Microbiol. Immunol. Vol. 21 (11), 611-619, 1977
Immunological
Properties of Vibrio cholerae Lipopolysaccharides
Masayasu NAKANO,Masao J. TANABE,Hideko HORI, Seiichi KONDO,and Kazuhito HISATSUNE Departmentof Microbiology,Jichi Medical School, Tochigi, and Departmentof Microbiology,Schoolof PharmaceuticalScience,Johsai University,Sakado, Saitama (Received for publication, May 10, 1977)
Abstract Immunological effects of wall lipopolysaccharide (LPS) preparations obtained from VibriocholeraeInaba 569B, Ogawa NIH 41 and NAG 4715 strains by the hot phenol-water procedure were examined in mice. Although these LPS lack KDO, which are basic components of the core region of most gram-negative LPS, they still have potencies as B-cell mitogens, adjuvants, immunosuppressants, polyclonal B-cell activators and phagocytic stimulants for macrophages. The activities of these V. choleraeLPS on murine immune system seemed to be weaker than those of Salmonella typhimurium LT2-LPS. Among these V. cholerae LPS, NAG 4715-LPS showed the strongest mitogenic activity and phagocytic stimulation, while the potencies of this NAG 4715-LPS for the induction of polyclonal B cell activation, adjuvant effects and immunosuppression did not seem to be greater to those of the other LPS.
Lipopolysaccharides (LPS) are structural components in the cell walls of gram negative bacteria and these components are easily extractable from many kinds of bacteria, especially Enterobacteriaceae,by physicochemical procedures (14, 23, 24). Similar LPS components could be extracted from Vibrio choleraeorganisms (7). However, V. choleraeLPS lack 2-keto-3-deoxyoctonate (KDO) and galactose, which are components of the core region of most gram-negative LPS (10, 14, 23) among their constituents (12, 21). The lipid A in V. choleraeLPS contains unusually large amounts of glycin and ammoniac radicals in comparison with other gram-negative LPS (10). Inaba type vibrio LPS contain considerable amounts of the odd numbered fatty acids, while those of Ogawa and NAG vibrios do not (11). These findings suggest that cholera vibrios may have a fundamentally different LPS structure from that of Salmonellaand other gram-negative bacteria, and, furthermore, structural differences of lipid A components in LPS may exist among V. choleraestrains. Recently, much attention has been focused on the effects of LPS on immune phenomena (1, 2, 4, 15, 22). Almost all of the experiments to examine the immunological properties of LPS have been performed using LPS prepared from gram-negative rods, but not from vibrios. Therefore, it may be worthwhile to examine whether or not the structurally different V. choleraeLPS are capable of stimulating immune 611
612
M.
NAKANO
ET AL
phenomena as other endotoxins do. In this paper, we describe the effects of V. choleraeLPS on the murine immune system as follows: a) mitogenic stimulation of cultured lymphocytes, b) enhancement and suppression of antibody response against sheep erythrocyte antigen, c) polyclonal B cell activation, and d) stimulation of the phagocytic function of the reticuloendothelial system. MATERIALS
AND
METHODS
LPS. V. choleraeLPS used in this study were isolated from V. cholerae569B Inaba type, NIH 41 Ogawa type and NAG 4715 by Westphal's phenol-water technique (24). They were highly purified by repeated ultracentrifugations and treatments with RNase and cetabrone (5). LT2-LPS was used in the experiments as a control. LT2-LPS was extracted by Westphal's technique from the LT2 strain (smooth type) of S. typhimurium, and purified by repeated ultracentrifugations. Mouse lethal doses (LD50: intraperitoneal injections into young adult DD strain mice) of these Inaba 569B-, Ogawa NIH 41-, NAG 4715-, and LT2-LPS were 90, 110, 90, and 13 mg per kg respectively. Animals. colony
AKR
were
or
used
in
Incorporation
of
removed
and
teased
through
a fine
nylon
and
subsequently
with
10%
of
air
10%
the
vested
onto
dried.
end
of
counter
serum
(ATS)
incubated
with
pig
complement
the
incubation,
number
of and
One-tenth each
and ml of
Determination
cell-rich
spleen by
in
order
to
cells
were
cells
in
for
the
diluted
the
the
cultures
immunization.
of the the
from
or
thymuses
in
in
1640
the
our
own
a sterile cultured
of
incubation GF/F)
by
into
the
cell
suspension.
72
hr,
was
the
medium
supplemented to of
the
desired
these
cell
sus-
(methyl-3H-
to
the
cultured
culture cells
and
assessed
passing
tube (12 •~75 mm, C in an atmosphere
added
aspiration,
cells
of
3H-thymidine was
for
ml
each
culture at 37
sterile
Tokyo)
50
medium ml
were
After
medium
culture One
into were
Ltd.,
medium.
times
suspensions.
(Whatman
prepared
adjusted
Antigen
three
microcurie
After
adequately
viable
age,
then
by
a
20
were washed
Packard
hr
harand scintil-
3320). B
was
of
Spleens
1640
(RPMI
dissolved
Co.,
radioactivity
(Model
washed
cell
One
filters of
of
weeks
cells.
RPMI
medium
was
Chemicals
fiber
8-12
lymphoid
cells) was poured and the cells
CO2.
uptake
sexes,
ice-cold were
the
incubation.
glass
The
Preparation
into
into
Pure
cultured
culture
LPS
added
and
of both
in cells
in
serum).
Daiichi
before
test,
the
(2 •~ 106 nucleated Plastics, Los Angeles)
90%
into
forceps
mesh,
and
thymidine,
lation
3H-thymidine with
human
mice
experiments.
suspended
concentration, pensions Falcon
C57BL/6
the
SRBC
kill
method ATS
of for
45
Rabbit Blanden
min
thymus-derived
washed suspension (2 •~ Sheep
suspension
suspended
was 106
counted
viable red (108
at cells
and
cells
blood
cells
erythrocytes)
by in
anti-mouse
(3).
