, and centrifuged.
A to ta l o f ;2 x 10
c e lls was resuspended in 0.1 ml o f appropriate serum or in Hank's
BSS containing 5 percent FCS to serve as c o n tro ls, allowed to
incubate 10 minutes a t room temperature, and in jected i . p . in to
Balb/c neonates.
C.
"Enriched" c e ll populations.
Spleens were removed from
normal or LPS-treated a d u lt mice and placed in 5.0 mis Hank's BSS
plus FCS and held on ic e .
Spleens were gently teased in to single
c e ll suspensions, p ip e tte d , passed through an 80 mesh stain less
steel screen, and d is trib u te d in to fiv e 60 x 15 mm p la s tic Falcon
7
p e tri dishes per spleen.
A fte r incubation fo r one hour a t 37C,
nonadherent (Nonad) c e lls were decanted, washed two times w ith BSS,
and then adherent (Adh) c e lls were scraped fre e w ith a rubber
policeman.
I f c e lls were to be in je c te d a f t e r th is i n i t i a l separa­
tio n procedure, c e lls were washed, counted and 5 x 10® adherent
c e lls per 0.1 ml or 2 x IO^ nonadherent c e lls per 0.1 ml were
in jected i . p . in to Balb/c neonates.
A lte rn a tiv e ly , i f possible absorptive removal o f GVH
r e a c tiv ity by adherent c e lls was desired, 5 x 10
6
adherent c e lls
were returned to clean p e tri dishes and allowed to re-adhere fo r
30 minutes a t 37C.
A fte r th is tim e, 2 x IO^ normal spleen c e lls
were added to each dish and incubated an additional one and a h a lf
to two hours, a t 37C.
Nonadherent c e lls were removed, centrifuged
and the nonadherent content of one dish in 0.1 ml was in je c te d i . p .
in to Ba!b/c neonates.
Carbon clearance assay.
The procedure followed was modified
from the Handbook of Experimental Immunology (2 1 ).
Normal and LPS-
treated mice, four mice per te s t group, 10-13 weeks o f age, were
used to assay in vivo phagocytosis.
Mice receiving LPS were injected
i . v . with 20 yg in 0.1 ml 72 hours before assay.
Carbon from
Gunther, Pelikan Werke, Hanover, Germany, was d ilu te d in phosphate
buffered s a lin e (PBS) to 16 mg/ml.
Each animal was weighed and the
weight divided by 100 was the amount in m i l l i l i t r e s fo r in je c tio n .
;
8
Mice were in je c te d i . v .
in to the t a i l vein.
Tw enty-five lambda
of blood were drawn from the r e tr o -o r b ita l plexus a t three minute
in te rv a ls from time 0 minutes to 15 minutes w ith heparinized 25
lambda micropipets and placed in four ml o f s t e r ile s in g le d is t ille d
water.
Optical density (O .D .) was read on a K le tt spectrophotometer
using a red f i l t e r .
The O.D. readings were converted to a
logarithm ic scale and p lo tte d versus time.
The slope o f th is lin e
is the phagocytic c o e ffic ie n t K, which is a measure o f the ra te of
phagocytosis.
. Mixed leukocyte c u ltu re .
The follow ing protocol fo r mouse
spleen mixed leukocyte c u ltu re was only s lig h tly modified from the
one received from Dr. F r it z Bach, U n iversity of Wisconsin, Madison,
Wisconsin.
RPMI 1640 (Gibco) c u ltu re medium was supplemented with
p e n ic illin , 100 u n its /m l, and streptomycin, 100 pg/m l.
Frozen
human plasma, obtained from a large pool collected a t Bozeman
Deaconess H o s p ita l, was thawed and hard-spun a t 1650x g fo r 10
minutes.
A fte r heat in a c tiv a tio n a t 56C fo r 30 minutes, the plasma
was hard-spun tw ice, and added to a fin a l concentration o f 5 percent
plasma in RPMI where in dicated.
To prepare responding and stimu­
la tin g c e lls , spleens were removed a s e p tic a lly in to 60 x 15 mm
p la s tic p e tri dishes (Falcon #3002) held on icq.
Approximately 1.0 ml
o f c u ltu re medium without plasma was in je c te d in to each end o f the
splenic capsule to release c e lls .
The spleen was then gently teased
9
with curved forceps u n til a ll the c e lls had been removed from w ithin
the capsule.
Cell clumps were separated by repeated p ip e ttin g with
a 5 ml serological pi pet ( Falcon #7543).
The suspension was passed
one time through a 27 gauge needle in to a 15 ml ce n trifu g e tube
(Falcon #2095) and then centrifuged a t 180x g fo r 10 minutes in
the cold.
The supernatant was discarded and the c e ll p e lle t was
resuspended in 0 .5 ml of PBS.
Red blood c e lls were lysed by a 10
second exposure to 4 .0 ml o f s t e r i le , sin g le d is t ille d w ater, p rio r
to returning the d ilu e n t to is o to n ic ity using 0 .5 ml o f 10X PBS.
Cells were c e n trifu g ed , supernatant decanted, and p e lle t resuspended
in 5 ml c u ltu re medium.
. At th is point,, responding c e lls were counted in a hema­
cytometer and d ilu te d to appropriate concentration by adding the
amount o f plasma needed to bring the fin a l concentration to 5 percent.
These c e lls were then kept cold u n til added to p late s .
To the
stim ulatin g c e lls , mitomycin C was added a t a concentration of
25 yg per ml c e ll suspension and mixed thoroughly.
placed in a 37C water bath fo r 25 minutes.
The mixture was
Cells were then c e n tri­
fuged, supernatant decanted, and p e lle t resuspended in medium.
Cells were then washed two times by c e n trifu g a tio n and decantation.
A fte r fin a l wash, c e lls were counted and d ilu te d to appropriate
concentration, making the fin a l plasma concentration 5 percent.
10
The responding c e lls a t a concentration o f I x IO^ and
I x 10® stim ulatin g c e lls per well were then d is trib u te d
in d iv id u a lly in 0.1 ml volumes to Linbro m icroti t e r plates (Falcon
#3040 and #3041) using an 100 lambda Eppendorf pi pet with s t e r ile
tip s .
Cultures were incubated a t 37C in a humidified 5 percent
CO2 , 95 percent a i r atmosphere incubator.
incubation, 2 yCi
A fte r 72 hours of
H-thymidine (s p e c ific a c t iv it y 20 Ci/mmole)
was added to each w ell in 0.05 ml volumes.
Sixteen hours la t e r ,
cultures were p re c ip ita te d onto Whatman glass fib e r f i l t e r s with
s a lin e , 5 percent tric h lo ro a c e tic a c id , and methanol using an Otto
H ill e r semi-automatic m u ltip le sample p re c ip ita to r.
Uptake o f the
isotope, was determined by placing the f i l t e r paper discs containing
the c e ll p re c ip ita te s in to v ia ls with Aquasol (New England Nuclear)
and counting in a Beckman LS-100C Liquid S c in t illa t io n System.
