Download Effect of Boar Seminal Immunosuppressive Fraction on B

BIOLOGY OF REPRODUCTION 55, 194-199 (1996)
Effect of Boar Seminal Immunosuppressive Fraction on B Lymphocytes and on
Primary Antibody Response'
Leopold Veselsk ,2,3 Jaromir Dostl,4 Vladimir Holfii,3 Josef Soucek,5 and Blanka Zelezna 3
Institute of Molecular Genetics,3 Academy of Sciences of the Czech Republic, 166 37 Prague, Czech Republic
Institute of Animal Physiology and Genetics, 4 Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech
Republic Institute of Haematology and Blood Transfusion,5 128 20 Prague, Czech Republic
cytes [11]. In vitro immunosuppressive activity can be attributed to spermine [12], and, as has been shown, the in
vivo suppression of T lymphocytes requires the presence
of spermine or related enzymes locally [13]. Seminal plasma impairs the ability of macrophages and neutrophils to
generate oxygen species after being triggered with opsonized zymosan and phagocyte opsonized bacteria [141.
The physico-chemical characterization of seminal plasma has provided evidence that transglutaminase together
with rat seminal immunosuppressive component, and prostaglandins are the principal molecules contributing to seminal plasma immunosuppression of macrophages and NK
cells [15-17]. Kelly et al. [18] have demonstrated that prostaglandins of the E series are almost entirely responsible
for the inhibition of immunosuppression of the NK cell
system. Nevertheless, seminal plasma is a complex mixture,
and there is evidence of other immunosuppressive components present in seminal secretions. A protein of high molecular mass, isolated from human seminal plasma and
identified as transforming growth factor , inhibited both
DNA synthesis and killing activity of interleukin 2-stimulated murine lymphocytes [19]. It has also been documented that extracellular organelles of prostate-origin prostasomes may play a complementary role to other immunosuppressive factors contained in human semen [20].
Recently we have investigated the absorption of the immunosuppressive component (immunosuppressive fraction,
ISF) isolated from boar seminal vesicle secretion on the
surface of white blood cells after its in vivo application to
male BALB/c mice. It was demonstrated that seminal ISF
caused a decrease in lymphocyte numbers in the blood of
treated mice, but the concentration of the granulocytes was
not changed [21]. To better understand the role of seminal
immunosuppressor in the regulation of immune reactions
in the organism, it is important to determine which cell
subset of the immune system absorbs the immunosuppressor and which cells are inhibited after the in vivo application of ISF The aim of this study was to determine the
direct immunosuppressive effect of seminal ISF administered in vivo on separated T and B lymphocytes. The ability
of ISF to inhibit the primary antibody response to soluble
or corpuscular antigens was also studied.
ABSTRACT
Repeated i.p. or rectal treatment of male and female mice
with an immunosuppressive component isolated from boar seminal vesicle secretion reduced responses of B lymphocytes to
mitogen as evaluated by [3 H]thymidine or bromo-deoxyuridine
incorporation. The proliferative activity of T lymphocytes was
not affected. By means of the immunofluorescence method, the
seminal immunosuppressive component was detected on the
membranes of B lymphocytes separated from the spleens of mice
treated in vivo with immunosuppressor. An i.p. injection or rectal infusion of the immunosuppressive component also led to a
suppression of primary antibody response to soluble and particulate antigens. These findings indicate that in vivo deposition of
semen may compromise some aspects of the immune system and
may be an important cofactor in the development of viral and
bacterial infections in homosexual men.