The
37
C in
in
the
cell
in
the
culture
the
thymocyte
spleen
the
presence
cells
population.
After
medium.
trypan
blue
were
of guinea
dye
The exclusion
1 ml). (SRBC)
were
used
was
inoculated
suspensions
were
as an
antigen.
intravenously
mice. of
immune
response.
Spleen
cell
prepared
from
IMMUNOLOGICAL
PROPERTIES
OF
V. CHOLERAE
LPS
613
individual mice and the number of plaque forming cells (PFC) in the suspension was counted by the technique of localized hemolysis in agar (13). As a rule, five mice were used for the determination of the PFC-response in one group, and the mean PFC of the five mice and the standard error were calculated. The effect of LPS was estimated by comparison between PFC-numbers of LPS-treated mice and of controls. Estimation of the phagocyticfunction of macrophages. The phagocytic function of macrophages was measured by the rate of carbon clearance from the circulating blood of mice according to the method of Freedman (8). RESULTS Mitogenic
Effect
of
Mitogenic thymidine LPS
into
is
while
with
than
in that
stimulating
of
cells
NAG
were
cells
in
Inaba
Lymphocytes examined
vitro.
569-
4715-LPS.
of
probably was
treated
20
shown
uptake
of
and
radioactive
,ƒÊg of
by
As
Ogawa
However,
and
act
were
Table
cultured
1.
Mitogenicity
on
with
lymphocytes
cells
Cultured
3H-thymidine of
Inaba-LPS
increase
thymus-derived spleen
on LPS
the
Table
the
addition
of
incorporation
to
spleen
cells,
into
the
are
obviously
adequate
the
doses
cultures
cells
3H-
4714-
41-LPS
Ogawa-LPS)
of
1, NAG
cultured
NIH
the
incorporation
in
in
of
always
comparison
controls. LPS
spleen
LPS
cholerae
lymphoid
,ƒÊg of
some in
V.
effects
those (80
These of
of
mitogenic
LPS
resulted
cholerae of
cultured
capable
the
weaker these
Vibrio
effects
bone ATS
(T with
marrow-derived in
cells). or
the
Both
without
of V. cholerae
LPS
lymphocytes
presence these
the
addition
on
cultured
of
complement
ATS-treated of
spleen
V.
(B
cells).
in
order
and cholerae
cells
LPS,
of AKR-mice
Part to
kill
non-treated and
then
614
M. Table
2.
Table
Mitogenicity in cultured
3.
NAKANO
ET AL
of V. cholerae LPS on B lymphocytes spleen cells of AKR mice
Mitogenicity thymocytes
of V. cholerae LPS of AKR mice
on cultured
3H-thymidine uptake of the cultured cells was examined (Table 2). After the treatment with ATS, 3H-thymidine uptake (background) of these cultured cells was reduced to about one half and most of the mitogenic response of these cells to Con A, known to be a mitogen for T cells (9), disappeared. However, the mitogenic responses of these ATS-treated cells to NAG- and LT2-LPS increased when compared with those of untreated cells, suggesting that these LPS have the potency to act as B cell mitogen and elimination of T cells from the spleen cell population results in the enhancement of the mitogenic response of the cell population. Since the mitogenic potencies of Ogawa- and Inaba-LPS were weaker than that of NAG-LPS, the increased response of ATS-treated cells to these LPS was not obvious, but these LPS seemed to have mitogenic potency on ATS-treated spleen cells. These V. choleraeLPS, like LT2-LPS, did not have any stimulatory effect on 3H-thymidine incorporation into cultured thymus cells (Table 3) .
IMMUNOLOGICAL Adjuvant
and
LPS mice
antigen
producing first and mune
in
and
their
with
days
were
showed
or
mice,
V. CHOLERAE
LPS
on
suppression
injection several
with days
Anibody of
later
we
Response
the
antigen
615
LPS
antibody
(19). may
response
If
find
we
of
inject
a lot
LPS
of antibody
(18). Conversely, if the mice are injected the antigen in several days later, we find
10
after
the
As
than
the
mice,
on
with a poor
LPS im-
of
day
LPS
they
4,
in PFC
were Table
and
108
killed 4,
all
the
in
the
numbers
of
SRBC
and
the
simultaneousnumbers
groups
showed
spleen
these
groups
ten
injected
with
these
spleens
than
LPS-minus-control
of
of
mice
when
times
PFC
injected compared
or
more
PFC
controls.
which
showed
V. cholerae
shown
increases
Especially
spleens
,ƒÊg of injection,
examined.
significant
control.
However, munization,
into
injected 4
spleens
the
V. cholerae
of
spleens with
OF
(19).
Two
LPS
of
stimulation
time-relation
simultaneously
were
with
the
for
cells in their then immunized
response
their
Effects
effects
to
Mice
with
in
dual
according
and
ly.
Immunosuppressive
has
PROPERTIES
less
had PFC
been in
their
LPS
2 days
before mice
the
im-
(Table
5).
Polyclonal An xenogenic
Activation injection
of B Lymphocytes of
LPS
erythrocytes
Table 4.
in
Table 5.
alone mouse
by
V. cholerae
nonspecifically spleens
(20).
LPS increases Under
the these
number
circumstances,
Adjuvant effect of V. choleraeLPS on anti-SRBC PFC responses in the spleen of C57BL/6 mice
Immunosuppressive effect of V. choleraeLPS on anti-SRBC PFC responses in the spleen of C57BL/6 mice
of
PFC antibody
to
616
M.
