Purified Porcine Seminal Plasma Protein Enhances In Vitro Immune Activities of Porcine Peripheral Lymphocytes

^a Department of Health and Human Services, Food and Drug Administration, Rockville, Maryland 20857 ^b Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602 ^c Department of Pathology and Laboratory Medicine, Albert Einstein Medical Center, Philadelphia, Pennsylvania 19141

	ABSTRACT

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The porcine seminal plasma protein (PSP) accounts for much more than 50% of the total proteins in seminal plasma. PSP has been previously purified and its biochemical properties characterized. However, the biological functions of PSP remain to be elucidated. We hypothesize that PSP is involved in the regulation of uterine immune activity. In the current study, effects of PSP on in vitro lymphocyte activities and the presence of PSP binding sites on lymphocytes were examined. In mitogen-induced proliferation assay, lymphocytes from peripheral blood of gilts were cultured with pokeweed mitogen (PWM), phytohemagglutinin (PHA), or concanavalin A (Con A) in the presence or absence of PSP. PSP at 50, 125, and 250 ng/well augmented PWM-induced [³H]thymidine uptake in a dose-responsive manner by 152.8 ± 8.1%, 225.9 ± 35.2%, and 274.8 ± 53.6%, respectively, compared with that of control. PSP did not alter lymphocyte proliferation in the absence of PWM. Similarly, PSP had little or no effect on PHA- or Con A-induced lymphocyte proliferation. In one-way mixed lymphocyte reactions, PSP at 50, 125, and 250 ng/well enhanced [³H]thymidine uptake in a dose-responsive manner by 181.5 ± 16.5%, 339.9 ± 48.2%, and 600.1 ± 84.8% of control, respectively. Using biotinylated PSP-I, PSP binding sites were localized on approximately 3–5% of the lymphocyte population. In summary, we have demonstrated that PSP itself is not a mitogen/antigen to porcine lymphocytes but that it has a stimulatory effect on lymphocyte activities initiated by PWM or surface antigens of lymphocytes. PSP may exert its functions by interacting with PSP binding sites on a subpopulation of porcine lymphocytes. The high potency of PSP on lymphocyte activities and the abundance of PSP in seminal plasma have suggested that PSP may play an important role in regulating immune responses in the porcine uterine environment.

	INTRODUCTION

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Seminal plasma is the liquid portion of semen that contains a large quantity of proteins. Many of these seminal proteins have been structurally elucidated and functionally studied. For example, seminal plasmin, a protein from bovine seminal plasma, has been shown to be bacteriocidal to a variety of Gram-positive and Gram-negative bacteria [1,2]. This was later found to be identical to caltrin, which was purified from bovine seminal plasma on the basis of its ability to inhibit Ca²⁺ transport into bovine spermatozoa [3, 4]. ß-Microseminoprotein, a 94-residue protein from human seminal plasma, has been shown to bind immunoglobulin [5]. Spermadhesins AQN-1, AQN-3, and AWN were isolated from boars and were capable of stabilizing zona pellucida binding sites to allow sperm-egg interaction [6,7]. It is noteworthy that although these seminal proteins are quite similar in size, they do not share much sequence homology.

In pigs, the porcine seminal plasma protein (PSP) accounts for much more than 50% of the total proteins in seminal plasma. It has been found that PSP is composed of two monomers, PSP-I and PSP-II, and is present in seminal plasma in the forms of homodimers and heterodimers [8]. PSP-I and PSP-II are glycoproteins of 13–14 kDa [8], are found within the epithelium of seminal vesicles [9], and have distinctly different amino acid sequences [10]. They share about 50% sequence homology with a family of spermadhesins purified from boar spermatozoa [10]. PSP-I and PSP-II also share 47% and 35% sequence homology, respectively, with acidic seminal fluid protein from bovine seminal plasma [11], which appears to be a growth factor for ovarian granulosa cells [12].