INTRODUCTION
The female reproductive tract has the capacity to mount
an immune response to environmental stimuli. Intravaginal
immunization with antigens introduced into the female genital tract can elicit a specific humoral response, demonstrating that the female genital tract is not an immunoprivileged
site. Antibodies have been induced by direct immunization
of the vagina with protein antigens or as a result of natural
or experimental infection of the lower reproductive tract
with a variety of pathogens [1, 2]. In the male and female
genital tracts, many cells belonging to the immune system
are physiological residents [3]. However, insemination with
histoincompatible sperm normally does not invoke an antisperm immune response. Seminal plasma may provide a
physiological protective environment to prevent immune
recognition of highly antigenic sperm. The abrogation of
the immune response to sperm is important for successful
conception [4], but at the same time other essential immunological events are suppressed. In vitro studies have
demonstrated that seminal plasma components can impair
the generation of cytotoxic T cells, the response of B cells
to a variety of antigens [5, 6], and the cytotoxic effect of
natural killer (NK) cells and of activated human antitumor
effector cells [7, 8]. The inhibition of immunocompetent
cell activity has been demonstrated in a number of species,
but the effect of seminal immunosuppressors on macrophages and lymphocytes is not species-specific [9,10]. Human seminal plasma components can also decrease the antibacterial activity mediated by peripheral blood lympho-
MATERIALS AND METHODS
Isolation of ISF
ISF was isolated from boar seminal vesicle secretion according to the procedure described by Dostal et al. [21].
Seminal vesicle secretion was precipitated in 8% ethanol
(pH 7.2) at -2.5°C. A sample of 100 mg of the precipitate,
which had been dialyzed and dissolved in PBS (pH 7.2),
was applied to a Sephacryl S-200 column (2.4 x 63 cm;
Pharmacia, Uppsala, Sweden) equilibrated with PBS (pH
7.2) at a flow rate of 4.2 ml per 15 min. The fractions with
Accepted March 11, 1996.
Received September 25, 1995.
'Supported by grant No. 310/93/0307 from the Grant Agency of the
Czech Republic.
2Correspondence: Leopold Veselsky, Institute of Molecular Genetics,
Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37
Prague 6, Czech Republic. FAX: 42224310 955.
194
EFFECT OF BOAR IMMUNOSUPPRESSOR ON B LYMPHOCYTES
inhibitory activity on porcine and murine lymphocytes were
pooled and run through a Sephadex G-75 column (1.4 x
94 cm; Pharmacia) equilibrated with PBS (pH 7.2) at a flow
rate of 1.5 ml per 20 min. The active fraction was further
purified by HPLC on a Vydac C-18 RPc column (Vydac,
Hesperia, CA) in a gradient of 20-40% acetonitrile in
0.01% trifluoroacetic acid at a flow rate of 1 ml/min. The
gradient was set according to a high-pressure LKB system
(LKB, Bromma, Sweden) with two 2150 pumps and a 2152
controller. The yield of ISF after a final step of purification
was 100-200 pxg/ml of boar seminal secretion. For the analysis of molecular mass, a 1 mM solution of ISF was prepared in 70% acetonitrile with 1% trichloroacetic acid. An
aliquot of 0.2 1d (2 pmol ISF) of sample solution was
mixed with 0.1 p1 of protein matrix (1 mM sinapinic acid)
on a disposable sample slide. The droplet was allowed to
dry, and the slide was loaded into a Lasermat Mass Analyzer (Finnigam MAT, San Jose, CA).
Immunization
The ability of ISF to suppress the primary antibody response to a soluble antigen keyhole limpet hemocyanin
(KLH; Sigma, St. Louis, MO) and sheep red blood cells
(SRBC) was evaluated. Fifteen male and 15 female BALB/
c mice each received an i.p. injection of 0.2 mg ISF in 0.1
ml PBS, and another group of 15 male and 15 female mice
each received a rectal infusion of 0.4 mg ISF in 0.1 ml
saline, on Days 0 and 1. On Day 2, the mice were immunized i.p. with 250 jIg KLH. The same-day schedule of ISF
rectal or i.p. treatment was used in mice immunized with
SRBC (1 x 108 cells in 0.1 ml PBS). Saline instead of ISF
was administered as a control, with the same immunization
procedure. Antibody titers to KLH or SRBC were measured
by ELISA in the blood serum of experimental and control
mice on Day 10 after the immunization. The technique of
rectal infusion of ISF used was described previously [21,
22].