The data represent the re su lts o f t r ip l ic a t e samples from
which the sum o f the counts o f the responding c e lls alone and the
stim ulatin g c e lls alone have been subtracted.
E ffe c t o f trypan blue on LPS LD^q .
Trypan blue from Kallestad
Labs, In c ., Minneapolisj Minnesota, was dissolved in double d is t ille d
water and.dialyzed against d is t ille d water fo r 72 hours a t 4C.
The
d ia ly s a te was lyo p h iI i zed and stored in a tig h tly stoppered b o ttle
a t room temperature u n til used.
At time o f assay, a fresh sample
was resuspended to 10 mg/ml in 0.85 percent s a lin e .
The mice
n
receiving trypan blue were given 4 .0 mg i . p . 24 hours before and
1.0 mg i . p . two hours before LPS in je c tio n .
A ll animals were then
given the specified dose o f LPS i . p . and observed fo r m o rta lity
w ith in 72 hours.
In most cases, the animals were two to three
months old and four or fiv e mice were injected, per dose group.
The LDgg in each assay was computed using the Reed-Muench technique
( 2 2 ).
S ta tis tic a l a n a ly s is .
The data presented as means of
in divid ual samples were analysed using the student t te s t.
The
te s t was modified fo r groups o f unequal sizes where appropriate.
Al I m o rta lity values were compared using the z t e s t, derived from
the normal approximation to the binom ial.
In a ll cases, data were
tested fo r s ig n ific a n c e a t the p=0.05 level (2 3 ).
RESULTS
GVH assayed using spleen weight in d ic e s .
To determine
whether spleen indices are useful fo r measuring GVH r e a c t iv it y ,
neonatal Balb/c mice were given e ith e r 2 x 10
7
whole normal (WN)
6
or nonadherent (Nonad) c e lt s , or 5 x 10° adherent (Adh) c e lls from
normal CBA/J or C3H/HeJ ad u lts.
Other neonates received 2 x IO^
whole spleen c e lls from CBA/J or C3H/HeJ adults given LPS fo r seven,
successive days (LPS-).
Spleen indices were then calculated on
c e ll or BSS in je c te d mice 10 days p o s t-in je c tio n .
The number of
animals in each group having an index greater than or equal to
1.50 a t day 10, and the average index fo r each te s t group are shown
in Table I .
Both the WN CBA/J and Nonad CBA/J spleen preparations
caused 100 percent GVH disease in the in je c te d Balb/c neonates as
judged by th is c r ite r io n .
Although the average indices o f the two
groups were d if fe r e n t , they were not s ig n ific a n tly so.
The average
index fo r mice given Adh CBA/J was s ig n ific a n tly lower than the
index calculated fo r mice given whole normal spleen c e lls .
The
spleen c e lls from the LPS-CBA/J mice gave spleen weight indices
th a t were e s s e n tia lly the same as nontreated c e lls .
A wider range o f spleen indices was seen w ith various types
o f c e lls from the C3H/HeJ mice.
High incidence o f GVH disease was
caused by both the WN and Nonad-C3H/HeJ c e ll populations, as indicated
13
TABLE I
Graft-versus-Host disease in Balb /c neonates assayed using spleen
weight indices
Cell O rig in3
WN-CBA/J
Nonad-CBA/J
I>1.50 / T o ta lb
Average Index
7 / 7
2.18
14 / 14
2.00
1 .69* C
Adh-CBA'/J
8 / 12
LPS-CBA/J
14 / 17
1.97
WN-C3H/HeJ
29 / 32
2.17
Nonad-C3H/HeJ
16 / 19
1.95
Adh-C3H/HeJ
2 / 11
1 .34* +
C3H/HeJ kidney
0 / 8
1.06* + X
LPS-C3H/HeJ
12 / 19
1.97*
O
a2 x IO7 MN, Non, or LPS c e lls or 5 x 10® Adh c e lls injected
i . p . in to Ba!b/c neonates 24 hr old. See M aterials and Methods or
te x t fo r explanation of abbreviations.
^Balb/c mice were s a c rific e d 10 days p o s t-in je c tio n . Spleen
index equals spleen weight/body weight of experimental neonates
divided by spleen weight/body weight of control neonates. T h irty four BSS controls were included. Those neonates dead between days
7-10 were given an a rb itra ry index of 2 .0 . Numerator designates
number o f mice with an index > 1.50. Denominator designates to ta l
mice in each group.
c* = s ig n ific a n t from WN, + = s ig n ific a n t from Nonad, x =
s ig n ific a n t from Adh, and o = s ig n ific a n t from kidney using the
student t te s t fo r unequal group sizes a t p = 0.05 le v e l.
14
by the number o f mice experiencing enlarged spleens and by mean
spleen index.
With th is mouse s tr a in , no s ig n ific a n t disease was
caused by adherent c e lls or by kidney c e lls , the la t t e r being a
volume e ffe c t control with c e lls o f nonlymphoid o rig in .
The LPS-
treatm ent o f C3H/HeJ mice resulted in a mean spleen index th a t was
s ig n ific a n tly d iffe r e n t only from the adherent and kidney c e ll
preparations.
GVH assayed by m o rta lity .
A comparison of GVH r e a c tiv ity
o f normal or LPS-treated spleen c e lls as estimated by m o rta lity is
shown in Table I I .
I t is c le a rly seen th a t p rio r LPS treatment of
CBA/J animals decreases the a b il it y o f th e ir spleen c e lls to cause
fa ta l GVH disease in Balb/c neonates.
In c o n tra s t, no d iffe re n c e
was found between in je c tio n o f normal or LPS-treated C3H/HeJ spleen
c e lls .
Mixed leukocyte c u ltu re .
The mouse spleen mixed leukocyte
cultu re.h as been reported to be an in v itr o c o rre la te to the in vivo
G raft-versus-H ost process (2 4 ).
As such, the mixed leukocyte cultures
were set up in the same c e ll combinations as had been used in the
GVH assay to determine e ffe c t of LPS on GVH r e a c tiv ity .
Analysis
o f Table I I I shows th a t p rio r LPS treatm ent of C3H/HeJ mice did not
a lt e r the a b il it y of th e ir spleen c e lls to react against the
a l l d a n tigens present on the Balb/c lymphocytes as compared to
15
TABLE I I
Graft-versus-Host disease in Balb/c mice given spleen c e lls from
normal and LPS-treated CBA/J and C3H/HeJ mice
M o rta lity by Day 30 P o s t-in je c tio n
Cell O rig in3
WN-CBA/J
Dead / Total
6 /7
%M o rta lity
85.7
LPS-CBA/J
18/37
4 8 .6 * b
WN-C3H/HeJ
15/28
53.6
LPS-C3H/HeJ
22/37
59.5
BSS Controls
1/20
5. 0
aSee M aterials and Methods section or te x t fo r explanation
o f abbreviations. ATI BaTb/c neonates received 2 x 10' c e lls or
0.1 ml BSS i . p .
k* = s ig n ific a n t d iffe re n c e w ith in group using the z te s t
a t the p = 0.05 le v e l.