Despite the fact that PSP accounts for the bulk of the total proteins in porcine seminal plasma and that the biochemical properties of PSP have been elucidated, its biological functions are yet to be determined. Several lines of evidence have suggested that PSP may be involved in the regulation of uterine immune activity. First, it has been shown that PSP-I binds to a number of proteins including IgA and IgG [9]. Second, porcine seminal plasma is a potential mediator of inflammation in the uterus after mating in the pig [13, 14]. Third, many proteins that compose seminal plasma of various animal species are capable of suppressing lymphocyte activities in vivo and in vitro [15–20]. Taking these findings together, we hypothesize that PSP plays a physiological role in reproduction by regulating uterine immune activities. In this study, experiments were designed to examine whether PSP affects mitogen-induced lymphocyte proliferation and mixed lymphocyte reactions (MLR). The PSP binding sites on lymphocyte membrane were also identified by cytochemical localization studies.

	MATERIALS AND METHODS

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Materials

PSP dimer was purified from boar seminal plasma as previously described [8]. PSP-I was purified from the dimer by CM-cellulose chromatography in the presence of 8 M urea. Biotinylated PSP-I was prepared as described earlier [9].

Preparation of Porcine Lymphocytes

Peripheral blood mononuclear cells were obtained from the jugular vein of healthy gilts (n = 9) between 9 and 14 mo of age. Blood samples were collected in heparin (10 000 IU)-coated 35-ml syringes and mixed with equal volumes of RPMI-1640 medium containing 100 IU penicillin and 100 µg streptomycin per milliliter. Mononuclear cells were separated by gradient centrifugation (600 x g for 25 min) on Histopaque 1077 (Sigma Chemical Co., St. Louis, MO) [21], collected from the interface, and resuspended in RPMI-1640 medium containing 1% antibiotics (penicillin 100, IU/ml; streptomycin, 100 µg/ml; fungizone, 0.25 µg/ml). Cells were washed three times and reconstituted in RPMI-1640 medium containing 10% fetal bovine serum at a concentration of 2 x 10⁶ cells/ml. Cell viability was determined by the trypan blue exclusion method and was always greater than 95%. The collection of tissues was performed in accordance with procedures outlined in the Guidelines for Care and Use of Research Animals.

Lymphocyte Mitogenesis Test

Lyophilized PSP was reconstituted with saline and then filtering with a sterile Acrodisc 13 (Gelman Sciences, Ann Arbor, MI). The lymphocyte mitogenesis test was performed as previously described [22]. Porcine lymphocytes, 1 x 10⁶/50 µl, were placed into wells containing varying concentrations of PSP (50 µl of 1, 2.5, 5 µg/ml, equivalent to 50, 125, and 250 ng/well, respectively) or saline solution (50 µl), along with various mitogens (100 µl) or medium (100 µl) in a final volume of 200 µl. Mitogens used (except in appropriate controls) included pokeweed mitogen (PWM) (1:100 and 1:400 dilutions of stock solution prepared as indicated by manufacturer; Gibco BRL, Grand Island, NY), phytohemagglutinin (PHA) (1:1000 and 1:2000 dilutions of stock solution prepared as indicated by manufacturer; Difco, Detroit, MI), and concanavalin A (Con A) (5 and 10 µg/ml; Sigma). Three types of control cultures were used: 1) porcine lymphocytes + PSP, 2) porcine lymphocytes + mitogen, and 3) lymphocytes only. Cultures were performed in 96-well flat-bottom microtiter plates (Costar, Cambridge, MA) and incubated at 39°C with 5% CO₂ in air for 72 h. At 18 h before the termination of cultures, cells were pulsed with [³H]thymidine (1 µCi/well; ICN, Costa Mesa, CA). All cultures were harvested onto fiberglass filter paper disks with an automated cell harvester (Skatron, Sterling, VA). Filter disks were then placed in scintillation vials containing 3-ml scintillation cocktail, and the [³H]thymidine incorporated into DNA was determined by a liquid scintillation counter (Packard, Downers Grove, IL) having a counting efficiency of 67%.

MLR

Eight gilts were used in this experiment, with one being designated as a responder and the remaining as stimulators. Stimulating cells from all gilts were tested individually against responding cells. Responding cells (2 x 10⁵ cells) in 50 µl were added to wells containing 100 µl of varying dilutions of PSP and 50 µl of irradiated (3500 R) stimulating cells (2 x 10⁵ cells). These cultures were performed in triplicate and incubated at 39°C with 5% CO₂ for 7 days. Cells were labeled with [³H]thymidine (1 µCi/well) for 18 h before cultures were terminated; the cells were then harvested. The incorporated radioactivity was measured as described for mitogen-stimulated cultures.