Cell Preparation, and T and B Lymphocyte Separation
The effect of ISF on B or T lymphocyte proliferation
was evaluated by mitogen-induced lymphocyte proliferation and by an immunoassay system for detection of bromodeoxyuridine incorporation (Amersham kit; Amersham,
Little Chalfont, UK). For i.p. application, 3 groups of 5
mice each received injections of 0.2 mg of ISF on Days 0
and 1. The fifth day after the first injection of ISE splenocytes were obtained from the treated mice. For rectal infusion, 3 groups of 5 mice each were anesthetized with
ether and received infusions of 0.4 mg of ISF in 0.1 ml
saline on Days 0 and 1. The ninth day after the first infusion, splenocytes were tested. Controls received saline instead of ISF Spleens of the treated mice were homogenized,
and the cell suspension was passed through a 110-iLm
mesh, washed three times by centrifugation at 400 X g for
5 min each time, and resuspended in RPMI 1640 culture
medium. Purified T and B lymphocytes were prepared by
a two-cycle passing procedure of spleen cells through nylon
wool columns [23].
The proportion of T and B lymphocytes in T and B cellenriched cell populations was tested by a fluorescence activating cell sorter (FACS) analysis using anti-CD3 and
anti-Ig antibodies, respectively. T cell-enriched cell populations contained 85-90% of T cells and less than 5% of
Ig+ cells. B cell-enriched populations contained over 90%
of Ig+ cells and less than 5% of T cells. Separated lym-
195
phocyte subsets were used for measurement of lymphocyte
proliferation. The absorption of ISF in vivo on the surfaces
of T and B lymphocytes was determined by an immunofluorescence assay.
Measurement of Lymphocyte Proliferation
Separated T and B lymphocyte suspensions from mice
treated rectally or i.p. with ISF were adjusted to 2 x 106
cells ml in RPMI 1640 medium (Serva, Heidelberg, Germany) supplemented with 10% fetal calf serum (FCS), 2
mmol/L L-glutamine, 100 IU/ml penicillin, and 0.1 g/ml
streptomycin. The B lymphocytes, stimulated with 5 [Ig
pokeweed mitogen (PWM; Sigma) per ml RPMI 1640 medium, and the T lymphocytes, stimulated with 5 ig concanavalin A (Con A; Serva, Heidelberg, Germany) per ml
RPMI 1640 medium, were cultured in triplicate in culture
microtiter plates. Nonseparated lymphocyte cultures were
stimulated with 10 g phytohemagglutinin (PHA; Wellcome Research Laboratories, Dartford, UK) per milliliter of
RPMI 1640 medium. The lymphocytes were cultured at
37°C for 72 h. After 48 h of culture, 37 kBq [3 H]thymidine
(spec. act. 840 GBq/ml; Institute for Research, Production
and Application of Radioisotopes, Prague, Czech Republic)
per well was added. The cells were collected onto glassfiber discs by use of a semiautomatic harvester, and the
radioactivity was measured according to a standard liquid
scintillation technique. The viability of the lymphocytes
was greater than 95% as determined by trypan blue dye
exclusion.
In the immunoassay system for detection of bromo-deoxyuridine incorporation, the proliferation of lymphocytes
was accomplished according to instructions supplied with
the cell proliferation assay kit (Amersham). Suspensions of
T and B lymphocytes separated from rectally or i.p. treated
mice were adjusted to 1.5 x 106 cells/ml in RPMI 1640
medium supplemented with 10% FSC, L-glutamine, penicillin, and streptomycin. Triplicate cultures of B lymphocytes stimulated with PWM, of T lymphocytes stimulated
with Con A, and of nonseparated lymphocytes stimulated
with PHA were incubated in 100 p,1 of culture medium in
culture microtiter plates at 37°C for 72 h. The cells were
incubated during the last 2 h of culture with 100 1 of
labeling medium containing a solution of 5-bromo-2'-deoxyuridine and 5-fluoro-2'-deoxyuridine in a ratio of 10:1
(v:v). The T-cell plates were centrifuged at 1500 X g for
10 min and dried at 37°C for 3 h after medium was removed. The B cells were washed with PBS. The B and T
cells were fixed in a solution of ethanol:acetic acid:water
(9:5:5 v:v:v) for 30 min and washed in 0.1% Tween 20 in
PBS. The plates were blocked with 1% nonfat milk for 15
min at 22°C, and 50 l1 of anti-5-bromo-2'-deoxyuridine,
containing the nuclease to denature DNA, was added into
the wells. After the plates were washed, 50 p,1 of antimouse IgG2a serum conjugated with peroxidase was added.