NAKANO
ET AL
Table 6. Nonspecific elicitation of anti-SRBC PFC in the spleen of C57BL/6 mice after the injection of V. choleraeLPS
forming
cell
lated
by
against
of
the
numbers
one
hundred
mice
which
plaques
in
B
LPS
unrelated
,ƒÊg each
viously
precursor
the
to
lymphocytes
manifest
antigens
V. cholerae of
PFC
Three their
background had their
weaker
been
PFC
spleens than
Fig.
the of
1. function mice toneally vance.
after were
the
with
than those
days
in
of the (carbon injected
of
(2).
the
of
clearance with
these
the
two
effects
LT2-LPS.
LPS
test). 10ƒÊg
on
phagocytic
system Mice of
were LPS
in
AKR
intraperi48hr
in
ad-
killed were
However,
or of
These
stimu-
with
were
There
controls.
showed
be
antibodies
injected
mice
6).
untreated
although
cholerae
were
(Table
LPS
reticuloendothelial
might specific
Mice
injection,
cholerae
V.
population
synthesizing
examined
typhimurium
Effects
cell of
spleen
V.
controls
S.
spleen
activation)
spleens
injected
the
capabilities
(polyclonal
LPS. in
in
their
three
these results
times LPS
were
suggest
10 and
about the more obthat
IMMUNOLOGICAL
PROPERTIES
OF
V. CHOLERAE
LPS
617
V. choleraeLPS are capable of maturing nonspecifically precursor B lymphocytes to antibody producing cells. Stimulationof the PhagocyticFunctionof the Reticuloendothelial Systemby V. choleraeLPS Mice were injected with 10 ,ug each of V. choleraeLPS. Forty eight hours after the LPS-injection, they were injected with carbon-ink through their tail veins. Five, 10 and 15 min after the injection, small amounts of blood samples were collected from the retroorbital plexus of the mice, and the concentrations of carbon in the blood samples were photometrically determined. As shown in Fig. 1, mice injected with these LPS showed a marked acceleration of clearance, indicating that these LPS were capable of stimulating the phagocytic function of the reticuloendothelial system. DISCUSSION
The effects of LPS or endotoxins obtained from gram-negative bacteria on the murine immune system have been extensively studied in vivoand in vitro. LPS exert various effects in susceptible hosts. For example, injections of LPS into mice in conjunction with xenogeneic erythrocytes as the antigen result in marked increases in the numbers of anti-erythrocyte antibody PFC generated in the spleens (adjuvant effect), while under some experimental conditions, LPS is known to have reverse potency as an immunosuppressant (6, 15, 19). Furthermore, even without the administration of any antigen, an injection of LPS alone increases the phagocytic function of the reticuloendothelial macrophages (8, 16) and nonspecifically generates PFC to xenogeneic erythrocytes in the spleens of mice (polyclonal B cell activation) (4, 20). The addition of LPS into in vitrocultured spleen cells appears to cause DNA synthesis in B-cells, resulting in cell division (mitogenicity) (2). However, it should be pointed out that the effects of V.choleraeLPS on the immune response have scarcely been studied. LPS of gram-negative bacteria consist of a specific polysaccharide covalently linked through a KDO to the lipid A. Lipid A is thought to be the endotoxic center of the LPS, being responsible for many biological activities (1, 14, 23). However, another part of LPS, which may act as a carrier to solubilize hydrophobic lipid A, seems to play some role in the expression of endotoxic activity of lipid A (16, 17). Jann et al (12) reported that LPS extracted from microorganisms of Inaba 568B and Ogawa NIH 41 contained glucose, heptose, glucosamine,fructose and quinovosamine, but lacked KDO. Our LPS-preparations, of Inaba 569B-, Ogawa NIH 41 and NAG 4715 used in the present experiments also did not contain any detectable KDO (10, 11). Salmonellalipid A contains glucosamine, phosphate, and long chain fatty acids consisting of lauric, myristic, palmitic and 3-D(-)hydroxymyristic acids (14). Investigations of LPS of genera other than Salmonellarevealed that the nature and distribution of fatty acids may vary (14). All the preparations of V. chole7ae LPS used for the present experiments contained normal fatty acids (C14 and C16) and hydroxyfatty acids (C13 3-hydroxymyristic acid and C16 hydroxyfatty acid) (11). Furthermore, Inaba 569B-LPS contained some additional (normal and 3-
618
M.