Localization of PSP Binding Sites

Mononuclear cell suspensions were prepared as for lymphocyte proliferation assays. These were then smeared to form a thin layer on gelatin-coated slides and air-dried or gently heat-dried. The cells were fixed to the slides by incubation in 1.8% paraformaldehyde or Zamboni's fixative [23] for 20 min, followed by three 5-min washes in Tris-buffered saline (TBS) at a pH of 7.4. They were then incubated with biotinylated PSP-I (final concentration of 1 µg/ml in TBS) for 30 min, washed twice with TBS, and finally washed with Tris buffer without saline before incubation in avidin-horseradish peroxidase (HRP) conjugate in Tris buffer for 30–60 min (4 µl avidin-HRP in 10 ml Tris buffer without saline). After three washes in Tris buffer without saline (5 min each), the cells were incubated in diaminobenzidine-hydrogen peroxidase (DAB-H₂O₂) for 3–10 min (50 µg DAB in 0.05 M Tris with 0.01% H₂O₂). Color development was frequently monitored microscopically. Finally, slides were washed three times in Tris buffer (5 min each). Controls included incubation with increasing concentrations of nonbiotinylated PSP together with biotinylated PSP (nonbiotinylated PSP at concentrations greater than 10-fold the concentration of biotinylated PSP). Additional controls included incubation with biotin (Sigma) substituted for biotinylated PSP or with only avidin-HRP, skipping the biotinylated PSP step. Some slides were counterstained with Wright's stain.

Statistical Analysis

The data were analyzed after converting mean cpm of [³H]thymidine for each treatment group to a percentage of control values. To determine control responses, cpm of [³H]thymidine were averaged for the three wells of the zero-dose PSP within each mitogen dose group for each pig's cells. Mean value for the three wells for each dose of PSP within each mitogen treatment was then expressed as a percentage of its appropriate control value. These percentages for each pig's cells were subjected to two-factor (mitogen dose, PSP dose) ANOVA. Further analysis of differences between mitogen doses or PSP doses was examined by the Student-Neuman-Keuls test. An effect of the mitogen was initially determined from Student's t-test between wells with and without mitogen present.

	RESULTS

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Lymphocyte Mitogenesis Test

Without the addition of mitogens, PSP did not influence [³H]thymidine incorporation into porcine lymphocyte DNA. However, in the presence of PWM (1:400), PSP enhanced the proliferation activity in a dose-dependent manner. The three PSP dosages of 50, 125, and 250 ng/well significantly increased (p < 0.05) [³H]thymidine incorporation by 152.8 ± 8.1%, 225.9 ± 35.2%, and 274.8 ± 53.6% of the control value, respectively. A higher amount of PWM (1:100 dilution) did not result in difference in the responsiveness to PSP stimulation (Fig. 1).

View larger version (38K): [in this window] [in a new window]   FIG. 1. Left) Total amount of [3H]thymidine uptake in culture wells with lymphocytes only (denoted as cells) and lymphocytes plus PWM (average value from groups of 1:100 and 1:400 dilutions) as a reference for the culture condition. Right) Effects of increasing dosages of PSP on PWM-induced lymphocyte proliferation. The control group, without the addition of PSP, contained only PWM and lymphocytes. The mean [3H]thymidine uptake of the control group was designated as 100%. Treatment differences are indicated by dissimilar letters, and similar letters over different bars indicate that those treatments were not different (p < 0.05). Error bars represent SEM within the group.

PSP did not significantly alter the PHA-induced lymphocyte proliferation (Fig. 2). Similarly, lymphocyte proliferation activity induced by Con A was not affected by the addition of PSP (Fig. 3).

View larger version (44K): [in this window] [in a new window]   FIG. 2. Left) Total amount of [3H]thymidine uptake in culture wells with lymphocytes only (denoted as cells) and lymphocytes plus PHA (average value from groups of 1:1000 and 1:2000 dilutions) as a reference for the culture condition. Right) Effects of increasing dosages of PSP on PHA-induced lymphocyte proliferation. The control group, without the addition of PSP, contained only PHA and lymphocytes. The mean [3H]thymidine uptake of the control group was designated as 100%. Error bars represent SEM within the group.