Bound peroxidase activity was detected by use of 2-2'azino-bis(3-ethylbenzthiazoline sulfonate) and H2 0 2 as substrate. Incorporation of 5-bromo-2'-deoxyuridine into replicating DNA was determined at 410 nm.
The effects of ISF on the proliferation of normal human
lymphocytes and on two human tumor cell lines, ML-1
(non-T, non-B cells) and K 562 (erythroleukemia) were also
tested. The effect of ISF on proliferation of normal human
lymphocytes was evaluated by mixed lymphocyte culture
(MLC). Lymphocytes from two unrelated persons, isolated
on a Ficoll-Paque gradient (Pharmacia, Uppsala, Sweden),
VESELSKY ET AL.
196
FIG. 1. Antibody response of male and female mice treated rectally or i.p. with ISF
two days before KLH immunization. Antibodies to KLH were measured by ELISA
on Day 10 after immunization. Values are
expressed as mean + SD from three different experiments with 5 male or female
mice. Comparable titer values were obtained when mice were immunized with
SRBC. *Difference in suppression of primary antibody response to challenging antigen between ISF treated and control
mice, p < 0.01. +Difference in suppression of primary antibody response to challenging antigen between ISF-treated male
and female mice, p < 0.01.
KLH
rectal ISF KLH
F
1~~~*
rectal ISF KLHJ
i. p. ISF KLH2
i, p. ISF KLHd
g+
were mixed at a ratio of 1:1. The final lymphocyte concentration was adjusted to 1 x 106 cells/ml RPMI 1640 medium supplemented with 20% human AB serum and antibiotics.
The effect of ISF on the growth of the two tumor cell
lines was evaluated by inhibition of spontaneous cell proliferation. The tumor cell concentration was adjusted to 2
x 105 cells/ml RPMI 1640 medium supplemented with
10% FCS and antibiotics.
All cell cultures (100 1I per well) were done in triplicate
in microtiter plates. Seminal ISF (100 I 1 per well) was
added at concentrations of 50, 100, and 150 l.ug into wells.
The cells were cultured at 37C for 6 days. Four hours
before the termination of incubation, the cells were pulsed
with 37 kBq [3 H]thymidine. The values were measured by
a standard liquid scintillation technique [24].
10
Antibody
Titer
3
(x 10 )
pensions (6 x 106 cells/ml) were incubated with an equal
amount of LIS-4 (120 jig lyophilized antibody/ml PBS) at
22°C for 20 min, were thoroughly washed with 1% BSA
in PBS and 1% non-fat milk in PBS, and were incubated
at 22°C for 20 min with fluorescein isothiocyanate conjugated to swine anti-mouse immunoglobulin diluted 1:40,
containing 0.02% sodium azide (USOL). A droplet of cell
suspension was applied to a glass slide and examined under
the Orthoplan fluorescence microscope (Leitz, Wetzlar, Germany) with an excitation filter of 450-490 nm and barrier
filter of 515 nm.
Statistical Analysis
The significance of differences between experimental
and control groups was analyzed by means of Student's ttest.
ELISA
The microtiter plates were coated with 1 g of KLH and
incubated at 4°C for 18 h. After the plates were washed in
PBS-Tween (PBS containing 0.1% Tween 20 and 1%
BSA), serially diluted antisera to KLH from mice treated
rectally or i.p. with ISF were added and incubated at 22°C
for 2 h. The plates were washed, and porcine anti-mouse
IgG serum conjugated with peroxidase (USOL, Prague,
Czech Republic) diluted 1:3000 was added. Bound peroxidase activity was detected with o-phenylenediamine and
H20 2 used as substrate. The absorbance was determined at
492 nm.