NAKANO
ET AL
hydroxy) fatty acids with odd numbers (C11 and C13) of carbon atoms (11). Because chemical studies on the nature and structure of V. choleraeLPS have not progressed as rapidly as studies on the immunochemistry of the LPS of Salmonellaand other Enterobacteriaceae,the sequential linkages of these saccharides, covalent linkage of the polysaccharides to lipid A without KDO and the structure of lipid A have not been clarified as yet. The experiments presented here clearly demonstrate that, although the structure of V.choleraeLPS may differ greatly from those of other gram-nagative bacteria, these LPS have still some potencies as B cell mitogens, adjuvants, immunosuppressants, polyclonal B cell activators and phagocytic stimulants for macrophages. However, the activities of these V. choleraeLPS on the immune phenomenon seemed to be weaker than those of S. typhimuriumLT2-LPS. Among these V. choleraeLPS which have similar LD50 potencies for mice (see MATERIALSAND METHODS),NAG 4715LPS showed stronger mitogenic activity and phagocytic stimulation when compared with either Inaba 569B- or Ogawa NIH 41-LPS (Tables 1 and 2, and Fig. 1). However, the potencies of this NAG 4715-LPS for the induction of polyclonal B cell activation, adjuvant effect and immunosuppression seemed not to be superior to those of other LPS (Tables 4, 5 and 6). Unfortunately, we do not have enough knowledge to discuss the basic nature and chemical structure of V. choleraeLPS which make the effects of these LPS weaker than those of S. typhimuriumLT2-LPS and the mitogenic and phagocytosis-stimulating effects of NAG 4715-LPS stronger than those of other V. choleraeLPS. To obtain an exact answer to these questions, further chemical and immunological studies on V. choleraeLPS will be necessary. REFERENCES
1)
2) 3) 4) 5) 6) 7) 8) 9) 10)
Andersson, J., Melchers, F., Galanos, C., and Luderitz, O. 1973. The mitogenic effect of lipopolysaccharide on bone marrow-derived mouse lymphocytes. Lipid A as the mitogenic part of the molecule. J. Exp. Med. 137: 943-953. Andersson, J., Sjoberg, O., and Mailer, G. 1972. Mitogens as probes for immunocyte activation and cellular cooperation. Transplant. Rev. 11: 131-177. Blanden, R.V. 1970. Mechanisms of recovery from a generalized viral infection :mousepox. I. The effects of antithymocyte serum. J. Exp. Med. 132: 1035-1054. Coutinho, A., and Moller, G. 1975. Thymus-independent B-cell induction and paralysis. Adv. Immunol. 21: 113-236. Fensom, A.H., and Gray, G.W. 1969. The chemical composition of the lipopolysaccharide of Pseudomonasaeruginosa. Biochem. J. 114: 185-196. Finger, H., Fresenius, H., and Angerer, M. 1971. Bacterial endotoxins as immunosuppressive agents. Experientia 27: 456-458. Finkelstein, R.A. 1975. Immunology of cholera. Curr. Top. Microbial. Immunol. 69: 137-200. Freedman, H.H. 1960. Reticuloendothelial system and passive transfer of endotoxin tolerance. Ann. N.Y. Acad. Sci. 88: 99-106. Hirst, J.A., Beverley, P.C.L., Kisielow, P., Hoffmann, M.K., and Oettgen, H.F. 1975. Ly antigens : Markers of T cell function of mouse spleen cells. J. Immunol. 115: 1555-1557. Hisatsune, K., Kondo, S., Kobayashi, K., Ikekawa, N., Morisaki, M., Ishikawa, T., Tanabe, M., Nakano, M., and Takeya, K. 1976. Lipopolysaccharides of Vibriocholerae. Chemistry and immunology. The 12th Joint Conference of the US-Japan Cooperative Medical Science Program Cholera Panel, October 6-8, Sapporo, Japan.
IMMUNOLOGICAL
11) 12) 13)
14)
15)
16) 17)
18)
19) 20) 21)
22) 23) 24)
PROPERTIES
OF
V. CHOLERAE
LPS
619
Hisatsune, K., Kondo, S., Tanabe, M.J., and Nakano, M. 1977. Lipopolysaccharides of Vibrio cholerae.Chemical and immunological properties. Japan. J. Med. Sci. Biol. 30: 61-64. Jann, B., Jann, K., and Beyaert, G.O. 1973. 2-amino-2, 6-dideoxy-D-glucose (D-quinovosamine) : a constituent of the lipopolysaccharides of Vibriocholerae. Eur. J. Biochem. 37: 531-534. Jerne, N.K., Nordin, A., and Henry, C. 1963. The agar plaque technique for recognizing antibody-producing cells, p. 109-122. In Amos, B., and Koprowski, H. (eds.), Cell-bound antibody, Wister Inst. Press, Philadelphia. Luderitz, O., Galanos, C., Lehmann, V., Nurminen, M., Rietschel, E.T., Rosenfelder, G., Simon, M., and Westphal, O. 1973. Lipid A: Chemical structure and biological activity. J. Infect. Dis. 128: s17-s29. Hoffmann, M., Weiss, O., Koenig, S., Hirst, J.A., and Oettgen, H.F. 1975. Suppression and enhancement of the T cell-dependent production of antibody to SRBC in vitro by bacterial lipopolysaccharide. J. Immunol. 114: 738-741. Nakano, M., Asou, H., and Yamamoto, I. 1975. Stimulation of phagocytic activity in the reticuloendothelial systems of mice by lipid A complexed with homologous or heterologous proteins. Infect. Immunity 11: 592-594. Nakano, M., Saito, T., and Asou, H. 1975. Adjuvant effect of lipid A obtained from Salmonella typhimuriumon antibody response in mouse spleen. The role of carriers conjugated to lipid A. Japan. J. Microbiol. 19: 403-406. Nakano, M., Shimamura, T., and Saito, K. 1971. Cellular mechanisms of adjuvant action of bacterial lipopolysaccharide in anti-sheep red blood cell antibody response. Japan. J. Microbiol. 15: 149-158. Nakano, M., Tanabe, J.M., Saito, T., and Shimizu, T. 1976. Immunosuppressive effect of bacterial lipopolysaccharide on antibody response. Japan. J. Microbiol. 20: 53-58. Nakano, M., Uchiyama, T., Tanabe, M.J., Saito, K. 1975. Nonspecific elicitation of antibodyforming cells in mouse spleen by bacterial lipopolysaccharide. Japan. J. Microbiol. 19: 141-148. Redmond, J.W., Korsch, M.J., and Jackson, G.D.F. 1973. Immunochemical studies of the Oantigens of Vibriocholerae.Partial characterization of an acid-labile antigenic determinant. Aust. J. Exp. Biol. Med. Sci. 51: 229-225. Skidmore, B.J., Chiller, J.M., and Weigle, W.O. 1977. Immunologic properties of bacterial lipopolysaccharide (LPS). IV. Cellular basis of the unresponsiveness of C3H/HeJ mouse spleen cells to LPS-induced mitogenesis. J. Immunol. 118: 274-281. Westphal, O. 1975. Bacterial endotoxins. Int. Arch. Allergy Appl. Immunol. 49: 1-43. Westphal, O., and Liideritz, O. 1954. Chemische Erforschung von Lipopolysacchariden gramnegativer Bakterien. Angew. Chem. 66: 407-417.
