(02,. '02, H202 and OH) during their normal pattern of oxidative metabolism .... Analysis. Differences between two means were assessed by. Student's t-test.
Journal
Glycolipid
of Leukocyte
Biology
43:11-17
(1988)
Induced Proliferation of Lipopolysaccharide Hyporesponsive C3H/HeJ Splenocytes Mark A. Tomai,
Department
Arthur
G. Johnson,
and Edgar
Ribi
of Medical Microbiologylimmunoiogy (MAT., A.G.J.), University of Minnesota, Duluth, and Ribi immunochem Research, Inc. (ER.), Hamilton, Montana
Although the C3H/HeJ mouse is hyporesponsive to lipopolysaccharides (LPS), certain forms of the lipid A fraction have been shown to stimulate cells from this mouse strain. To determine the role of the oligosaccharide chain length on the lipid A-induced proliferation of C3H/HeJ splenocytes, a panel of glycoiipids from R-chemotypes (Re, Rc, and Rd) and a nontoxic monophosphoryl lipid A (MPL) were tested. The MPL cells isolated from the MPL of Salmonella minnesota, Salmonella typhimurlum, and the Reglycoiipids isolated from Escherichia coil were found to be effective at stimulating the LPS-hyporesponsive spleen cells. A Re-glycolipid isolated from a different strain of E. coil cells was inactive, as were the S. minnesota Rc and Rd chemotypes. Proliferation induced by MPL and the active Re preparations was dose dependent and was inhibited by polymyxin B. Thus, if contamination of the Re-LPS or MPL with lipid A-associated protein occurred, it was below functional levels. The data suggest that the C3H/HeJ spleen cells are capable of responding to certain glycolipids, but they may lack the ability to convert native LPS into a stimulatory signal. In addition, a monosaccharide precursor of lipid A (lipid X), and a monoacyl glucosamine phosphoiipid derivative of lipid X (MaGP), were capable of inhibiting the proliferation induced by the MPL and Re-glycolipids. These data are compatible with the existence of a spleen cell receptor for lipid A. Key words:
endotoxin,
mitogenicity,
C3H/HeJ
INTRODUCTION Mice
Sultzer
from
[22]
polysaccharides tive bacterial revealed that
the
C3H/HeJ
studies
LPS
are
also
In particular, shown
in early
were
shown
in 1968
outer membrane. Subsequent the abnormality associated with
toxin low-responder an autosomal gene ther
strain
by
to be resistant to the toxic effects of Lipo(LPS), a component of the gram-nega-
have
strains located shown
is due to a single on chromosome that
defective in this cells from the studies
other
activities
hyporesponsive C3H/HeJ strain
to be incapable
of being
by [11]. been
stimulated
by the lipid A portion of the LPS molecule in vivo as well as in vitro [13] For example, LPS was unable to enhance antibody production [27], protect against x-irradiation [30], activate macrophages to secrete interleukin1 (IL-i) and prostaglandins of the E series (PGE) [10,35], .
or induce serum interferon (S-IFN) and colony-stimulating factor (CSF) [1,2] in the C3H/HeJ strain. One of the more studied responses induced by LPS the stimulation [13]. Several preferentially
© 1988
Alan
R. Liss,
Inc.
C3H/HeJ that ability
cells,
the defect to proliferate
[2 1 ,37]
Using C3H/HeN .
Skidmore
et
al.
mixtures and low [20]
of re-
demon-
in the endotoxin low-responder in response to LPS is due
to a
defect in the B lymphocyte itself. Glode et al [5] have confirmed these results. However, studies performed by Williamson et al. [39] indicate that macrophages may also
play
an
important
induced mitogenesis. Recently, several
role investigators
in certain have
forms noted
of that
forms of lipid A were able to bypass the lesions ated with the low-responder strains and stimulate eration and other responses in cells from these [28,32,33]. Thus, Vogel et al. [32] demonstrated lipid A precursor from Salmonella typhimurium togenic for LPS-hyporesponsive splenic cultures. dition, they showed that the precursor activated
LPScertain associprolifstrains that a
was miIn adC3H/
is
of proliferation of spleen cell cultures studies have demonstrated that LPS acts by stimulating B lymphocytes to divide [4,19].
As with the other properties genic response induced by
sponder
strains responder
.
mutation in 4 [36]. Furstrain have
LPS-hyporesponsive the histocompatible strated cells’
analysis the endo-
induced
mouse
described above, this mitoLPS is also defective in the
Received Edgar
February
24,
1987;
accepted
iune
16, 1987.
Ribi is deceased.
Reprint requests: biology/Immmunology,
Arthur
G. iohnson, Department University of Minnesota.
of Medical MicroDuluth, MN 55812.
12
Tomai,
HeJ
Johnson,
macrophages
Vukajlovich lipid
to secrete
iL-i
and Morrison
A-rich,
graphic
and Ribi
polysaccharide-
fractions
and
and
isolated
from
able to stimulate proliferation Likewise, we recently found
that certain
Inc. and procedure
chromatoR595 were
cells of the Re-mutant of Escherichia was a generous gift from E. Rietschel
PGE.