For erythrocyte ELISA, the plates were incubated with
0.01% poly L-lysine (Sigma) in PBS at 22 0C for 1 h. After
being washed, the plates were filled with 100 p.1 of SRBC
(1 X 106 cells/ml) and incubated at 22°C for 1 h. After the
plates were washed in PBS, serially diluted antisera to
SRBC from mice treated rectally or i.p. with ISF were added and incubated at 22°C for 2 h. The same procedure was
used as for detection of KLH antibodies in ELISA. Antisera
of control mice immunized with KLH or SRBC but not
treated with ISF served as positive controls. Normal sera
of mice treated with saline alone were used as negative
controls.
Indirect Immunofluorescence Technique
Indirect fluorescence was used in studies of ISF absorption on separated T and B lymphocytes isolated from ISFtreated mice 5 days after i.p. injection and 9 days after
rectal infusion of ISE Production of the monoclonal antibody to ISE LIS-4, used in the immunofluorescence assay
for detection of ISF absorption on lymphocytes was described previously [21]. Separated T and B lymphocyte sus-
RESULTS
Isolation of ISF
The ISF separated from boar seminal vesicle secretion
by gel filtration on a Sephadex G-75 column [10] was further purified by reversed-phase HPLC. The inhibitory activity was estimated by inhibition of mitogen-induced lymphocyte proliferation on porcine lymphocytes. One milliliter of seminal vesicle secretion yielded 100-200 g of ISE
The molecular mass of ISF estimated on a Lasermat Mass
Analyzer (Finningam-MAT; San Jose, CA) was 14 kDA
[21].
Suppression of Primary Antibody Response
We examined the ability of ISF to suppress the antibody
response to soluble and particulate antigens. The i.p. or
rectal administration of ISF two days before immunization
of male or female mice significantly suppressed the primary
antibody response to both KLH and SRBC (Fig. 1). In ISFtreated females immunized with KLH, the antibody response suppression was 81% after i.p. injection and 69%
after rectal infusion of ISF (p - 0.01). In ISF-treated males
immunized with KLH, the antibody response suppression
was 98% after i.p. injection and 96% after rectal infusion
of ISF (p
0.01). The difference in the suppression of
primary antibody response to challenging antigens between
ISF-treated male and female mice was statistically significant (p < 0.01).
Comparable antibody titers were obtained when male
and female mice were immunized with SRBC. Antibody
titers to KLH and SRBC were measured by ELISA on Day
10 after immunization.
EFFECT OF BOAR IMMUNOSUPPRESSOR ON B LYMPHOCYTES
197
TABLE 1. Effect of i.p. and rectal administration of ISF on T and B lymphocytes evaluated by mitogen-induced
lymphocyte proliferation test.a
Treatment
B lymphocytes
PWM 5 .g/ml
%
Inhibition
T lymphocytes
Con A 5 ig/ml
%
Inhibition
Nonseparated
lymphocytes PHA
10 g/ml
%
Inhibition
Peritoneal
Saline
ISF
68 080 + 4272
10 104 t 2546
85*
44 840 ± 5240
45 971 + 4081
-
27 457 ± 3820
10 782 ± 1672
61*
Rectal
Saline
ISF
72 936 + 5637
16 235 + 1206
78*
56 262 ± 2635
51 320 ± 3960
-
31 672 ± 2968
13 303 + 1012
58*
Data represent the mean ± SD from 3 different experiments with 5 mice in each group. The lymphocyte
proliferation was expressed as incorporation of [3H]thymidine.
* p < 0.01.
Suppression of the Proliferative Response of Lymphocytes
To examine whether administration of ISF affected the
mitogenic activity of T and B lymphocytes, mice were pretreated i.p. or rectally with ISF On Day 5 after i.p. injection
and on Day 9 after rectal infusion, spleen cells from the
treated mice were prepared and assayed for cell proliferation upon in vitro stimulation with mitogens. In the mitogen-induced proliferation test, the blastogenic response of
B lymphocytes stimulated by PWM was significantly reduced (p - 0.01). The proliferative activity of T lymphocytes stimulated with Con A was not affected (Table 1). A
similar inhibitory effect of ISF on B lymphocytes was observed in the immunoassay system for detection of bromodeoxyuridine incorporation. The proliferative activity of B
lymphocytes was decreased by 76% after the rectal infusion
and by 79% after the i.p. injection of ISE The proliferative
activity of T lymphocytes was not affected (Table 2).
