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(Journal of Leukocyte Biology. 2002;72:512-521.)
© 2002 by Society for Leukocyte Biology

Human pregnancy-specific glycoprotein 1a (PSG1a) induces alternative activation in human and mouse monocytes and suppresses the accessory cell-dependent T cell proliferation

Claudia Cristina Motrán*, Fernando López Díaz*, Adriana Gruppi*, Daniela Slavin*, Bruno Chatton{dagger} and José Luis Bocco*

* Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina; and
{dagger} Ecole Supérieure de Biotechnologie de Strasbourg, Université Louis Pasteur, Illkirch, France

Correspondence: Claudia C. Motrán, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, Córdoba (5000), Argentina. E-mail: cmotran{at}bioclin.fcq.unc.edu.ar


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been proposed that pregnancy-specific factors induce the suppression of a specific arm of the maternal response accompanied by activation of the nonspecific, innate immune system. The aim of this study was to determine whether pregnancy-specific glycoprotein 1a (PSG1a), the major variant of PSG polypeptides, is able to modulate the monocyte/macrophage (Mo) metabolism to regulate T cell activation and proliferation. Using the recombinant form of this glycoprotein (rec-PSG1a), expressed in mammalian cells with a vaccinia-based expression vector, we have demonstrated that human PSG1a induces arginase activity in peripheral blood human Mo and human and murine Mo cell lines. In addition, rec-PSG1a is able to induce alternative activation because it up-regulates the arginase activity and inhibits the nitric oxide production in Mo activated by lipopolysaccharides. We also observed that rec-PSG1a is an important accessory cells-dependent T cell suppressor factor that causes partial growth arrest at the S/G2/M phase of the cell cycle. Additionally, an impaired T cell proliferative response induced by mitogens and specific antigen was observed in BALB/c mice upon in vivo expression of PSG1a. Our results suggest that PSG1a function contributes to the immunomodulation during pregnancy, having opposite effects on maternal innate and adaptative systems.

Key Words: arginase • iNOS • immunosuppression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several years ago, Medawar proposed the concept of "fetal allograft" to describe the immune relationship between mother and fetus [1 ]. This model compares pregnancy with tissue transplantation where immunological interaction between mother and fetus is suppressed through lack of fetal-antigen presentation or maternal lymphocyte functional suppression. In agreement, several reports have demonstrated that during the normal pregnancy, a state of systemic suppression of the maternal immune system seems to be present [2 3 4 ]. Subsequently, the results of Wegmann et al. [5 ] suggested that normal pregnancy is characterized by a lack of strong maternal, cell-mediated, antifetal immunity [T helper cell type 1 (Th1)-type reactivity] and that instead, a dominant humoral immune response is prevalent (Th2-type reactivity). Both hypotheses supported the view that pregnancy is an immunosuppressive state, although pregnant women are not significantly more susceptible to infections than nonpregnant. More recently, Sacks et al. [6 ] proposed that pregnancy-specific factors induce the suppression of a specific arm of the maternal response accompanied by activation of the nonspecific, innate immune system in which the monocyte/macrophage (Mo) rather than lymphocyte assumes the central role in maternal immunological adaptation.