16
TABLE I I I
Mouse spleen mixed leukocyte cultures assaying r e a c tiv ity o f
normal or LPS-treated CBA/J and C3H/HeJ leukocytes against mitomycin
C-treated (Me) Balb/c c e lls 9
Cell Combinations^
C3H/HeJ+McBalb/c
.
Net Average cpmc
4855.42
LPS-C3H/HeJ+McBalb/c
4943.25
CBA/J+McBalb/c
4006.39
LPS-CBA/J+McBalb/c
2174.08
a In addition to appropriate controls (not shown), I x IO6
responding c e lls in 0.1 ml RPMI and I x IO^ stim ulating c e lls in
0 .1 ml RPMI were added to each w ell and cultured fo r 72 hr. Then
2 yCi
thymidine was added, c e lls were cultured an addition al 16
h r, harvested, and counted in a liq u id s c in t illa t io n system.
^See M aterials and Methods section or te x t fo r explanation
o f abbreviations.
cNet average cpm = average of t r ip l ic a t e samples o f stim ulated
c u ltu re minus (average counts o f responding c e lls alone + average
counts o f stim ulatin g c e lls alon e).
17
counts obtained w ith normal C3H/HeJ spleen c e lls .
In c o n trast,
r e a c tiv ity o f CBA/J spleen c e lls was decreased almost two fo ld by
the pretreatm ent w ith IPS.
These data then support the observations
found w ith s im ila r c e ll combinations in the GVH system.
GVH r e a c tiv ity a f t e r treatm ent w ith butanol-extracted IPS.
Since i t has been previously reported th a t the method used to e x tra c t
LPS a ffe c ts it s a b il it y to evoke a mitogenic response in C3H/HeJ
mice (2 5 ), i t was postulated th a t method o f e xtractio n might lik e ­
wise a ffe c t suppression o f GVH by spleens from LPS-treated donors.
The data reported in Table IV in d ic a te th a t no s ig n ific a n t d iffe re n c e
was found between any o f the experimental groups, regardless of
s tra in or previous treatment o f spleen donors.
Thus, there was no
suppression o f GVH disease in Balb/c neonates a fte r treatm ent of
spleen donors of e ith e r CBA/J or C3H/HeJ o rig in w ith butanolr.
extracted LPS (B-LPS).
Effects o f serum on GVH induction by normal c e lls .
It
has been re c e n tly shown th a t in je c tio n o f normal CBA/J spleen c e lls
together w ith serum from LPS-treated CBA/J animals in to Ba!b/c
neonates re s u lts in suppression o f the a b il it y o f those spleen
c e lls to cause f a ta l GVH disease (2 6 ).
Table V displays, results
obtained employing various combinations of sera and c e lls in a
s im ila r system.
In oculatio n o f whole normal CBA/J c e lls with normal
18
TABLE IV
Graft-versus-Host r e a c tiv ity o f spleens from mice treated with
butanol-extracted LPS
M o rta lity by Day 30 P o s t-in je c tio n
Cell O rig in8
WN-CBA/J
B-LPS-CBA/J
Dead / Total
8 /8
10/11
% M o rta lity
100
9 0 .9b
WN-C3H/HeJ
2 /3
66.7
B-LPS-C3H/HeJ
9 / 11
i—I
CO
BSS Controls
0 /6
0.
aSee M aterials and Methods section or te x t fo r an explanation
o f abbreviations.
Each Balb/c neonate received 2 x IO^ c e lls or 0.1
ml BSS i. p .
k|\|o s t a t is t ic a l d iffe re n c e between experimental groups using
the z te s t a t the p = 0.05 le v e l.
19
TABLE V
Effects of serum on G raft-versus-Host r e a c tiv ity of WN c e lls when
in je c te d together in to Balb/c neonates
M o rta lity by Day 30 P o s t-in je c tio n
C ells 3
WN-CBA/J
WN-C3H/HeJ
Source o f Seruma ,b
Normal CBA/J
18 / 30
% M o rta lity
60.0
LPS-CBA/J
5 7 34
Normal C3H/HeJ
9/26
LPS-C3H/HeJ
6 / 16
37.5
Hank's BSS
12 / 19
63.2
Normal CBA/J
10 / 23
4 3 .5 *
LPS-CBA/J
10 / 22
4 5.5*
Normal C3H/HeJ
4 / 22
18.2*
LPS-C3H/HeJ
4 / 25
16.0*
Hank's BSS
BSS Controls
Dead / Total
-
20/26
I / 59
14.7* c
-
3 4 .6 ** d
76.9
IJ
aSee M aterials and Methods or te x t fo r explanation of
abbreviations.
bEach Ba!b/c neonate received 2 x IO^ WN c e lls in 0.1 ml
serum or in 0 .1 ml BSS, or BSS alone, i . p . as in dicated.
c* = s ig n ific a n tly d iffe r e n t from WN of th a t s tra in using
the z te s t a t the p = 0.05 le v e l.
d ** = s ig n ific a n tly d iffe r e n t from WN controls a t the
p = 0.06 le v e l.
20
CBA/J serum re su lts in a percent GVH disease s im ila r to inoculation
o f the same type o f c e lls in Hank's balanced s a lt solu tion (WN
c o n tro ls ).
The in oculatio n o f WN c e lls together with LPS-CBA/J
serum y ie ld s a s ig n ific a n t decrease in m o rta lity .
CBA/J c e lls
in a combination with e ith e r normal or LPS-C3H/HeJ serum caused
m o rta lity s im ila r to each other.
Although values are lower than
the CBA/J c e lls in BSS c o n tro ls , they are not s ig n ific a n tly
d iffe r e n t a t the p = 0.05 le v e l.
When in je c tin g WN C3H/HeJ c e lls
together w ith serum, the c r it ic a l fa c to r appears to be. the serum
source ra th e r than p rio r treatm ent o f the serum donors with LPS
or not.
A ll values reported fo r combining C3H/HeJ c e lls w ith serum
are s ig n ific a n tly lower than in je c tio n of neonates w ith WN C3H/HeJ
c e lls in BSS.
The differences seen using sera from CBA/J versus
C3H/HeJ sources are s ig n ific a n t a t the p = 0.067 le v e l.
. Removal o f GVH r e a c tiv ity by adherent c e lls .
As described
in the M aterials and Methods section, spleen c e lls were allowed to
adhere to p la s tic to separate the adherent and nonadherent c e ll
populations.
Adherent c e lls were then counted and returned to p e tri
dishes and whole normal c e lls were added.
A fte r incubation, i t was
the nonadherent c e lls from these combinations th a t were in jected
in to the neonates.
As can be seen in Table V I, the number of mice
in each experimental group is very small and the re su lts are taken
21
TABLE V I
Graft-versus-Host r e a c tiv ity a f t e r layering WN c e lls on normal or
LPS-adherent c e lls
M o rta lity by Day 30 P o s t-in je c tio n
Cell O rigin
Dead / Total
%M o rta lity
on LPS-CBA/J adha ,b :
WN-CBA/J
WN-C3H/HeJ
33.3
33.3
I / 3
.1 / 3
.
on LPS-C3H/HeJ Adh:
WN-CBA/J
WN-C3H/HeJ
3 /3
3/3.