Requests for reprints should be addressed to Dr. Masayasu Nakano, Department biology, Jichi Medical School, Tochigi-ken 329-04, Japan.
of Micro-
Immunological
Properties of Vibrio cholerae Lipopolysaccharides
Masayasu NAKANO,Masao J. TANABE,Hideko HORI, Seiichi KONDO,and Kazuhito HISATSUNE Departmentof Microbiology,Jichi Medical School, Tochigi, and Departmentof Microbiology,Schoolof PharmaceuticalScience,Johsai University,Sakado, Saitama (Received for publication, May 10, 1977)
Abstract Immunological effects of wall lipopolysaccharide (LPS) preparations obtained from VibriocholeraeInaba 569B, Ogawa NIH 41 and NAG 4715 strains by the hot phenol-water procedure were examined in mice. Although these LPS lack KDO, which are basic components of the core region of most gram-negative LPS, they still have potencies as B-cell mitogens, adjuvants, immunosuppressants, polyclonal B-cell activators and phagocytic stimulants for macrophages. The activities of these V. choleraeLPS on murine immune system seemed to be weaker than those of Salmonella typhimurium LT2-LPS. Among these V. cholerae LPS, NAG 4715-LPS showed the strongest mitogenic activity and phagocytic stimulation, while the potencies of this NAG 4715-LPS for the induction of polyclonal B cell activation, adjuvant effects and immunosuppression did not seem to be greater to those of the other LPS.
Lipopolysaccharides (LPS) are structural components in the cell walls of gram negative bacteria and these components are easily extractable from many kinds of bacteria, especially Enterobacteriaceae,by physicochemical procedures (14, 23, 24). Similar LPS components could be extracted from Vibrio choleraeorganisms (7). However, V. choleraeLPS lack 2-keto-3-deoxyoctonate (KDO) and galactose, which are components of the core region of most gram-negative LPS (10, 14, 23) among their constituents (12, 21). The lipid A in V. choleraeLPS contains unusually large amounts of glycin and ammoniac radicals in comparison with other gram-negative LPS (10). Inaba type vibrio LPS contain considerable amounts of the odd numbered fatty acids, while those of Ogawa and NAG vibrios do not (11). These findings suggest that cholera vibrios may have a fundamentally different LPS structure from that of Salmonellaand other gram-negative bacteria, and, furthermore, structural differences of lipid A components in LPS may exist among V. choleraestrains. Recently, much attention has been focused on the effects of LPS on immune phenomena (1, 2, 4, 15, 22). Almost all of the experiments to examine the immunological properties of LPS have been performed using LPS prepared from gram-negative rods, but not from vibrios. Therefore, it may be worthwhile to examine whether or not the structurally different V. choleraeLPS are capable of stimulating immune 611
612
M.
NAKANO
ET AL
phenomena as other endotoxins do. In this paper, we describe the effects of V. choleraeLPS on the murine immune system as follows: a) mitogenic stimulation of cultured lymphocytes, b) enhancement and suppression of antibody response against sheep erythrocyte antigen, c) polyclonal B cell activation, and d) stimulation of the phagocytic function of the reticuloendothelial system. MATERIALS
AND
METHODS
LPS. V. choleraeLPS used in this study were isolated from V. cholerae569B Inaba type, NIH 41 Ogawa type and NAG 4715 by Westphal's phenol-water technique (24). They were highly purified by repeated ultracentrifugations and treatments with RNase and cetabrone (5). LT2-LPS was used in the experiments as a control. LT2-LPS was extracted by Westphal's technique from the LT2 strain (smooth type) of S. typhimurium, and purified by repeated ultracentrifugations. Mouse lethal doses (LD50: intraperitoneal injections into young adult DD strain mice) of these Inaba 569B-, Ogawa NIH 41-, NAG 4715-, and LT2-LPS were 90, 110, 90, and 13 mg per kg respectively. Animals. colony
AKR
were
or
used
in
Incorporation
of
removed
and
teased
through
a fine
nylon
and
subsequently
with
10%
of
air
10%
the
vested
onto
dried.
end
of
counter
serum
(ATS)
incubated
with
pig
complement
the
incubation,
number
of and
One-tenth each
and ml of
Determination
cell-rich
spleen by
in
order
to
cells
were
cells
in
for
the
diluted
the
the
cultures
immunization.
of the the
from
or
thymuses
in
in
1640
the
our
own
a sterile cultured
of
incubation GF/F)
by
into
the
cell
suspension.
72
hr,
was
the
medium
supplemented to of
the
desired
these
cell
sus-
(methyl-3H-
to
the
cultured
culture cells
and
assessed
passing
tube (12 •~75 mm, C in an atmosphere
added
aspiration,
cells
of
3H-thymidine was
for
ml
each
culture at 37
sterile
Tokyo)
50
medium ml
were
After
medium
culture One
into were
Ltd.,
medium.
times
suspensions.
(Whatman
prepared
adjusted
Antigen
three
microcurie
After
adequately
viable
age,
then
by
a
20
were washed
Packard
hr
harand scintil-
3320). B
was
of
Spleens
1640
(RPMI
dissolved
Co.,
radioactivity
(Model
washed
cell
One
filters of
of
weeks
cells.
RPMI
medium
was
Chemicals
fiber
8-12
lymphoid
cells) was poured and the cells
CO2.
uptake
sexes,
ice-cold were
the
incubation.
glass
The
Preparation
into
into
Pure
cultured
culture
LPS
added
and
of both
in cells
in
serum).
Daiichi
before
test,
the
(2 •~ 106 nucleated Plastics, Los Angeles)
90%
into
forceps
mesh,
and
thymidine,
lation
3H-thymidine with
human
mice
experiments.
suspended
concentration, pensions Falcon
C57BL/6
the
SRBC
kill
method ATS
of for
45
Rabbit Blanden
min
thymus-derived
washed suspension (2 •~ Sheep
suspension
suspended
was 106
counted
viable red (108
at cells
and
cells
blood
cells
erythrocytes)
by in
anti-mouse
(3).