Mo populations, in addition to having a role in innate immunity, participate as effector cells in adaptative immune responses. Two different subsets of macrophages had been described. Classically activated Mo occur in a type I cytokine environment [interferon-{gamma} (IFN-{gamma}) and tumor necrosis factor {alpha}] and are inhibited by type II cytokines [interleukin (IL)-4, IL-10, and IL-13]. They have cytotoxic and antimicrobial function, mainly based on their ability to secrete nitric oxide (NO) [7 ]. In contrast, anti-inflammatory agents, such as IL-4, IL-10, IL-13, transforming growth factor-ß (TGF-ß), and glucocorticoids, inhibit the expression of proinflammatory cytokines in Mo and preferentially induce the expression of innate immunity receptors with broad specificity for foreign antigens (e.g., macrophage mannose receptor, scavenger receptor, and CD163) [7 ], thus enhancing its capacity for endocytosis and antigen presentation. Therefore, this state of Mo activation has been called alternative immunologic activation of Mo [8 ]. Despite their enhanced ability for phagocytosis, alternatively activated Mo does not exert killing functions toward microbes. NO production is counteracted in these Mo by enhanced expression of the arginase enzyme, competing with inducible NO synthase (iNOS) for its substrate, L-arginine, to give the products L-ornithine and urea [9 ]. Alternative activated Mo secret anti-inflammatory molecules such as IL-10 and TGF-ß and seem to be the first defense line cell that is not dependent on a strong, Th1-mediated immune response to perform their function. In healthy organisms, these alternatively activated Mo are preferentially found in normal placenta and lung [10 ], and today, several lines of evidence indicate that these Mo and naturally occurring suppressor Mo isolated from these organs may at least partially overlap. In addition, it has been proposed that the alternatively activated Mo are able to induce differentiation of naïve T cells into antigen-specific Th2 cells and to accomplish Th2-associated functions [11 , 12 ]. The cytokine milieu generated during pregnancy suggests the involvement of alternatively activated Mo, whereas the presence of Th1 cytokines and inflammatory Mo is frequently associated with fetal loss [5 , 13 ].

The placenta is an essential organ for mammalian reproduction that links maternal and fetal compartments. It has been proposed that placenta is an anatomical site of "immune privilege," as it is not rejected by the maternal immune system, although this organ is mainly composed of extra embryonic cells. Several soluble factors and cells of placental origin have been considered important in contributing to marshaling the maternal immune system to support pregnancy [14 15 16 ]. Pregnancy-specific glycoproteins (PSG), the major placental glycoproteins, are a group of highly similar proteins synthesized in large amounts by placental trophoblasts that, together with the carcinoembryonic antigen, comprise a subfamily within the immunoglobulin (Ig) superfamily [17 , 18 ]. The PSG family is encoded by at least 11 different genes that give rise to 30 different proteins through alternatively spliced mRNAs [19 ]. During normal pregnancy, PSG molecules are released to maternal circulation reaching 200–400 µg/ml in serum at the end of gestation [20 , 21 ]. Low levels of PSG have been associated with certain human, pathological conditions, such as spontaneous abortion, intrauterine growth retardation, and pre-eclampsia [22 23 24 25 ]. In addition, the PSG spatiotemporal expression pattern is conducive to hypothesize that these glycoproteins play a crucial role in supporting gestation and fetus protection against the maternal-immune system [26 ]; however, their biological function is not yet thoroughly defined. It had been reported that a peptide derived from human PSG 11 binds to a human monocyte receptor and to the cell surface of promonocyte lineage, but not to T or B cells [27 ], and recently, the receptor for murine PSG 17 has been reported in murine Mo as the tetraspanin molecule CD9 [28 ]. Besides, it has been demonstrated that human PSG 1, PSG 6, and PSG 11 as well as murine PSG 18 induce the secretion of anti-inflammatory cytokines by human and murine monocytes [29 , 30 ]. Both results suggest that PSG may have the ability to modulate the macrophage activity.