.
100
100
WN-CBA/J on C3H/HeJ Adh
WN-C3H/HeJ on CBA/J Adh
3 / 3
3 /3
100
100
Nonad LPS-CBA/JC
Nonad LPS-C3H/HeJ
2 /3
2 /2
66.7
100
WN-CBA/J
WN-C3H/HeJ
57 7
5 / 7
71.4
71.4
BSS. Controls
0 / 7
0
aSee M aterials and Methods section or te x t fo r an explanation
o f abbreviations.
^2 x IO7 WN c e lls .were incubated on 5 x 10® adherent c e lls
fo r ih hr a t 37C. The subsequent nonadherent c e lls were in jected
i . p . in to Baib/c neonates.
g2
x
IO7 c e lls in je c te d i . p . in to BaIb/c neonates.
22
as in d ic a tiv e o f a trend ra th e r than as s t a t i s t ic a l ly s ig n ific a n t
data.
Broadly speaking, th e re fo re , layering normal spleen c e lls
on adherent c e lls taken from LPS-treated CBA/J animals re su lts in
loss of GVH r e a c tiv ity o f the subsequent nonadherent preparation
whereas incubation w ith adherent c e lls from LPS-treated C3H/HeJ
mice or normal allo geneic adherent c e lls produced no such decrease
in GVH r e a c t iv ity .
I t can also be seen th a t the nonadherent c e lls
from both LPS-treated CBA/J and C3H/HeJ are capable o f inducing
fa ta l GVH disease in a manner comparable to normal spleen c e lls .
Carbon clearance.
To examine y e t another parameter of
c e llu la r r e a c tiv ity influenced by LPS treatm ent. Table V ila presents
the calculated phagocytic index (K) fo r B alb/c, CBA/J, and C3H/HeJ
mice w ith and w ithout p rio r treatm ent w ith 20 yg phenol-extracted
LPS i . v .
The ra te o f phagocytosis is s ig n ific a n tly increased in
the Balb/c and CBA/J mice 72 hours a f t e r in oculation o f LPS.
No
stim u latio n is seen in ra te of carbon uptake in the C3H/HeJ animals.
To determine i f LPS mitogenic fo r C3H/HeJ B c e lls also had
a stim ulatory e ffe c t on th e ir re tic u lo e n d o th e lia l system, a carbon
clearance assay was also conducted on C3H/HeJ mice 72 hours a fte r
i . v . in je c tio n of 20 yg butanol-extracted LPS.
As can be seen from
Table V IIb , the value o f K was not changed from normal by the LPS
in je c tio n .
23
TABLE V I I
Rate o f phagocytosis in normal or LPS-treated mice measured by a
carbon clearance assay3
Experiment
A. phenol-LPS
B. butanol-LPS
S tra in
Ave K - Normal
Ave K - LPS-treated
Balb/c
-0.0375
-0 .0 5 9 0 * b
CBA/J
-0.0263
-0 .0 5 1 8 *
C3H/HeJ
-0.0201
-0.0228
C3H/HeJ ,
-0.0336
-0.0303
aMice in the LPS groups received 20 pg LPS in 0.1 ml PBS
72 hr before conducting the carbon clearance assay. Each mouse
received (body weight divided by 100) ml o f 16 mg/ml carbon i . v .
and was bled a t 3 minute in te rv a ls time 0-15 minutes. The slope
o f a lin e a r regression p lo t o f the log o f the o p tic al density
readings versus time is the phagocytic index K.
. k* = LPS-treated is s ig n ific a n tly d iffe r e n t from normal of
same s tra in using the student t te s t a t the p = 0.05 le v e l.
24
LDgQ o f LPS alone or a f t e r pretreatment o f mice with trypan
b lu e .
Since the C3H/HeJ are known to be re s is ta n t to the le th a l
e ffe c ts o f LPS, i t was necessary to establish the r e la tiv e LDgQ
to LPS in the mouse s tra in s employed.
As seen in Table V I I I , the
CBA/J mice had a LDgQ o f 460 yg and the B alb /c, 750 ug.
This
exhibits the w ell known v a ria tio n between s tra in s in s u s c e p tib ility
to LPS.
Great resistance is shown, however; by the C3H/HeJ mice
which had a LDgQ o f 9165 yg, a dose 12 times higher than fo r
Balb/c mice and almost 20 times higher than fo r the CBA/J mice.
The.macrophage is known to be a major processing center fo r
LPS w ith in the animal so trypan blue was administered to m ice.prio r
to LPS to estab lish the e ffe c t o f in a c tiv a tio n of lysosomal enzymes
on s u s c e p tib ility to LPS.
In the case o f both the CBA/J and the
Balb/c mice, the LD50 was decreased by greater than 12 fo ld .
No
data are shown fo r the C3H/HeJ mice treated with trypan because
re su lts did not in d ic a te a real influence o f the dye on suscepti­
b i l i t y to LPS.
Rather, th is mouse s tra in exhibited m o rta lity in
eigh t week old mice treated only with trypan blue, and death in
animals receiving both trypan blue and LPS was random in re la tio n
to dose o f LPS received.
No such s e n s itiv ity to the dye was
observed in C3H/HeJ mice four months o f age or older.
Work is
now underway to fu rth e r characterize the reaction of trypan w ithin
the C3H/HeJ re tic u lo e n d o th e lia l system.
25
TABLE V I I I
A comparison o f LPS LDgQ in normal or trypan blue .pretreated mice
Mouse S tra in
LPS LD5Qa
LPS LD50
a f t e r Trypan Blue
CBA /J
460 pg
37.5 pg
Balb/c
750 p g .
60.0 pg
C3H/HeJ
9165 pg
aLD5 0 Was' calculated using the Reed-Muench technique
^Mice in the trypan blue groups were in jected i . p . with
4 mg trypan 24 hr before and 1.0 mg trypan blue 2 hr before in je c tio n
of LPS.
DISCUSSION
Although in divid ual s tra in differences in reactions to LPS
had been recognized before, S u ltze r f i r s t reported the opposite
pattern o f leukocyte accumulation a f t e r i . p . in je c tio n o f LPS and
the great resistance to the le th a l e ffe c ts o f LPS by the C3H/HeJ
mouse s tra in in 1968 ( 5 ) .
Since then, much work has been performed
u t iliz in g the C3H/HeJ mice in studies to help elucidate possible
c e llu la r and genetic mechanisms in LPS r e a c t iv ity .
In contrast
to re s u lts found w ith other mice, LPS does not function as a B
c e ll mitogen (6 ,7 ) or an adjuvant in these mice, and the s p e c ific
immune response e lic it e d is tra n s ie n t ( 7 ) .