View larger version (42K): [in this window] [in a new window]   FIG. 3. Left) Total amount of [3H]thymidine uptake in culture wells with lymphocytes only (denoted as cells) and lymphocytes plus Con A (average value from groups of 10 and 5 µg/ml concentrations) as a reference for the culture condition. Right) Effects of increasing dosages of PSP on Con A-induced lymphocyte proliferation. The control group, without the addition of PSP, contained only Con A and lymphocytes. The mean [3H]thymidine uptake of the control group was designated as 100%. Error bars represent SEM within the group.

MLR

In MLR, PSP exhibited an even higher stimulatory effect. After 7 days of coculture with mixed porcine lymphocytes, PSP significantly enhanced [³H]thymidine incorporation into lymphocytes in a dose-responsive manner. Dosages of 50, 125, and 250 ng PSP per well significantly increased (p < 0.05) [³H]thymidine incorporation by 181.5 ± 16.5%, 339.9 ± 48.2%, and 600.1 ± 84.8% of the control value, respectively (Fig. 4).

View larger version (34K): [in this window] [in a new window]   FIG. 4. Effects of PSP on MLR. Lymphocytes from responders and designated stimulator were incubated for 7 days. Control group (100%) represents cultures of stimulator and responder lymphocytes, without the addition of PSP. Treatment differences are indicated by dissimilar letters (p < 0.05). Error bars represent SEM within the group.

Localization of PSP Binding Sites

A small percentage (3–5%) of cells from lymphocyte preparation were stained after incubation with biotinylated PSP-I (Fig. 5). The staining of biotinylated PSP-I could be replaced with excess nonbiotinylated PSP-I. There were no stained cells if incubation was carried out with biotin alone, or with deletion of one of the staining reagents (Fig. 5).

View larger version (81K): [in this window] [in a new window]   FIG. 5. Localization of PSP binding sites on porcine lymphocytes. A) Cytochemically stained cells (brown) after incubation with biotinylated PSP-I. Calibration bar = 10 µm. B) Absence of stained cells after coincubation with biotinylated PSP-I and excess nonbiotinylated PSP-I. C) Cells counterstained with Wright's (purple-pink) with positive cytochemically stained cells (arrowheads). Bars = 10 µm.

	DISCUSSION

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PSP is known to comprise more than 50% of all seminal plasma proteins in the boar, and this suggests significant physiological role(s). In this in vitro study, we have found an immunostimulatory effect of PSP on PWM-induced lymphocyte proliferation and MLR. In the absence of mitogens, PSP alone did not initiate lymphocyte proliferation. This observation is an indication that PSP is not an antigen or mitogen to porcine lymphocytes and that the stimulatory effect detected here is not attributable to direct immune reactions against PSP. The dosages of PSP used (50–250 ng) were relatively small compared to the total amount of PSP per ejaculation (at the gram level), indicating that PSP is highly potent in augmenting porcine lymphocyte activities. Localization staining studies using biotinylated PSP-I showed the distinct presence of binding sites for PSP on approximately 3–5% of porcine lymphocytes. This evidence supports the postulation that PSP is involved in initiating an intracellular chain of events in a subpopulation of lymphocytes leading to proliferation.

In mitogen-induced lymphocyte proliferation, two events occur in succession: the binding of mitogen to cell surface structures, followed by intracellular events leading to DNA synthesis. PSP enhanced PWM-induced lymphocyte proliferation but had little or no effect on PHA- or Con A-induced reactions. For porcine lymphocytes, PHA and Con A are T cell-specific mitogens, whereas PWM is a mitogen for both T and B cells [22]. Therefore, the effect of PSP appeared to be mainly on the mitogen-activated B cells. The one-way MLR was an examination of the ability of responders to react with histocompatibly distinct determinants present on the cell surface of stimulators. Addition of PSP to mixed lymphocyte cultures increased the antigenic effect of stimulators in a dose-responsive manner. These findings suggest that PSP enhances both mitogen- and antigen-activated lymphocyte activities.