The
37
C in
in
the
cell
in
the
culture
the
thymocyte
spleen
the
presence
cells
population.
After
medium.
trypan
blue
were
of guinea
dye
The exclusion
1 ml). (SRBC)
were
used
was
inoculated
suspensions
were
as an
antigen.
intravenously
mice. of
immune
response.
Spleen
cell
prepared
from
IMMUNOLOGICAL
PROPERTIES
OF
V. CHOLERAE
LPS
613
individual mice and the number of plaque forming cells (PFC) in the suspension was counted by the technique of localized hemolysis in agar (13). As a rule, five mice were used for the determination of the PFC-response in one group, and the mean PFC of the five mice and the standard error were calculated. The effect of LPS was estimated by comparison between PFC-numbers of LPS-treated mice and of controls. Estimation of the phagocyticfunction of macrophages. The phagocytic function of macrophages was measured by the rate of carbon clearance from the circulating blood of mice according to the method of Freedman (8). RESULTS Mitogenic
Effect
of
Mitogenic thymidine LPS
into
is
while
with
than
in that
stimulating
of
cells
NAG
were
cells
in
Inaba
Lymphocytes examined
vitro.
569-
4715-LPS.
of
probably was
treated
20
shown
uptake
of
and
radioactive
,ƒÊg of
by
As
Ogawa
However,
and
act
were
Table
cultured
1.
Mitogenicity
on
with
lymphocytes
cells
Cultured
3H-thymidine of
Inaba-LPS
increase
thymus-derived spleen
on LPS
the
Table
the
addition
of
incorporation
to
spleen
cells,
into
the
are
obviously
adequate
the
doses
cultures
cells
3H-
4714-
41-LPS
Ogawa-LPS)
of
1, NAG
cultured
NIH
the
incorporation
in
in
of
always
comparison
controls. LPS
spleen
LPS
cholerae
lymphoid
,ƒÊg of
some in
V.
effects
those (80
These of
of
mitogenic
LPS
resulted
cholerae of
cultured
capable
the
weaker these
Vibrio
effects
bone ATS
(T with
marrow-derived in
cells). or
the
Both
without
of V. cholerae
LPS
lymphocytes
presence these
the
addition
on
cultured
of
complement
ATS-treated of
spleen
V.
(B
cells).
in
order
and cholerae
cells
LPS,
of AKR-mice
Part to
kill
non-treated and
then
614
M. Table
2.
Table
Mitogenicity in cultured
3.
NAKANO
ET AL
of V. cholerae LPS on B lymphocytes spleen cells of AKR mice
Mitogenicity thymocytes
of V. cholerae LPS of AKR mice
on cultured
3H-thymidine uptake of the cultured cells was examined (Table 2). After the treatment with ATS, 3H-thymidine uptake (background) of these cultured cells was reduced to about one half and most of the mitogenic response of these cells to Con A, known to be a mitogen for T cells (9), disappeared. However, the mitogenic responses of these ATS-treated cells to NAG- and LT2-LPS increased when compared with those of untreated cells, suggesting that these LPS have the potency to act as B cell mitogen and elimination of T cells from the spleen cell population results in the enhancement of the mitogenic response of the cell population. Since the mitogenic potencies of Ogawa- and Inaba-LPS were weaker than that of NAG-LPS, the increased response of ATS-treated cells to these LPS was not obvious, but these LPS seemed to have mitogenic potency on ATS-treated spleen cells. These V. choleraeLPS, like LT2-LPS, did not have any stimulatory effect on 3H-thymidine incorporation into cultured thymus cells (Table 3) .
IMMUNOLOGICAL Adjuvant
and
LPS mice
antigen
producing first and mune
in
and
their
with
days
were
showed
or
mice,
V. CHOLERAE
LPS
on
suppression
injection several
with days
Anibody of
later
we
Response
the
antigen
615
LPS
antibody
(19). may
response
If
find
we
of
inject
a lot
LPS
of antibody
(18). Conversely, if the mice are injected the antigen in several days later, we find
10
after
the
As
than
the
mice,
on
with a poor
LPS im-
of
day
LPS
they
4,
in PFC
were Table
and
108
killed 4,
all
the
in
the
numbers
of
SRBC
and
the
simultaneousnumbers
groups
showed
spleen
these
groups
ten
injected
with
these
spleens
than
LPS-minus-control
of
of
mice
when
times
PFC
injected compared
or
more
PFC
controls.
which
showed
V. cholerae
shown
increases
Especially
spleens
,ƒÊg of injection,
examined.
significant
control.
However, munization,
into
injected 4
spleens
the
V. cholerae
of
spleens with
OF
(19).
Two
LPS
of
stimulation
time-relation
simultaneously
were
with
the
for
cells in their then immunized
response
their
Effects
effects
to
Mice
with
in
dual
according
and
ly.
Immunosuppressive
has
PROPERTIES
less
had PFC
been in
their
LPS
2 days
before mice
the
im-
(Table
5).
Polyclonal An xenogenic
Activation injection
of B Lymphocytes of
LPS
erythrocytes
Table 4.
in
Table 5.
alone mouse
by
V. cholerae
nonspecifically spleens
(20).
LPS increases Under
the these
number
circumstances,
Adjuvant effect of V. choleraeLPS on anti-SRBC PFC responses in the spleen of C57BL/6 mice
Immunosuppressive effect of V. choleraeLPS on anti-SRBC PFC responses in the spleen of C57BL/6 mice
of
PFC antibody
to
616
M.
