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)
M.J. TANABE ET AL
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SERUM FACTOR FOR ADJUVANT EFFECT OF ITS
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Requests for reprints should be addressed to Dr. Masao J. Tanabe, Department of Micro-
biology, Jichi Medical School, Tochigi-ken 329-04, Japan.