Absorption of ISF on B Lymphocytes
For the in vivo absorption of ISF on separated T or B
lymphocytes of the treated mice, an indirect fluorescence
method was used. The positive reaction with LIS-4 antibody was found on the membranes of 40-60% of the nonseparated lymphocytes and on membranes of 95-100% of
the B lymphocytes separated from spleens of mice treated
i.p. or rectally with ISF (Fig. 2, a and b). No positive reaction of LIS-4 with T lymphocytes separated from spleens
of i.p. or rectally treated mice was found (Fig. 2c). The
reaction of lymphocytes from control mice with LIS-4 was
also negative. Negative results were also obtained when
separated T lymphocytes were incubated in vitro with 100,
200, and 300 Rg of ISF per 1 ml media for 3 days at 37°C.
Immunofluorescence of these ISF-treated T lymphocytes
incubated with monoclonal antibody to ISF was equally as
negative as the reaction shown in Figure 2c. Immunofluorescence of B lymphocytes was positive.
Effect of ISF on Human Leukocytes
The effect of ISF on human peripheral blood leukocytes
and tumor cell lines ML-1 and K562 was evaluated by
MLC and a spontaneous cell proliferation assay. The results
from three separate experiments indicated that ISF suppressed only lymphocytes of healthy men. The proliferation
of tumor cell lines was not affected (Fig. 3).
DISCUSSION
Seminal plasma suppresses a variety of immunological
functions in vitro and in vivo [10, 25]. It has been concluded in studies of NK cell function that prostaglandins in
primate semen are responsible for the inhibition of cell
function [26]. Recent studies have indicated that rectal insemination of semen had several consequences. Significantly lower titers of IgM, IgG, and IgA antibodies to KLH
were found in immune sera from KLH-immunized rabbits
inseminated with homologous semen [27]. It was also demonstrated that cellular immune functions, including NK cell
activity and mitogen stimulation of lymphocytes, were not
reduced after a single rectal insemination of human seminal
plasma into rhesus monkeys. However, metabolite values
of blood plasma prostaglandin E2 did become elevated in
the inseminated monkeys but not in controls [28]. Anal
infusion of prostaglandin E2 or D 2 into male rats reduced
in vivo response of T lymphocytes to PHA, but the T-cell
response of female rats was not significantly changed [29].
TABLE 2. Effect of Intraperitioneal and intrarectal administration of ISF on T and B lymphocytes evaluated by
an immunoassay system for detection of bromodeoxyuridine incorporation.a
Treatment
B cells
PWM 5 g/ml
%
Inhibition
T cells
Con A 5 g/ml
%
Inhibition
Nonseparated
lymphocytes PHA
10 i.g/ml
%
Inhibition
Peritoneal
Saline
ISF
0.235
0.049
0.049 ± 0.007
79*
0.296 ± 0.029
0.279 ± 0.049
-
0.286 ± 0.057
0.112 ± 0.021
61*
Rectal
Saline
ISF
0.290 ± 0.052
0.074 ± 0.005
76*
0.317 ± 0.056
0.298 + 0.036
-
0.263 + 0.046
0.113 ± 0.027
57*
Data represent the mean
SD from 3 different experiments with 5 mice in each group. Lymphocyte proliferation was expressed as incorporation of 5-bromo-2'deoxyuridine into replicated DNA determined by ELISA
using specific monoclonal antibody.
* p < 0.01.
198
VESELSKY ET AL.
CPI
ISF pg/ml
FIG. 3. Effect of seminal immunosuppressor on proliferation of healthy
human lymphocytes (circles), tumor cell line K 562 (triangles), and tumor
cell line ML-1 (squares). Lymphocyte proliferation was expressed as incorporation of [3H]thymidine - SD from three different experiments.
