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.

Microbiol. Immunol. Vol. 21 (11), 611-619, 1977 Immunological Properties of Vibrio cholerae Lipopolysaccharides Masayasu NAKANO,Masao J. TANABE,Hid...
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