NAKANO
ET AL
Table 6. Nonspecific elicitation of anti-SRBC PFC in the spleen of C57BL/6 mice after the injection of V. choleraeLPS
forming
cell
lated
by
against
of
the
numbers
one
hundred
mice
which
plaques
in
B
LPS
unrelated
,ƒÊg each
viously
precursor
the
to
lymphocytes
manifest
antigens
V. cholerae of
PFC
Three their
background had their
weaker
been
PFC
spleens than
Fig.
the of
1. function mice toneally vance.
after were
the
with
than those
days
in
of the (carbon injected
of
(2).
the
of
clearance with
these
the
two
effects
LT2-LPS.
LPS
test). 10ƒÊg
on
phagocytic
system Mice of
were LPS
in
AKR
intraperi48hr
in
ad-
killed were
However,
or of
These
stimu-
with
were
There
controls.
showed
be
antibodies
injected
mice
6).
untreated
although
cholerae
were
(Table
LPS
reticuloendothelial
might specific
Mice
injection,
cholerae
V.
population
synthesizing
examined
typhimurium
Effects
cell of
spleen
V.
controls
S.
spleen
activation)
spleens
injected
the
capabilities
(polyclonal
LPS. in
in
their
three
these results
times LPS
were
suggest
10 and
about the more obthat
IMMUNOLOGICAL
PROPERTIES
OF
V. CHOLERAE
LPS
617
V. choleraeLPS are capable of maturing nonspecifically precursor B lymphocytes to antibody producing cells. Stimulationof the PhagocyticFunctionof the Reticuloendothelial Systemby V. choleraeLPS Mice were injected with 10 ,ug each of V. choleraeLPS. Forty eight hours after the LPS-injection, they were injected with carbon-ink through their tail veins. Five, 10 and 15 min after the injection, small amounts of blood samples were collected from the retroorbital plexus of the mice, and the concentrations of carbon in the blood samples were photometrically determined. As shown in Fig. 1, mice injected with these LPS showed a marked acceleration of clearance, indicating that these LPS were capable of stimulating the phagocytic function of the reticuloendothelial system. DISCUSSION
The effects of LPS or endotoxins obtained from gram-negative bacteria on the murine immune system have been extensively studied in vivoand in vitro. LPS exert various effects in susceptible hosts. For example, injections of LPS into mice in conjunction with xenogeneic erythrocytes as the antigen result in marked increases in the numbers of anti-erythrocyte antibody PFC generated in the spleens (adjuvant effect), while under some experimental conditions, LPS is known to have reverse potency as an immunosuppressant (6, 15, 19). Furthermore, even without the administration of any antigen, an injection of LPS alone increases the phagocytic function of the reticuloendothelial macrophages (8, 16) and nonspecifically generates PFC to xenogeneic erythrocytes in the spleens of mice (polyclonal B cell activation) (4, 20). The addition of LPS into in vitrocultured spleen cells appears to cause DNA synthesis in B-cells, resulting in cell division (mitogenicity) (2). However, it should be pointed out that the effects of V.choleraeLPS on the immune response have scarcely been studied. LPS of gram-negative bacteria consist of a specific polysaccharide covalently linked through a KDO to the lipid A. Lipid A is thought to be the endotoxic center of the LPS, being responsible for many biological activities (1, 14, 23). However, another part of LPS, which may act as a carrier to solubilize hydrophobic lipid A, seems to play some role in the expression of endotoxic activity of lipid A (16, 17). Jann et al (12) reported that LPS extracted from microorganisms of Inaba 568B and Ogawa NIH 41 contained glucose, heptose, glucosamine,fructose and quinovosamine, but lacked KDO. Our LPS-preparations, of Inaba 569B-, Ogawa NIH 41 and NAG 4715 used in the present experiments also did not contain any detectable KDO (10, 11). Salmonellalipid A contains glucosamine, phosphate, and long chain fatty acids consisting of lauric, myristic, palmitic and 3-D(-)hydroxymyristic acids (14). Investigations of LPS of genera other than Salmonellarevealed that the nature and distribution of fatty acids may vary (14). All the preparations of V. chole7ae LPS used for the present experiments contained normal fatty acids (C14 and C16) and hydroxyfatty acids (C13 3-hydroxymyristic acid and C16 hydroxyfatty acid) (11). Furthermore, Inaba 569B-LPS contained some additional (normal and 3-
618
M.