By using various c e ll
combinations in v itr o and by tra n s fe r experiments in v iv o , the B
lymphocyte o f the C3H/HeJ was shown to be the c e ll responsible fo r
the lack o f response.
A possible membrane defect was postulated
because, although the C3H/HeJ B c e ll is capable o f binding LPS
as w ell as B c e lls from LPS-responsive mouse s tra in s , no a c tiv a tio n
resu lts ( 9 ) .
In f a c t , the binding o f LPS to the C3H/HeJ B lymphocyte
membranes a c tu a lly leads to th e ir subsequent in a c tiv a tio n , even
causing a dose- and time-dependent suppression of B c e ll stim ulation
by another B c e ll m ito g e n ,p o ly I (1 0 ).
The genetics governing the
LPS unresponsiveness continues to be an unresolved issue.
S ultzer
o r ig in a lly reported th a t the d if fe r e n t ia l leukocyte accumulation
27
and the high resistance to the le th a l e ffe c ts o f LPS was under
polygenic control ( 8 ) .
And although Watson and R ib le t demonstrated
th a t the mitogenic and immune responsiveness were under control of
a single dominant autosomal gene ( 6 ) , S u ltzer has ju s t published
a report contending th a t the lymphocyte a c tiv a tio n is governed by.
a p a ir of autosomal co-dominant genes (2 7 ).
For the most p a rt, C3H/HeJ T lymphocytes and macrophages .
have been disregarded as fa r as playing any ro le in LPS unresponsive­
ness.
In the case of the I c e l l , th is outlook is seemingly ju s t if ie d
since T c e lls do not re a d ily respond to LPS and appear not to in te ra c t
in LPS responses by B c e lls , thus leading to the designation of
LPS as a thymus-independent antigen (2 ,2 8 ,2 9 ).
The macrophage,
however, can pinocytize LPS and pass the processed molecule to
lymphocytes (1 6 ).
Macrophages are also known to be ac tiv ate d by
in te ra c tio n w ith LPS (3 0 ).
Chedid e t al have recently reported
th a t C3H/HeJ mice did not e x h ib it increased nonspecific resistance
to in fe c tio n with K le b s ie lla pneumoniae a fte r LPS treatm ent and
th a t, using a tumor c e ll growth in h ib itio n assay, th e ir macrophages
were re fra c to ry to in v itr o stim ulatio n by LPS (3 1 ).
The current study was undertaken to in ve s tig ate whether other
b io lo g ic a l reactions influenced by LPS were s im ila rly impaired in
the C3H/HeJ mice.
Research was conducted in systems involving a ll
three o f the tr a d itio n a l immunologicalIy a c tiv e c e ll types:
the
28
GVH assay and the mixed leukocyte c u ltu re are usually considered
tests o f I c e ll function; serum immunoglobulins are considered
evidence o f B c e ll function; and adherent c e lls and the
re tic u lo e n d o th e lia l system (RES) are re la te d to macrophage c e ll
types, although other c e lls are obviously involved in the la t t e r
populations.
The CBA/J mouse s tra in was chosen fo r comparison with
r e a c tiv ity o f the C3H/HeJ s tra in because the CBA/J are LPS-sensitive
whereas the C3H/HeJ are not.
Also an important consideration is
th a t both s tra in s are H-2 .
Although they are d iffe r e n t a t minor
l o c i , the mouse major h is to c o m p a tib ility complex is the most.
important in determining reactions against the a l l o a n tigens of
the H-2
d
Balb/c mice used as targ ets in the studies.
An increase in spleen weight index is generally regarded
as an accurate in d ic a tio n of a GVH re ac tio n .
For these studies,
however, th is assay fa ile d to d istingu ish between spleen c e lls from
normal or LPS-treated donors o f e ith e r the CBA/J or the C3H/HeJ
mouse s tra in s .
Thus, an index in d ic a tiv e o f an ongoing GVH process,
a t day 10 was not an accurate predictor, o f possible eventual survival
o f s im ila r ly treated Balb/c neonates.
On the other hand, employing death by day 30 as the c rite rio n
o f GVH disease resulted in a c le a r d is tin c tio n between experimental
groups of the CBA/J s tra in .
As reported, by Thomson and J u tila (1 5 ),
29
p rio r LPS treatm ent of CBA/J a d u lt spleen donors s ig n ific a n tly
decreases m o rta lity o f in jected Balb/c neonates as compared with
in je c tio n o f WN CBA/J c e lls .
This was a ttrib u te d to a c tiv a tio n of
the adherent population by the LPS and these c e lls in turn nons p e c ific a lly removed the lymphocytes capable o f reacting against
a l l o a n tigens when placed in to the immature foreign host.
Such a
suppressive e ffe c t is not evoked by p rio r LPS treatm ent o f C3H/HeJ
spleen donors so the c e llu la r elements, presumably I c e lls , involved
in GVH induction remain unaltered by the presence o f LPS.
S im ila r observations were made from c e lls reacting in the
mixed leukocyte c u ltu re s .
This would seem to in d icate th a t e ith e r
the a llo a n tig e n re a c tiv e c e lls were present in the LPS-CBA/J system
but had been in ac tiv a te d or th a t they were removed during laboratory
manipulations p rio r to addition to the mixed c u ltu re s .
Since many
c e lls are removed.in a l l samples by clumping w ith in tissue elements
during c e n trifu g a tio n , the la t t e r p o s s ib ility is very li k e l y , thus
leaving only lymphocytes unable to re ac t to the foreign antigens
to be counted and placed in the c u ltu re s .
Since method o f e x tra c tio n o f LPS from the b a c te ria a ffec ts
the a b il it y o f the re s u ltin g LPS preparation to stim ulate B c e lls
from C3H/HeJ mice in to a mitogenic response, i t was postulated
th a t suppression of GVH r e a c tiv ity might be s im ila rly a ffe c te d .
Even though butanol-extracted LPS (B-LPS) is mitogenic fo r B c e lls
30
o f the C3H/HeJ mice, as w ell as fo r LPS-responsive mouse s tra in s (2 5 ),
no suppressive e ffe c t was exerted on the GVH r e a c tiv ity o f B-LPS
treated C3H/HeJ spleen c e lls .
This indicates th a t stim ulatio n a t
the B c e ll level, does not likew ise a c tiv a te the c e lls involved in
suppression o f allo geneic reco gnition.
The addition al evidence of
no decrease in m o rta lity o f neonates in je c te d w ith B-LPS-CBA/J
c e lls shows th a t b u tan o l-extractio n o f LPS a ffe c ts more o f it s chemical
properties than had been expected.
I t should be noted a t th is point
th a t, even u t iliz in g 120 yg of B-LPS d a ily , none o f the to x ic e ffe c ts
o f phenol-extracted LPS usually shown by the CBA/J animals, such as
diarrhea and some death, were observed.
There was also only a s lig h t
splenomegaly a f t e r s a c r ific e , a crude visual observation o f in a c tiv ity
of the B-LPS preparation even in the LPS-sensitive s tra in .