FIG. 2. a) Immunofluorescent detection of immunosuppressive factor on
membranes of nonseparated lymphocytes in mice infused rectally with
immunosuppressor. b) Immunofluorescent detection of immunosuppressive factor on membranes of B lymphocytes in mice infused rectally with
immunosuppressor. c) Nonpositive immunofluorescent reaction of LIS-4
antibody with T lymphocytes in mice infused rectally with immunosuppressor. a-c, x800.
Nevertheless, it has been found that boar seminal plasma
contains an extremely low concentration of prostaglandins
(0.01 mg/ml) as compared with that in humans (0.578 mg/
ml) [30].
Seminal plasma expresses a number of semen-specific
antigens that can elicit the immune response in sexual
partners [31 ]. The lymphocyte function may be paralyzed
by seminal plasma immunosuppressive factors, but semen macrophages can retain phagocytic, adherent, and
motile functions [3]. No effect of boar ISF was found on
cells involved in transplantation events including NK cell
activity [32]. Moreover, seminal ISF did not affect the
proliferation of human tumor cell lines ML-1 and K 562,
but it inhibited the proliferation of MLC-stimulated normal human lymphocytes. It can be concluded that the
effect of ISF on the immune system cells is not speciesspecific but that its antiproliferative activity depends on
the presence of complementary receptors on the leukocyte membranes. The present study also demonstrated
ISF on membranes of the B-cell lymphocyte subpopulation isolated from spleens of mice treated i.p. or rectally
with ISE In vivo treatment with ISF significantly reduced
the mitogenic activity of B lymphocytes stimulated with
PWM. However, seminal immunosuppressor was not detected on T-cell membranes; likewise, T-cell proliferation
was not abrogated by ISE The effect of ISF on white
blood cells depends, we assume, on its half-life. In a preliminary study, we determined the duration of immune
suppression induced by ISF treatment after primary and
secondary immunizations with soluble and corpuscular
antigens. The results indicated that the treatment with
ISF leads to a prolonged immunosuppression but not to
a permanent tolerance to antigens. These preliminary
data suggest that prolonged immunosuppression is associated with the continued presence of ISF in blood serum.
It can also be assumed that the capability of the receptors
to bind ISF on the cell surface is renewed.
The most important result of our study is the evidence
that ISF inhibited the antibody response to challenging
antigens. The finding of the primary antibody inhibition
to antigens indicated the sex-related difference of the
sensitivities to ISE Even when the capacity of ISF to
suppress the antibody response in females was also significant, the antibody titers in the female sera were higher
than those found in male sera. It seems reasonable to
conclude that females are less sensitive to male seminal
immunosuppressor.
These data indicate that the boar seminal immunosuppressor might impede the development of humoral im-
EFFECT OF BOAR IMMUNOSUPPRESSOR ON B LYMPHOCYTES
mune response via reduction of lymphocyte proliferation
and inhibition of antibody response, both in males and
females, after its in vivo deposition. We do not suppose
that rectal infusion of ISF traumatizes the mucosal barrier. If there were a breach of the intestinal wall after
rectal infusion, the suppressive activity of ISF on white
blood cells would be simultaneous with the i.p. injection.
The results of our previous paper [21] indicated that the
suppressive effect of ISF on white blood cells reached a
maximum on Day 5 after the i.p. application. However,
maximum reduction of the lymphocyte number was
achieved on Day 9 after rectal infusion of ISE Also, inspection (after they were killed) of the mice treated rectally with ISF excluded physical damage of the colon,
including the rectal colon. The finding that seminal ISF
infused rectally inhibited the primary antibody response
suggests that seminal immunosuppressive components
may interfere with immunological functions associated
with a number of pathological states including AIDS, and
may be a factor that decreases an immune response. Excessive exposure to seminal plasma at sites other than the
genital tract may have a systemic effect. Seminal components may gain access to the circulation if they are
deposited in the gastrointestinal tract, especially if it has
been traumatized [4].
We have postulated that seminal components deposited in the rectum may be a factor in the etiology of AIDS
and other viral and bacterial infections in males and females. Immunosuppressive molecules might reduce the
host defense mechanisms, especially after repeated exposure.
ACKNOWLEDGMENTS
The authors thank Mrs. M. Ho~kovd and Mrs. L. KoberovA for their
excellent technical assistance.
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