NAKANO
ET AL
hydroxy) fatty acids with odd numbers (C11 and C13) of carbon atoms (11). Because chemical studies on the nature and structure of V. choleraeLPS have not progressed as rapidly as studies on the immunochemistry of the LPS of Salmonellaand other Enterobacteriaceae,the sequential linkages of these saccharides, covalent linkage of the polysaccharides to lipid A without KDO and the structure of lipid A have not been clarified as yet. The experiments presented here clearly demonstrate that, although the structure of V.choleraeLPS may differ greatly from those of other gram-nagative bacteria, these LPS have still some potencies as B cell mitogens, adjuvants, immunosuppressants, polyclonal B cell activators and phagocytic stimulants for macrophages. However, the activities of these V. choleraeLPS on the immune phenomenon seemed to be weaker than those of S. typhimuriumLT2-LPS. Among these V. choleraeLPS which have similar LD50 potencies for mice (see MATERIALSAND METHODS),NAG 4715LPS showed stronger mitogenic activity and phagocytic stimulation when compared with either Inaba 569B- or Ogawa NIH 41-LPS (Tables 1 and 2, and Fig. 1). However, the potencies of this NAG 4715-LPS for the induction of polyclonal B cell activation, adjuvant effect and immunosuppression seemed not to be superior to those of other LPS (Tables 4, 5 and 6). Unfortunately, we do not have enough knowledge to discuss the basic nature and chemical structure of V. choleraeLPS which make the effects of these LPS weaker than those of S. typhimuriumLT2-LPS and the mitogenic and phagocytosis-stimulating effects of NAG 4715-LPS stronger than those of other V. choleraeLPS. To obtain an exact answer to these questions, further chemical and immunological studies on V. choleraeLPS will be necessary. REFERENCES
1)
2) 3) 4) 5) 6) 7) 8) 9) 10)
Andersson, J., Melchers, F., Galanos, C., and Luderitz, O. 1973. The mitogenic effect of lipopolysaccharide on bone marrow-derived mouse lymphocytes. Lipid A as the mitogenic part of the molecule. J. Exp. Med. 137: 943-953. Andersson, J., Sjoberg, O., and Mailer, G. 1972. Mitogens as probes for immunocyte activation and cellular cooperation. Transplant. Rev. 11: 131-177. Blanden, R.V. 1970. Mechanisms of recovery from a generalized viral infection :mousepox. I. The effects of antithymocyte serum. J. Exp. Med. 132: 1035-1054. Coutinho, A., and Moller, G. 1975. Thymus-independent B-cell induction and paralysis. Adv. Immunol. 21: 113-236. Fensom, A.H., and Gray, G.W. 1969. The chemical composition of the lipopolysaccharide of Pseudomonasaeruginosa. Biochem. J. 114: 185-196. Finger, H., Fresenius, H., and Angerer, M. 1971. Bacterial endotoxins as immunosuppressive agents. Experientia 27: 456-458. Finkelstein, R.A. 1975. Immunology of cholera. Curr. Top. Microbial. Immunol. 69: 137-200. Freedman, H.H. 1960. Reticuloendothelial system and passive transfer of endotoxin tolerance. Ann. N.Y. Acad. Sci. 88: 99-106. Hirst, J.A., Beverley, P.C.L., Kisielow, P., Hoffmann, M.K., and Oettgen, H.F. 1975. Ly antigens : Markers of T cell function of mouse spleen cells. J. Immunol. 115: 1555-1557. Hisatsune, K., Kondo, S., Kobayashi, K., Ikekawa, N., Morisaki, M., Ishikawa, T., Tanabe, M., Nakano, M., and Takeya, K. 1976. Lipopolysaccharides of Vibriocholerae. Chemistry and immunology. The 12th Joint Conference of the US-Japan Cooperative Medical Science Program Cholera Panel, October 6-8, Sapporo, Japan.
IMMUNOLOGICAL
11) 12) 13)
14)
15)
16) 17)
18)
19) 20) 21)
22) 23) 24)
PROPERTIES
OF
V. CHOLERAE
LPS
619
Hisatsune, K., Kondo, S., Tanabe, M.J., and Nakano, M. 1977. Lipopolysaccharides of Vibrio cholerae.Chemical and immunological properties. Japan. J. Med. Sci. Biol. 30: 61-64. Jann, B., Jann, K., and Beyaert, G.O. 1973. 2-amino-2, 6-dideoxy-D-glucose (D-quinovosamine) : a constituent of the lipopolysaccharides of Vibriocholerae. Eur. J. Biochem. 37: 531-534. Jerne, N.K., Nordin, A., and Henry, C. 1963. The agar plaque technique for recognizing antibody-producing cells, p. 109-122. In Amos, B., and Koprowski, H. (eds.), Cell-bound antibody, Wister Inst. Press, Philadelphia. Luderitz, O., Galanos, C., Lehmann, V., Nurminen, M., Rietschel, E.T., Rosenfelder, G., Simon, M., and Westphal, O. 1973. Lipid A: Chemical structure and biological activity. J. Infect. Dis. 128: s17-s29. Hoffmann, M., Weiss, O., Koenig, S., Hirst, J.A., and Oettgen, H.F. 1975. Suppression and enhancement of the T cell-dependent production of antibody to SRBC in vitro by bacterial lipopolysaccharide. J. Immunol. 114: 738-741. Nakano, M., Asou, H., and Yamamoto, I. 1975. Stimulation of phagocytic activity in the reticuloendothelial systems of mice by lipid A complexed with homologous or heterologous proteins. Infect. Immunity 11: 592-594. Nakano, M., Saito, T., and Asou, H. 1975. Adjuvant effect of lipid A obtained from Salmonella typhimuriumon antibody response in mouse spleen. The role of carriers conjugated to lipid A. Japan. J. Microbiol. 19: 403-406. Nakano, M., Shimamura, T., and Saito, K. 1971. Cellular mechanisms of adjuvant action of bacterial lipopolysaccharide in anti-sheep red blood cell antibody response. Japan. J. Microbiol. 15: 149-158. Nakano, M., Tanabe, J.M., Saito, T., and Shimizu, T. 1976. Immunosuppressive effect of bacterial lipopolysaccharide on antibody response. Japan. J. Microbiol. 20: 53-58. Nakano, M., Uchiyama, T., Tanabe, M.J., Saito, K. 1975. Nonspecific elicitation of antibodyforming cells in mouse spleen by bacterial lipopolysaccharide. Japan. J. Microbiol. 19: 141-148. Redmond, J.W., Korsch, M.J., and Jackson, G.D.F. 1973. Immunochemical studies of the Oantigens of Vibriocholerae.Partial characterization of an acid-labile antigenic determinant. Aust. J. Exp. Biol. Med. Sci. 51: 229-225. Skidmore, B.J., Chiller, J.M., and Weigle, W.O. 1977. Immunologic properties of bacterial lipopolysaccharide (LPS). IV. Cellular basis of the unresponsiveness of C3H/HeJ mouse spleen cells to LPS-induced mitogenesis. J. Immunol. 118: 274-281. Westphal, O. 1975. Bacterial endotoxins. Int. Arch. Allergy Appl. Immunol. 49: 1-43. Westphal, O., and Liideritz, O. 1954. Chemische Erforschung von Lipopolysacchariden gramnegativer Bakterien. Angew. Chem. 66: 407-417.
Requests for reprints should be addressed to Dr. Masayasu Nakano, Department biology, Jichi Medical School, Tochigi-ken 329-04, Japan.
of Micro-