Recent
work on B-LPS has revealed the presence o f a low molecular weight
polypeptide t ig h t ly bound to the lip id A, which is removable by
treatment w ith phenol (3 2 ).
I t is the polypeptide which is responsible
fo r s tim u latio n o f the C3H/HeJ lymphocytes and i t is thought that
the peptide blocks a portion of the li p id in much the same manner as
polymixin B does.
Therefore, the polypeptide on the lip id A in
B-LPS does not seem to a c tiv a te the C3H/HeJ adherent c e lls responsible
fo r removing GVH r e a c tiv ity even though mitogenic a c t iv it y o f B c e lls
is stim ulated.
Also, the c r it ic a l portion o f a phenol-extracted IPS
molecule responsible fo r a c tiv a tio n of adherent c e lls in the
31
LPS-responsive CBA/J mice is not a v a ila b le fo r reaction on a B-LPS
molecule.
Thus, with no adherent c e ll a c tiv a tio n , m o rta lity is the
same as w ith untreated normal c e lls .
The observation by Thomson (26) th a t serum from LPS-treated
CBA/J mice is capable o f abrogating a GVH reaction by normal CBA/J
c e lls brings in a humoral fa c to r also involved in suppression.
Since CBA/J mice do respond Immunolo g ic a lly to LPS as an antigen,
serum from mice given seven d a ily in je c tio n s o f LPS contains a n tiLPS antibody (u s u a lly w ith a HA t i t e r of. 1:64 or g re a te r, data not
shown).
I t is th is a n t i-LPS component th a t is proposed to be
responsible fo r the reduced m o rta lity in the Balb/c neonates given
both the serum and the WN CBA/J c e lls .
Endotoxemia is a usual
phenomenon in animals undergoing a GVH reaction so a possible
mechanism fo r the p ro te c tiv e e ffe c t could be the prevention o f LPS
shock w ith in the neonates by the binding o f the LPS-serum with LPS
during a p relim inary GVH re ac tio n .
I t has been shown (33) th at
treatm ent o f neonates w ith a n tib io tic s decreases incidence o f GVH
disease by ju s t such an endotoxemia prevention mechanism.
Normal
serum from CBA/J animals given in conjunction w ith WN CBA/J c e lls
has no abrogating e ffe c t.
The data presented in the curren t study
include samples repeating those observations.
In a d d itio n , i t
was shown th a t there was no d iffe re n c e in re s u ltin g GVH disease
by giving e ith e r normal or LPS-C3H/HeJ serum. . The C3H/HeJ mice
I
32
respond poorly and tra n s ie n tly to LPS as an antigen (7 ) so i t would
be expected th a t no s ig n ific a n t antibody element would be produced
under these circumstances (HA t i t e r less th a n .1 :4 , data not shown).
I t must be noted, however, th a t ju s t the presence o f the C3H/HeJ
serum with the CBA/J c e lls reduced fin a l m o rta lity markedly, even
though the numbers were not s t a t i s t ic a l ly s ig n ific a n t a t the
p = 0.0.5 le v e l.
The question may be raised whether th is could be
due to a nonspecific fa c to r th a t has a suppressive e ffe c t or i f i t
is a phenomenon seen using nonsyngeneic serum.
Experiments using
other allogeneic sera or even heterologous sera seem to in dicate
th a t the la t t e r is not the case (data not shown).
\
With c e lls from normal C3H/HeJ mice, th e ir in je c tio n in
conjunction w ith any o f the s e ra .te s te d causes a s ig n ific a n t decrease
in GVH disease of the Balb/c neonates.
Even so, LPS-CBA/J serum does
not fu rth e r lower the level o f r e a c tiv ity from th a t obtained from
normal CBA/J serum.
This e ffe c t cannot then be a ttrib u te d to an
a n ti-LPS component found only in the LPS-serum, but ra th e r seems
to be re la te d to the CBA/J o rig in o f the serum.
The most surprising
re su lts are found w ith the combination o f C3H/HeJ c e lls with normal
or LPS serum o f syngeneic o rig in .
I t is hard to b elieve th a t humoral
elements could have such a suppressive e ffe c t on the allogeneic
r e a c tiv ity o f th e ir own c e lls , and no explanation is re a d ily
apparent.
Although attempts have been made to compare changes
33
evoked in r e a c tiv ity w ith in each mouse s tr a in , i t may deserve
mention th a t the drop in r e a c tiv ity from CBA/J c e lls in normal
CBA/J serum to the level seen o f CBA/J c e lls in the p a ir o f
C3H/HeJ sera is comparable to the drop from the r e a c tiv ity of
C3H/HeJ c e lls in the CBA/J sera down to the values found with
C3H/HeJ c e lls in the C3H/HeJ sera.
The contention th a t the adherent c e ll population o f LPS-CBA/J
mice was responsible fo r removal of GVH re a c tiv e c e lls (15) was
supported and fu rth e r extended by work in th is study.
I t is seen
th a t LPS-CBA/J adherent c e lls are capable of reducing m o rta lity not
only of WN CBA/J c e lls but also o f WN C3H/HeJ c e lls .
In co ntrast,
r e a c tiv ity of WN c e lls o f e ith e r s tra in is unaltered by incubation
w ith LPS-C3H/HeJ adherent c e lls .
The important controls o f layering
WN c e lls on the adherent c e lls o f the allogeneic s tra in show th at
the removal o f GVH r e a c tiv ity is the unique property o f the LPS-CBA/J
adherent c e lls .
The mechanism o f nonspecific absorptive removal
and/or cytotoxic e ffe c t l?y activated c e lls o f the adherent population
on allo a n t igen re a c tiv e c e lls is also strengthened because o f the
suppression o f GVH by the allogeneic C3H/HeJ c e lls as w ell as the
syngeneic CBA/J c e lls .
This is in contrast to the suppression caused
by the LPS-CBA/J serum because it s e ffe c t is only seen w ith WN c e lls
o f CBA/J o rig in .
That T lymphocytes o f both strain s remain
34
unaffected by the LPS treatment is seen by comparing r e a c tiv ity of
the nonadherent LPS-cells w ith m o rta lity caused by WN c e lls .
.
As another way o f te s tin g whether LPS had any a c tiv a tin g
e ffe c t on macrophage a c t iv it y w ith in the C3H/HeJ mice, carbon
clearance assays were conducted, u t iliz in g increase in ra te of
phagocytosis as in d ic a tiv e o f s tim u la tio n .
The phagocytic index
of both LPS-sensitive mouse s tra in s was s ig n ific a n tly increased
by pretreatm ent w ith LPS.
No stim ulatio n o f the phagocytic a c tiv ity
by the RES o f the C3H/HeJ mice was accomplished by e ith e r phenolextracted LPS or butanol-extracted LPS.
Since the la t t e r m aterial
is mitogenic fo r C3H/HeJ B lymphocytes, the low molecular weight
polypeptide responsible fo r the mitogenesis is again seen not to
be a stim ulatory m aterial fo r the macrophage.