Previously, observations have been reported on the presence of immunostimulatory substances in porcine seminal plasma. A low molecular weight fraction of porcine seminal plasma enhanced the growth of lymphocytes induced by PWM [24]. Kovacs and coworkers [25] demonstrated that porcine seminal plasma contains a potent mitogen that induces proliferation in monocyte-enriched leukocytes. After mating, porcine seminal plasma has been shown to mediate an inflammatory response in the female reproductive tract [13] and increased expression of interleukin-2 receptors in the uterine lymph nodes [14]. A 15-kDa protein isolated from porcine seminal plasma was found to activate cell-cell adhesion of porcine lymphocytes [26]. Similar observations have been reported in other animal species. For example, bovine seminal plasma contains two active factors that enhance PHA-induced lymphocyte proliferation [27]. Incubation of human lymphocytes with seminal plasma significantly increased percentages of cells expressing immunoglobulin G receptors [28]. These findings are parallel with our observation that the major protein of porcine seminal plasma, PSP, is immunostimulatory on porcine lymphocyte proliferation.

In contrast, results from our studies apparently differ with reports that fractions of porcine seminal plasma inhibited proliferation of porcine [24] and mouse [29–31] lymphocytes. In fact, abundant evidence has demonstrated the presence of immunosuppressive activities in seminal plasma from several animal species. For instance, a fraction of bovine seminal plasma possessed inhibitory effects on antibody response, Con A-induced lymphocyte proliferation, and MLR in mice [32]. Similarly, bovine seminal plasma was capable of inhibiting mitogen-induced proliferation of bovine and human lymphocytes and phagocytic activity of bovine neutrophils [33]. Mouse seminal plasma suppressed humoral immune responses induced by various antigens [16]. The immunosuppressive effect of human seminal plasma has been widely investigated. It has been shown that human seminal plasma inhibited lymphocyte activities induced by several mitogens and antigens [15, 34–36], depressed the functional activity of complement components [37, 38], and inhibited antibody complement-mediated bactericidal and opsonic effects against Gram-negative bacteria [39]. Human seminal plasma was found to contain a potent suppressor of human natural killer cell cytotoxic activity [17, 18, 40]. Natural and activated human antitumor cytotoxicity was suppressed by human seminal plasma [41–44]. Many of the compounds responsible for the immunosuppressive activities have been identified. For example, a 100- to 110-kDa fraction of porcine seminal plasma has the capability to inhibit immune functions in vitro and in vivo [29]. Yet the seminal immunosuppressive factor reported by Dostal and coworkers [30] was only 14 kDa. Prostaglandins [45, 46], transforming growth factor ß [47, 48], and a 16-kDa protein [49] of human seminal plasma have been demonstrated to inhibit a wide variety of immune activities. Bovine seminal ribonuclease has been found to suppress PHA- or Con A-induced proliferation of human lymphocytes and MLR [50–52]. Rat seminal vesicle protein SV-IV has been shown to inhibit interleukin-1 release and phagocytic activity [20, 53].

Although it is clear that multiple immunomodulatory factors are present in the seminal plasma fluid, the question remains regarding the biological significance of the coexistence of substances with seemingly contradictory functions. Nevertheless, our novel results provide an encouraging and interesting starting point for future characterization of the biological functions of PSP. The fact that PSP itself is not an antigen/mitogen and its high potency imply a role of PSP in the regulation of local immune activity in the uterine environment. It is possible that PSP may be pivotal in maintaining a sterile environment in the uterine lumen and may also be involved in clearing other cellular debris after fertilization has taken place. Further research is indicated to extend these findings on the basis of in vitro studies only, because conditions may differ in vivo.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge Dr. Fuller Bazer for providing the boar semen used in the purification of PSP-I and PSP-II.

	FOOTNOTES

 
¹ E.B. is a visiting scholar from Department of Animal Pathology, Faculty of Veterinary Medicine, Turin University, Torino, Italy.

² Correspondence. FAX: (706) 542–3015; oliverli{at}calc.vet.uga.edu

Accepted: March 9, 1998.

Received: October 1, 1997.

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