Vol. 21 (11), 653-665, 1977
Induced Serum Factor
to of Antibody
Masao J. TANABE, Yoshiko TSURUMI, and Masayasu NAKANO
Department of Microbiology, Jichi Medical School, Tochigi
(Received for publication, April 25, 1977)
injected with LPS several hours in advance, contained some active substance capable
of enhancing anti-sheep red blood cell (SRBC) antibody responses in mice. Ac-
tivity of the sera was still retained after passage through a rabbit anti-LPS antibody-
coated Sepharose 4B column, but greatly reduced by passage through a rabbit anti-
mouse thymocyte antibody-coated Sepharose 4B column.
the sera was eluted through a Sephadex G-200 column at the same position as the
The addition of this substance to B cell rich spleen cell cultures in vitro in the
presence of SRBC generated tremendous numbers of antibody forming cells 4 days
after the incubation, suggesting that this substance was able to take over the helper
function of T cells in thymus dependent antibody responses. However, this sub-
stance was not capable of stimulating 3H-thymidine-uptake into cultured spleen cells.
The possible role of this substance in the adjuvant effect of LPS is discussed.
The sera obtained from blood of the mice, which had been intravenously
The active substance in
Lipopolysaccharide (LPS) obtained from Gram-negative bacteria has recently
become a focal point of interest in immunology because of its various effects on the
murine immune system. This substance itself has been characterized as a mitogen or
polyclonal activator for B lymphocytes (2, 9, 10, 27). On the other hand, when
LPS is injected with antigens concomitantly, prominent adjuvant effects on antibody
responses to these antigens are observed (3, 15). Mechanisms of the adjuvant effect
of LPS have still not been clarified, but there are two opposite views on the target
cells of LPS in lymphoid cells; i.e., the adjuvant effect of LPS results from (a) its
direct effect on antibody producing B lymphocytes (27), or (b) its stimulatory effect
on helper T lymphocytes participating in antibody response via some unknown
mechanism (3, 15). Effects of LPS on both antigen-stimulated B and T lymphocytes
may participate with the elicitation of its adjuvant effects on antibody responses in
We have already shown (29) that a mediator or soluble factor capable of stimu-
lating antibody response is produced in circulating blood after the injection of ad-
juvants or secondary antigenic stimulation, and this factor may play some role in the
enhancement of antibody responses. In this paper, we report on the evidence of the
M. J. TANABE ET AL
participation of this soluble factor in adjuvant effects of LPS in primary antibody
response against erythrocyte antigen in mice, and some characteristics of this factor.
MATERIALS AND METHODS
Mice. C57BL/6 mice of both sexes, 8-12 weeks of age from our own colony,
were used in all the experiments.
Antigen and immunization. Sheep red blood cells (SRBC) in Alsever's solution
were obtained commercially. Before use in vivo, these erythrocytes were washed three
times with physiological saline and resuspended in the saline (1 •~ 109 erythrocytes per
ml). Two-tenths ml each of the SRBC suspension (2 •~ 108 SRBC) was inoculated
intraperitoneally (ip) into mice. For in vitro experiments, SRBC were washed three
times with Eagle's minimal essential medium (MEM) and resuspended in MEM to
an appropriate concentration for inoculation into cultures.
Bacterial lipopolysaccharide (LPS). LPS was extracted from Salmonella typhimurium
LT2 with hot phenol-water according to the method of Westphal et al (34, 35) or with
cold n-butanol-water according to the method of Morrison et al (21). The LPS
extracted with hot phenol-water was purified by repeated centrifugation at 100,000 •~
to remove contaminating
The LPS extracted with
was with (20 ƒÊg/ml of pronase
45,000 Tryosin units/g, Kaken Chemicals, Co., Ltd., Tokyo, Japan) at 37 C overnight
to digest contaminating proteins and then this solution was concentrated by a rotary
evaporator (butanol LPS). For further purification, the butanol LPS solution (20-
40 mg/ml) was loaded onto Sepharose 4B columns (90 •~ 2.6 cm). LPS content in
each eluate was estimated by the assay of KDO (32). Butanol-LPS rich eluates
(tube numbers 24 to 29) were pooled, condensed by a rotary evaporator and stocked
in a refrigerator before use for experiments (column-passed butanol-LPS).