This dichotomy of
response has also been re cen tly reported by Chedid (31) in th a t
clearance o f bacteria from the blood o f C3H/HeJ mice was not
increased a f t e r treatment with compounds th a t had been shown to
be stim ulatory fo r B c e lls o f the same s tra in .
While the C3H/HeJ mice are described as being LPS-resistant
and other s tra in s as LPS-sensitive, i t is necessary to estab lish
what level o f s e n s itiv ity or resistance is displayed w ith the
p a rtic u la r LPS source and preparation u tiliz e d in .th e study.
Of
the mouse s tra in s employed here, the CBA/J exhibited the greatest
s e n s itiv ity w ith a LPS LDgg o f 460 yg and the Balb/c were s lig h tly
35
more re s is ta n t with a LD50 o f 750 yg.
Apart from these s tra in
differences in LPS s u s c e p tib ility w ith in a lim ite d range, the
C3H/HeJ are tr u ly re s is ta n t w ith a dose o f 9165 yg required fo r
the median le th a l e ffe c ts .
This level is 12 times higher than fo r
the Balb/c and almost 20 times higher than fo r the CBA/J.
Hibbs (3 4 ,3 5) reported th a t trypan blue e ffe c tiv e ly blocks
the a c t iv it y o f macrophages by in a c tiv a tio n o f lysosomal enzymes
once the dye has been phagocytosed.
As an aside to the other
research on whether or not the C3H/HeJ macrophages were activated
by LPS, a possible mechanism fo r the C3H/HeJ's great resistance
to the le th a l e ffe c ts o f LPS was tested using trypan blue. . I t
was reasoned th a t i f the C3H/HeJ macrophage was capable o f exces­
sive overprocessing and d etoxifyin g LPS w ith in it s lysosomes,
in a c tiv a tio n o f these organelles should d ra s tic a lly decrease the
LPS LDgg o f the C3H/HeJ down to a level o f s im ila rly treated
s e n s itiv e s tra in s .
The macrophage indeed plays a substantial role
in trapping and deto xifyin g LPS because overwhelming the RES with
trypan blue p rio r to adm instration of the LPS leaves the animals
much more vulnerable to the to x ic LPS e ffe c ts .
Thus animals
succumb to much lower doses o f LPS than when an in ta c t RES is able
to absorb la rg e r amounts of the LPS before a le th a l le v e l is reached
w ith in the animals.
The RES
blockade could also be combined with
an increased perm eability o f lysosomes w ith in the re tic u lo e n d o th e lia l
36
c e lls , which has been reported to increase LPS s u s c e p tib ility in
RES stim u latio n experiments (3 6 ).
Indeed, p rio r in je c tio n with,
trypan blue decreases the LPS LD501S in both the CBA/J and the
Balb/c mice by greater than 12 fo ld .
No real increase in suscepti­
b i l i t y to LPS is produced in C3H/HeJ mice by pretreatm ent with
trypan blue.
An age-related s e n s itiv ity to the dye was revealed
and more experiments are planned to possibly elucidate the
mechanism o f th is s e n s itiv ity .
In an overview, a few key points have been elucidated in
th is study.
I t has been shown th a t LPS activates CBA/J adherent
c e lls n o n s p e cific a lly in th a t suppressive e ffe c ts are exerted on
normal lymphoid c e lls o f allogeneic o rig in as well as from the same
s tr a in , as had been previously reported.
In c o n tra s t, the abrogation
o f GVH by LPS-CBA/J serum was e ffe c tiv e only on the syngeneic c e lls
in th is p a rtic u la r system.
There is no a c tiv a tio n o f a suppressor
adherent c e ll or production o f a humoral antibody in the C3H/HeJ
th a t decreases GVH r e a c tiv ity when compared to normal c e lls or serum.
C3H/HeJ spleen c e lls also e x h ib it a lower r e a c tiv ity when in jected
in allogeneic sera, whether the donors were pretreated w ith LPS
or not.
Notably, C3H/HeJ serum i t s e l f possesses some nonspecific
suppressive element th a t decreases a c t iv it y of it s own spleen c e lls
as w ell as allogeneic c e lls .
37
The main theme emerging from th is work is th a t, in addition
to the in a c tiv a tio n o f C3H/HeJ B lymphocytes much reported in current
lit e r a t u r e , C3H/HeJ macrophages are also re fra c to ry to stim ulation
by LPS.
Even though B c e ll mitogenesis is seemingly not correlated
with macrophage a c tiv a tio n , the nonresponsiveness to the usual
a c tiv a tin g action LPS has on macrophages could be due to a membrane
defect, in the macrophages s im ila r to the one proposed fo r B c e lls .
The p o s s ib ility th a t macrophages have a c tu a lly been in a c tiv a te d by
contact w ith LPS would not seem to be the case because the phagocytic
index is not lowered by p rio r treatm ent with LPS.
I t may not be
v a lid to make th is supposition, however, since the duration of
in actio n has not been studied and the e ffe c t could e a s ily have
diminished by the time o f the 72 hour assay.
This is c e rta in ly
an area th a t deserves fu rth e r research a tte n tio n .
General observa­
tions a t the c e llu la r le v e l must pave the way fo r elu c id a tio n of
molecular mechanisms o f in teractio n s o f b a c te ria l LPS w ith in
eukaryotic systems.
REFERENCES
1.
M iln e r, K. C ., J. A. Rudbach, and E. R ib i. 1971. General
C h a ra c te ris tic s.
In_: M icrobial Toxins. Vol 4 , Edited by
G. Weinbaum, S. KadTs, and S. J. A j l . Academic Press, New
York. P. I .
2.
Andersson, J v, G. M o lle r, and 0. Sjoberg. 1972. S elective
induction o f DNA synthesis in T and B lymphocytes. C e ll.
Immunol. 4:381.
3.
Andersson, J . , 0. Sjoberg, and G. M o lle r. 1972. Induction
o f immunoglobulin and antibody synthesis in v itr o by
Iipopolysaccharides. Eur. J. Immunol. 2:349.
4.
H i l l , A. B ., J. M. HatswelI , and W. W. C. Topley. 1940.
The in heritan ce o f resistan ce, demonstrated by the develop­
ment o f a s tra in o f mice re s is ta n t to experimental inoculation
with a b a c te ria l endotoxin. J. Hygiene 40:538.
5.
S u ltz e r, B. M. 1968. Genetic control of leucocyte responses
to endotoxin. Nature 219:1253.
6.
Watson, J . , and R. R ib le t. 1974. Genetic control o f responses
to b a c te ria l lipopolysaccharides in mice. I .
Evidence fo r
a single gene th a t influences mitogenic and immunogenic
responses to lipopolysaccharides. J. Exp. Med. 140:1147.
7.
Skidmore, B. J . , J. M. C h ille r , D. C. Morrison, and W. 0.
Weigle. 1975. Immunologic properties of b a c te rial
I i popolysaccharide (IP S ): C orrelation between the m itogenic,
adjuvant, and immunogenic a c t iv it ie s . J. Immunol. 114:770.