Moreover,
[33] demonstrated protein-free
S. minnesota
of C3H/HeJ [28] that two
preparations of lipid (DPL) and a detoxified
A, a toxic diphosphoryl lipid A MPL isolated by Takayama et al. [26] and characterized by Ribi et al [ 18] were able to enhance antibody production significantly in both C3H/ HeJ mice and C57B1/lOScN mice, another LPS-lowresponder strain [31] However, LPS from a heptose-less mutant (LPS-Rd) was ineffective in enhancing antibody formation in both of the latter strains [28]. ,
.
strains [28]. Consequently, nents necessary
to clarify further the for signaling spleen
structural compocells from LPS-
hyporesponsive glycolipid proliferation
mice to proliferate, we preparations for their capacity of LPS-responder C3H/HeN
responder
C3H/HeJ
lipidic
structures
splenocytes. such
as MPL
eral Re-mutants were response in C3H/HeJ glycolipids was dose
lipid
X (2,3
a monosaccharide derivative
MPL-induced
fective in inhibiting associated protein
MATERIALS Mice
and splenic as well
mice Bar
originally
Harbor,
Lipid
Glycohipids
its monoacyl
capable
of inhib-
Re-LPS-induced
mitogenic
cultures. On the other hand, as polymyxin B, were inefinduced
by lipid
A-
obtained
ME,
were
from bred
Jackson in our
Labanimal
sister matings. C3H/HeJ mice Jackson Laboratories. All mice 2 and 5 months of age.
A’s from
[38].
MPL Ribi
from
Rd-
and
Rc-mutant
strains
of S. min-
S. minnesota characterization
strain
Research of
R595
Inc., the
was
obtained
Hamilton, MPL
have
by
elsewhere
to
the
from whole strain F515
coli (Forschunginstitut
Stock solutions by dissolving 2
of pyrogen-free
water
K.
Takayama,
Madison,
Additions
were
,
was
to Cultures
isolated
following
the
removal
of Galanos
[3] from the subjected to DNase
J5, previously tease.
WI,
Inc. Hamilton, MT. have been described
[24,25].
LPS
in the
of LPS
cell walls I, RNase
by the
of E. I, and
coli, pro-
ether
ex-
phenol:chloroform:petroleum
tract was precipitated by addition of water and removal by centrifugation. Crude LAP was precipitated from the supernatant fluid by 1 vol diethyl ether redissolved in phenol, precipitated the pellet freed of
again phenol
centrifuging
After
the
twice.
LAP
was
lized.
Final
TEA
solution
with by
ether, washed twice, suspending in ether
dialysis
solubilized
against
(v/v)
was achieved
twice
through
and and
distilled
in 1.3 % TEA
purification
water,
and
lyophi-
by passing
a ZETA
brand
a 0.2% (+)
filter
followed by lyophiization. The Chick Embryo Lethal Dose50 (CELD)50 was 0.25 pig. Stock working solutions of LAP were prepared by dissolving 2 mg quantities in 2 ml pyrogen-free water containing 0.05 % TEA. Polymyxin B (7, 900 U/mg) was purchased from Gibco Laboratories, Chagrin Click’s medium and
Falls, added
OH. It was suspended together with the
in test
compounds.
Mitogen Half
Assay a million
termed.
viable
medium
,
spleen
containing
cells
suspended
10%
FBS plates
96-well microtiter Roskilde, Denmark). Additions
in 0. 1 ml
were added (Nunclon
of the
to In-
glyco-
lipids to cultures were made in 50 jtl of Click’s medium supplemented with 10% FBS. The volume was subsequently 48
adjusted hours
to 0.2
MT.
for
been
CO2.
During
the
pulsed
with
1 Ci
described elsewhere [16,26]. Glycolipids from S. minnesota strain R595 (Re) and from the Re-mutant of S. typhimurium strain G30/C2l and from E. coli strain D3lm4 were provided by Ribi Immunochem Research,
according
of Germany). were prepared
in 1 or 2 ml volumes
isolated
round-bottom
Immunochem. and
MAGP,
Click’s
were prepared by A. Nowotny using the phenolextraction method described by Westphal and Jann
from Isolation
and
found
the proliferation (LAP).
facilities by brother and were also purchased from used were males between
nesota water
phosphate),
AND METHODS
C3H/HeN
LPS and
This stimulation by polymyxin B.
of LPS, were
sev-
cells
provided by Ribi Immunochem. Their isolation and characterization
LAP
certain
from
mg quantities
procedure
the mitogenic in contrast to
diacylglucosamine
[24, 25]
response in C3H/HeJ lipid X and MAGP,
oratories,
glycolipids
to stimulate cell cultures
precursor
(MAGP)
the
and
able spleen
that
from
et al. [3]. LPS isolated
containing 0.05% triethylamine (TEA). The solutions were clarified to slight opalescence after brief warming in a 50-60#{176}C water bath and sonication. These stock solutions were further diluted with Click’s medium contaming 10% fetal bovine serum (FBS). Lipid X and
Other
tested various to stimulate and LPS-non-
report
from Rc- and Rd-mutants. dependent and was inhibited
In addition,
iting
We
extracted
of Galanos,
Borstel, Federal Republic of the various compounds
splenocytes. highly purified
.
were
,
ml,
in a humidified
final
and
18 hours
3H-TdR
cultures
(specific
WI)
and collected
on glass
incubated
containing
of culture,
mmol; Amersham, Arlington Heights, then harvested with a microharvester Madison,
were
atmosphere
activity IL).
(Otto fiber
5%
cells
were
>40
Cultures
Hiier filters.
Ci/ were
Co., Radio-
Stimulation TABLE 1. Mitogenic and
Lipid
Response
A Preparations
From
of C3H/HeJ
of C3H/HeN
and C3H/HeJ
Salmonella
mlnnesotaa Mouse
Splenocytes
Splenocytes
a5
23,000 152,000 167,000 103,000 143,000 i#{248} splenocytes
X
mean
c.p.m.
were
± S.E.M.
± ± ± ± ±
C3H/HeJ P value
5,000 2,000 9,000 23,000 12,000
incubated
to Various LPS
strain
C3H/HeN cpm Medium MPL Re Rd Re
cpm 15,000 50,000 91,000 13,000 14,000
-
+002