In this study, the effects of the human PSG1a, the major variant of PSG polypeptides, on Mo activation pathways and T cell proliferation were investigated. For that, the glycosylated form of PSG1a was overproduced and secreted in mammalian cells, mimicking the natural process, using a recombinant (rec), vaccinia (Vac)-based expression vector harboring the complete open reading frame (ORF) of PSG1a cDNA. Our results indicate that the rec-PSG1a secreted to the culture supernatants of infected HeLa cells is able to induce the alternative activation of human peripheral blood Mo (PBMo) as well as human and murine Mo cell lines. In addition, rec-PSG1a up-regulates the arginase activity and inhibits the iNOS activity in Mo activated by lipopolysaccharides (LPS). We also observed that rec-PSG1a acts as an important, accessory cell-dependent T cell suppressor factor inducing a partial growth arrest at the S/G2/M phase of the cell cycle. Additionally, PSG1a is able to function as an important T cell modulator factor at the systemic level, as the in vivo expression of PSG1a in BALB/c mice markedly suppressed the T cell proliferative response induced by mitogens and a specific antigen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
All cell cultures were performed in RPMI-1640 medium supplemented with 10% endotoxin-free, heat-inactivated fetal calf serum (FCS) and 50 µg/ml gentamicin (completed RPMI medium). Purified anti-CD28 monoclonal antibody (mAb) 9.3 [31 ] was a gift from Dr. J. A. Ledbetter. Mouse anti-human CD3, phycoerythrin (PE)-labeled anti-human CD3, fluorescein isothiocyanate (FITC)-labeled anti-CD25, and human-rec IL-4 were purchased from PharMingen (San Diego, CA). LPS from Escherichia coli serotype 0111:B4, phorbol 12-myristate 13-acetate (PMA), phytohaemagglutinin (PHA), and calcium ionophore (ionomycin) was purchased from Sigma-Aldrich (St. Louis, MO).

rec-Vac viruses
A cDNA fragment encoding the complete ORF sequence of the human PSG1a (nucleotides -81 to +1260 with respect to the initiation codon) was cloned into the SalI/SacI sites of the polylinker present in the Vac thymidine kinase (tk) gene harbored by the pTG186 plasmid [32 ], thus placing the PSG1a sequences under the control of the tk promoter. Homologous recombinants with a disrupted tk gene were generated by transfection of Vac-infected cells with the resulting plasmid. Recombinants lacking tk activity were isolated after two steps of selection with bromodeoxyuridine (BrdU) of infected baby hamster kidney tk-negative cells followed by plaque purification [33 ]. Viruses isolated from BrdU-resistant cells were tested for their ability to express the PSG1a following infection of HeLa cells by Western blot analysis of culture supernatant and protein cell extracts using specific polyclonal antisera (Dako A/S Denmark). To scale-up the PSG1a production, HeLa cells (for human cell studies) or J774 cells (for murine cell studies) were infected with the rec-Vac virus at a multiplicity of 10 plaque-forming units (PFU)/cell, and the level of the PSG1a protein secreted to the culture medium (rec-PSG) was analyzed at different times post-infection by Western blot. Efficient PSG1a production was detected following 10 h of infection when no significant alterations in cell morphology were observed. Culture supernatant from HeLa or J774 cells infected with the wild-type (wt) Vac virus was used as a negative control supernatant (control) in all the experiments. The rec-PSG1a and control supernatants were filtered through a 0.22 µm nitrocellulose membrane and used at the doses indicated in the figure legends. The concentration of rec-PSG1a in culture supernatants from Vac virus-infected cells was estimated by densitometer analysis (ImmageGauge 3.12 Software, Fuji, Tokyo, Japan) of protein bands detected in Western blot. A fixed concentration of immunopurified rec-PSG1a (approximately 95% purity) quantified by Bradford’s method was used as standard. All the supernatants from rec-PSG1a Vac virus-infected cells used throughout this work contained roughly 15 µg/ml rec-PSG1a.

Western blot analysis
Whole cell extracts (100 µg) treated with RIPA buffer [phosphate-buffered saline containing 1% Nonidet P-40 (NP-40), 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] or culture supernatants (10 µl) from Vac wt- or PSG1a-infected cells were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. PSG protein was revealed by immunoblotting using the polyclonal anti-PSG antibodies. Immunocomplexes were visualized by a chemiluminescence reaction (Renaissance Western blot reagent, New England Nuclear, Boston, MA), according to the manufacturer’s recommendations.

Peptide N-glycosidase treatment
Supernatants containing rec-PSG1a were subjected to deglycosidation by peptide N- linked glycosidase F (PNGase F) from Flavobacterium meningosepticum (Sigma Chemical Co., St. Louis, MO) as described [34 ]. Briefly, rec-PSG1a from 1 ml culture supernatant was precipitated with amonium sulfate (50% P/V), pH 7.5, and resuspended in 1/10 vol phosphate buffer, pH 7.5, in the presence of 1 unit PNGase F (100 µl final vol). After 1 h of incubation at 37°C, the reaction was stopped by boiling in twofold-concentrated SDS-PAGE Laemmli sample buffer. The deglycosidase-treated rec-PSG1a was analyzed by Western blotting.