8.
S u ltz e r, B. M. 1972. Genetic control o f host responses to
endotoxin. In fe c t. Immun. 5:107.
9.
Watson, J v, and R. R ib le t. 1975. Genetic control o f responses
to b a c te ria l lipopolysaccharides in mice. I I .
A gene that
influences a membrane component involved in the a c tiv a tio n of
bone marrow-derived lymphocytes by lipopolysaccharides.
J. Immunol. 114:1462.
10.
Glode, L. M ., I . Scher, B. Osborne, and D. I . Rosenstreich.
1976. C e llu la r mechanism of endotoxin unresponsiveness in
C3H/HeJ mice. J. Immunol. 116:454.
39
11.
Murphy, J. B. 1916. The e ffe c t o f adult chicken organ
g ra fts on the chick embryo. J. Exp. Med. 24:1.
12.
B iIlingham 1 R. and W. S ilv e rs . 1971. The Immunobiology of
Transplantation. P re n tic e -H a ll, In c ., Englewood C l i f f s , New
Jersey. P. 151.
13.
Grebe, S. C ., and J. W. S t r e iIe in .
1976. G raft-versus-Host
reactio ns: A review. Adv. Immunol. 22:119.
14.
Chedid , I .
1973. Possible ro le o f endotoxemia during
immunologic imbalance. In B acterial Lipopolysaccharides.
Edited by E. H. Kass and S. M. W o lff. U niversity o f
Chicago Press. P. 104.
15.
Thomson, P. D ., and J . W. J u tila .
1974. The suppression of
Graft-versus-Host disease in mice by endotoxin-treated
adherent spleen c e lls . J. R eticuloendothel. Soc. 16:327.
16.
Bona, C. A. 1973. Fate o f endotoxin in macrophages:
B iological and u ltra s tru c tu ra l aspects. In B acterial
Lipopolysaccharides. Edited by E. H. Kass and S. M. W olff.
U n iversity o f Chicago Press. P. 66.
17.
M ichie, D. 1973. The biometrics o f the spleen weight assay.
In Handbook of Experimental Immunology. Second e d itio n .
V o l. 2. Edited by D. M. Weir. F. A. Davis C o., P hiladelp hia,
Ch. 30.
18.
Westphal, O ., and K. Jann. 1965. B acterial I i popolysaccharides:
Extraction w ith phenol-water and fu rth e r applications o f the
procedure. In Methods in Carbohydrate Chemistry. Edited by
R. L. W histler and M. L. WoI from. Academic Press, In c i ,
New York. P. 83.
19.
M iln e r, K. C ., and R. A. Finkels te in . 1966. Bibassay of
endotoxin: C orrelation between pyrpgenicity fo r rabbits and
le t h a lit y fo r chick embryos. J. In fe c t. Dis. 116:529.
20.
Morrison, D. C ., and L. Leive. 1975. Fractions o f
Iipopolysaccharide from. Escherichia c o li O l l l :.B4 prepared by .
two e xtractio n procedures. J. B io l. Chem. 250:2911.
40
21.
S tu a rt, A. E ., J. A. Habeshaw and A. E. Davidson: 1973.
Phagocytosis in v it r o . In Handbook o f Experimental
Immunology. Second e d itio n . Vol. 2, Edited by D. M. Weir.
F, A. Davis C o., P hiladelphia. Ch. 24.
22.
Reed, L. J . , and H. Muench. 1938. A simple method of
estim ating f i f t y per cent end-points. Am. J. Hyg. 27:493.
23.
Snedecor, G. W., and W. G. Cochran. 1967. S ta tis tic a l
Methods. Sixth e d itio n .
Iowa State U niversity Press. .
Ames, Iowa.
24.
Elves, M. W. 1969. The mixed leucocyte re ac tio n . An in
v itr o model fo r the homograft re ac tio n . Transplantation 8:44.
25.
Skidmore, B. J-., D. C. Morrison, J. M. C h ille r , and W. 0.
Weigle. 1975. Immunologic properties of b a c te ria l
lipopolysaccharide (LPS): I I .
The unresponsiveness of
C3H/HeJ mouse spleen c e lls to LPS-induced mitogenesis is
dependent on the method used to e x tra c t LPS. J. Exp. Med.
142:1488.
26.
Thomson, P. D ., J. W. J u t ila , and P. A. Rampy. A Mechanism
fo r the Suppression o f GVH w ith Endotoxin. Submitted to
In fe c t. Immun., 1976.
27.
S u ltz e r, B. M. 1976. Genetic analysis of lymphocyte
a c tiv a tio n by lipopolysaccharide endotoxin. In fe c t. Immun.
. 13:1579.
28.
M o lle r, G ., and G. Michael. 1971. Frequency o f antigens e n s itiv e c e lls to thymus-independent antigens. C e ll.
Immunol. 2:309.
29.
Andersson, B ., and H. Blomgren. 1971. Evidence fo r thymusindependent humoral antibody production in mice against
po lyvinylpyrrolidone and £. c o li lipopolysaccharide. C e ll.
Immunol. 2:411.
30.
Alexander, P ., and R. Evans. 1971. Endotoxin and double
stranded RNA render macrophages c y to to x ic . Nature (London)
New B io l. 232:76.
41
31.
Chedid9 L . , NI. Parant9 C. Damais9 F. Parant9 D. Juy9 and
A. G a le lli . .1976. F a ilu re o f endotoxin to increase non­
s p e c ific resistance to in fe c tio n o f lipopolysaccharide
low-responder mice. In fe c t. Immun. 13:722.
32.
Morrison, D. C ., S. J. B etz9 and D. M. Jacobs. 1976.
Is o la tio n o f a lip id A bound polypeptide responsible fo r
"L P S -in itia te d " mitogenesis o f C3H/HeJ spleen c e lls .
J. Exp. Med. in press.
33.
Keast9 D. 1973. Role o f b a c te ria l endotoxin in the G ra ftvs-Host syndrome. Ii% B acterial Lipopblysaccharides. Edited
by E. H. Kass and S. M. W olff. U niversity o f Chicago Press.
P. 96.
34.
Hibbs9 J. B. 1975. Activated macrophages as cytotoxic
e ffe c to r c e lls : I . In h ib itio n o f s p e c ific and nonspecific
tumor resistance by trypan blue. Transplantation 19:77.
35.
Hibbs9 J. B. 1975. Activated macrophages as cytotoxic
e ffe c to r c e lls .
II.
Requirement fo r local persistence of
inducing antigen. Transplantation 19:81.
36.
Lamperle9 G. 1966. E ffe c t o f RES stim ulation on endotoxin
shock in mice. Proc. Soc. Exp. B io l. Med. 122:1012.
IiHliiI
3 1762 10015335 O
N378
Rl47
cop.2
Raropy, P atricia A
The influence of
Iipopolysaccharide (LPS)
on c e llu la r a c tiv itie s
in LPS-unresponsive
C3H/HeJ mice
DATE
IS SU E D TO
v.
A/3?*
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