Preparation of anti-thymocyte-serum (ATS) and anti-LPS serum. Rabbit anti-mouse-
thymocyte-serum and anti-LPS-serum were obtained from blood of the rabbits 1
week after the last immunization, which had been intravenously (iv) immunized
repeatedly with either 5 •~ 108 C57BL/6 mouse thymocytes (5 times, 14 day intervals)
(6, 17) or 1010 heat killed organisms of S. typhimurium LT2 (7 times, 7 day intervals)
(22). These sera were inactivated at 56 C 30 min, and then adsorbed repeatedly
with one tenth volume of C57BL/6 mouse red blood cells until no hemagglutinin- or
hemolysin-titers to mouse erythrocytes could be detected in the sera.
Affinity column with ATS or anti-LPS serum. CNBr activated Sepharose 4B bead
gel (Pharmacia Fine Chemicals, Sweden) was swelled and washed by 1 mm HCl.
Anti-thymocyte- or anti-LPS-serum was coupled to the gel in 0.1 M NaHCO3-Na2-
CO3 buffer (pH 8.3, 0.5 M NaCl) for 2 hr at room temperature. The gels bound
around anti-thymocyte- or anti-LPS-antibodies were washed once with the same
buffer and then washed several times with acetate buffer (0.1 M NaCH3COO-CH3-
COOH pH 4, 0.5 M NaCl). Finally these gels were washed with phosphate buffered
saline (0.01 M K-PB pH 7.4, 0.15 M NaCl) and then packed into columns (1 •~ 10 cm)
SERUM FACTOR FOR ADJUVANT EFFECT OF LPS
" Endotoxin serum" and its chromatographic separation
. One hundred ƒÊg (in 0.2 ml
of saline) of column-passed butanol-LPS were injected intravenously (iv) into mice.
At indicated times after the injection, the blood of these mice were collected by car-
diac puncture. The sera were separated from the blood, pooled and inactivated at
C for 30 min.
Thus, the serum obtained from LPS-injected mice is termed
." To obtain an active substance from "endotoxin serum," the
serum was repeatedly chromatographed through a column (90 •~ 2.6 cm) of Sephadex
G-200 superfine at 4 C with 0.15 M phosphate buffered saline (PBS), pH 7.4, at a
pressure of 15 cm H2O (flow rate : 2.5 ml/hr).
Estimation of LPS content in "endotoxin serum." LPS content in the "endotoxin
serum" was estimated by the limulus test (36). Pregel solution, lysate of the ame-
bocyte of Limulus polyphemus (Teikokuzoki, Co., Ltd., Japan), was dissolved in 0.1 ml
of distilled water. One-tenth ml each of serially ten-fold diluted "endotoxin serum"
was dropped into the test tubes containing pregel solution and then the endpoint of
this serum in limulus gelation was determined after incubation at 37 C for 1 hour.
Spleen cell suspension and culture media for spleen cells. The spleen cell suspension was
prepared by teasing cells out of the splenic capsule in MEM containing with 10% of
FCS. After the elimination of undispersed cells by passage through fine nylon meshes
and placing the suspension in ice bath for 5 min, these cells were resuspended into
culture media. Nutrient mixture F-15 [Grand Island Biological Co., (Gibco),
Grand Island, N.Y.] supplemented with 10% heat-inactivated fetal calf serum was
employed as a routine culture medium to test for antibody responses. RPMI-1640
(Gibco) supplemented with 5% heat-inactivated human serum was employed as a
culture medium to test for the mitogenicity of spleen cells. If B cell rich suspensions
were desired for cultures, the spleen cells were treated for 45 min at 37 C with ade-
quately diluted ATS in the presence of fresh guinea pig complement (8) to kill T cells.
After this treatment, the cells were washed three times with fresh media and finally
suspended in the culture medium. The number of viable cells in the suspension was
calculated by the exclusion of trypan blue dye.