Immunoprecipitation of rec-PSG1a
Supernatants from HeLa cells expressing rec-PSG1a (250 µl) were immunoprecipitated as follows. After one step of preclearing the supernatant (~250 ng rec-PSG1a) with 100 µl protein A-sepharose (10% P/V, Pharmacia, Uppsala, Sweden), the anti-PSG (40 µg) was added, and the mixture was incubated for 1 h at 4°C. Then, 100 µl protein A-sepharose suspension was added and incubated for an additional hour. The rec-PSG1a-depleted supernatant was centrifuged at 5000 rpm for 5 min, and the rec-PSG1a content of the supernatant as well as the pelleted beads were assessed by SDS-PAGE followed by immunoblotting. The depleted medium was 0.22-µm filter-sterilized and used in the proliferation assays.

Mouse model
Inbred female BALB/c mice, aged 6–8 weeks (obtained from Comisión Nacional de Energía Atómica, Buenos Aires, Argentina), were injected intraperitoneally (i.p.) with 107 PFU of Vac-PSG1a (n=10) or Vac-wt (n=10). After 4 days, the mice were immunized with 50 µg ovalbumin (OVA; Sigma-Aldrich) emulsified in complete Freund’s adjuvant (CFA; Sigma-Aldrich) by subcutaneous (s.c.) injections at four different sites along the back and at the base of the tail (five mice from each group) or by i.p. injections (five mice from each group). Mice were maintained according to the National Research Council Guide for the Care and Use of Laboratory Animals and were killed on day 8 post-immunization.

Cells
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (Histopaque, Sigma-Aldrich) centrifugation of freshly drawn heparinized blood or buffy coat from healthy donors.

For T cell purification, monocytes were removed by two rounds of plastic adherence (1 h incubation at 37°C in 10-cm Petri dishes), and B cells were depleted by magnetic cell sorting using anti-B220-coated magnetic beads (Dynal, A. S., Oslo, Norway) following the manufacturer’s instructions. After this procedure, more than 95% of CD3+ cells were detected by flow cytometry.

Human PBMo were obtained from PBMC and posterior plastic adherence (2 h incubation at 37°C in 10-cm Petri dishes). The nonadherent cells were removed by washing three to four times with warm medium, and the adherent cells were cultured in different conditions, as indicated in the figure legends.

HeLa cells, RAW 264.7, and J774 murine macrophage cell lines and U937 human monocytic cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured under standard conditions in RPMI-1640 medium containing 10% FCS.

Spleens (from. i.p.-immunized mice) or draining lymph nodes (from s.c.-immunized mice) were removed, and cell suspensions were prepared by homogenization in a tissue grinder. Erythrocytes were lysed by brief incubation in erythrocyte lysing buffer (Sigma-Aldrich). Spleen mononuclear cells (SMC) or lymph nodes cells (LNC) were washed twice and resuspended in complete RPMI medium (RPMI-1640 medium supplemented with 10% endotoxin-free, heat-inactivated FCS and 50 µg/ml gentamicin).

Determination of arginase activity
All arginase assays were performed with cell lysates that had been cultured in different conditions, as indicated in the figure legends. Arginase activity was measured according to the already described procedure with slight modifications [35 ]. Briefly, 1 x 105 cells were lysed with 50 µl 0.1% Triton X-100 during 30 min at room temperature with gentle agitation. Then, 50 µl 50 mM Tris-HCl, 10 mM MnCl2, was added, and the enzyme was activated by heating for 10 min at 56°C. Arginine hydrolysis was initiated by the addition of 25 µl 0.5 M L-arginine, pH 9.7, at 37°C for 45 min. The reaction was stopped with 400 µl H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). The urea concentration was measured at 540 nm after addition of 25 µl alfa-isonitrosopropiophenone (dissolved in 100% ethanol) followed by heating at 95°C for 30 min. The results of arginase activity are expressed as µg urea.