Assay for helper activity of "endotoxin serum" on antibody response. The cells were
cultured by the technique of Marbrook (18) with minor modifications. The spleen
cell suspension (1 •~ 107 viable cells in 0.2 ml of culture medium) was poured into the
inner compartment (10 •~ 100 mm) of the culture vessel, and then 0.2 ml of SRBC
with or without
ml of chromatographic eluates of the
added to same
. The final volume of the
culture in the inner compartment was increased to one ml by the addition of culture
medium. Glass vial bottles for scintillation counting (Packard Instruments) were
used as the outer compartment for the nutrient culture medium and 15 ml of the
medium was poured into the outer compartment. Both the inner and outer com-
partments were separated by a visking membrane, but small molecular substances
could pass freely through the membrane. The cells were cultured at 37 C for 4 days
in an atmosphere of 95% air and 5% CO2. At the end of the cultivation, the num-
ber of plaque forming cells (PFC) against SRBC in the culture was determined by
Jerne's method (16). As a rule, duplicate tubes were used for the determination of
M. J. TANABE ET AL
in one group,
The effect of "endotoxin
and the means
of the PFC-
Assay for mitogenicity of "endotoxin serum." Two-tenths ml of spleen cell sus-
pensions (1 •~ 107 nucleated cells per ml of RPMI-1640 medium supplemented with
5% human serum) and 0.1 ml of chromatographic eluates of "endotoxin serum" or
of culture medium were poured into a disposable tissue culture tube (12 •~ 75 mm)
(Falcon Plastics, No. 2058, Los Angeles, Calif.). These cell suspensions were finally
adjusted to a one ml volume with the addition of culture medium and then cultured
at 37 C in an atmosphere of 5% CO2 in humidified air for 72 hr. The cultures
received 1 ,uCi of 3H-thymidine (thymidine-methyl-T, 11.6 Ci/m mole, Daiichi Pure
Chemicals, Co., Ltd., Tokyo, Japan) twenty hours before harvesting. Incorporation
of labeled 3H-thymidine into the acid insoluble precipitate was measured by the
standard method (1).
Assay for antibody response against SRBC in vivo. Immune response of mice to
SRBC was determined by counting the numbers of 19S plaque-forming cells (PFC)
in their spleens according to the direct technique of Jerne et al (16) 3 days after im-
munization. All the PFC values were expressed as the numbers of PFC per in-
dividual spleen. As a rule, five mice were used for the determination of PFC re-
sponse in one group, and the mean of the PFC for the five mice and its standard
error were calculated.
Preparation of Homogeneous and Macromolecular LPS
Butanol-LPS and phenol-LPS were loaded onto Sepharose 4B columns, and the
chromatographic profiles obtained are shown in Fig. 1. Butanol-LPS gave one
prominent peak while phenol-LPS gave two sluggish peaks. The peak fractions of
butanol-LPS (tube numbers from 24 to 29) were collected, pooled together and con-
densed by a vacuum evaporator. This preparation should contain homogeneous
and macromolecular LPS (approximately M.W. 1.5 •~ 106). Therefore, we used
this column-passed butanol-LPS for the following experiments.
Adjuvant Effect of "Endotoxin Serum" on Antibody Response
Mice were iv injected with column-passed butanol-LPS (100 ,ug in 0.2 ml of
saline) and the blood of these mice was collected 1, 2, 3, 4, and 5 hr after the injection.
The sera were separated from the blood, and termed "endotoxin serum" after
1, 2, 3, 4 or 5 hr. Then, these sera were injected ip with 2•~ 108 SRBC into mice.
PFC responses in the spleen of these mice were determined 3 days after the injection
(Table 1). When the mice were injected with the antigen and "endotoxin serum"
after 1 to 4 hr simultaneously, these mice showed greater numbers (about triple) of
anti-SRBC 19S PFC in their spleens than immune controls. The "endotoxin serum"
after 5 hr still has a significant enhancing effect on PFC response although the extent
SERUM FACTOR FOR ADJUVANT EFFECT OF LPS
of this effect seems to become weaker. These results indicate that "endotoxin serum"
has some adjuvant effects on anti-SRBC PFC responses in the spleen of mice.
Fig. 1. Chromatographic profiles of butanol-LPS and phenol-LPS through Sepharose 4B
LPS (100-200 mg in 5 ml) was loaded onto a column (90 •~ 2.6 cm) of Sepharose 4B and
eluted at 4 C with 0.15 M PBS, pH 7.4, at a pressure of 20 to 30 cm H2O (flow rate: 6 ml/
hr). Five ml each of eluate was collected in tubes and a 0.1 ml aliquot from each tube was
subjected to an assay for LPS content. The relative content of LPS in eluates was photo-
metrically determined by absorbance at 552 nm after the coloring of KDO with thio-
barbituric acid (12). 0, butanol-LPS; e, phenol-LPS.
Fi g. 2.
Chrom atographi c(● )and
rechrom atographi c(□ , ○ )profi l es of, "endotoxi n serum , ,
serum" (2 ml)
was loaded onto a column (90 •~ 2.6 cm) of superfine Sephadex
G-200 and chromatographed at 4 C with 0.15 M PBS, pH 7.4, at a pressure of 15 cm H20
rate; 2.5 ml/hr).
of each of the
in tubes and
29 in elution absorbance nm. Eluates in to 40
and 41 to 60 were pooled respectively, condensed and again rechromatographed through
superfine Sephadex G-200 under the same conditions mentioned above.
Table 1. Adjuvant effect of "endotoxin serum"