NO assay
NO was measured as nitrite using the Griess reagent. Culture supernatant was mixed with 100 µl 1% sulfanilamide, 0.1% N-(1-naphthyl) ethylene-diamine dihydrochloride, and 2.5% H3PO4. Absorbance was measured at 540 nm in a microplate reader (Bio-Rad, Hercules, CA). Optical density measurements were averaged and converted to µM nitrites using a standard curve of sodium nitrite.

Cell proliferation assays
Human cell cultures were set up in triplicate in 96-well microculture plates. Each culture contained 1.25 x 105 cells, and appropriate mitogen or stimulating antibody concentration [PHA: 5 µg/ml; anti-CD3: 2.5 µg/ml; anti-CD28: 1 µg/ml; PMA: 30 ng/ml; calcium ionophore (ionomycin): 300 ng/ml] was incubated for 72 h. Cell cultures maintained in a total volume of 0.20 ml were supplemented with different volumes of rec-PSG1a or control supernatant.

Murine cell cultures were carried out in RPMI complete medium. SMC or LNC (1x106/ml) from Vac-PSG1a- or Vac-wt-injected mice were cultured in triplicate in 96-well microculture plates for 72 h (mitogens) or 120 h (specific antigen) with concanavalin A (Con A: 5 µg/ml), anti-mouse CD3 (2.5 µg/ml), or different concentrations of OVA or medium alone.

All cultures were incubated at 37°C in 5% CO2 and pulsed with 1 µCi [3H] thymidine (TdR; specific activity, 6.7 Ci/mmol; New England Nuclear) during the last 18 h. Cultures were interrupted by harvesting, and the radioactivity was measured in a liquid scintillation counter (Beckman LS 7000).

Mixed lymphocyte reaction
The effect of rec-PSG1a on allogenic stimulation of lymphocytes was analyzed by the primary mixed leukocyte reaction. PBMC were isolated from normal human donors by Ficoll-Hypaque gradient centrifugation. Responder PBMC (2x105 cells) and 2 x 105-irradiated (30 Gy), stimulator PBMC were incubated with different doses of rec-PSG1a or a control supernatant during 6 days in 96-well microculture plates at 37°C in 5% CO2. [3H]TdR (1 µCi) was added for the last 18 h before harvesting the cells.

Cell viability assays
A colorimetric method to determine the number of viable cells in the proliferation assay was used (CellTiter 96TM AQueous nonradiactive cell proliferation assay, Promega Corporation, Madison, WI). In this assay, 3 x 105 cells were cultured in complete RPMI medium containing or not the mitogen and were supplemented with different volumes of rec-PSG1a or control supernatant. The cell viability was measured following 24, 48, and 72 h of culture according to the manufacturer’s instructions. The absorbance values at 490 nm were obtained in an enzyme-linked immunosorbent assay Reader Plate (Bio-Rad).

Flow cytometry determinations
PBMC cultured for 24 or 48 h with or without PHA in the presence or the absence of rec-PSG1a or control supernatant were washed three times with Hanks’ balanced salt solution (HBSS) containing 1% bovine serum albumin and 0.1% NaN3 and were preincubated with 1/10 autologous sera for 1 h at 4°C to block Ig, nonspecific trapping through Fc receptors. Following Fc blocking, cells were incubated with PE-labeled anti-human CD25 and FITC-labeled anti-CD3 for 30 min at 4°C using 1 µg each antibody/1 x 106 cells. The cells were washed three times with HBSS, fixed in 2% formaldehyde, and stored at 4°C in the dark until the cells were acquired in a Cytoron Absolute cytometer (Ortho Diagnostic System, Raritan, NJ).

The DNA content was determined as described Nicoletti et al. [36 ]. Briefly, after 48 h of culture with or without PHA in the presence of rec-PSG1a or control supernatant, the cells were stained with FITC-labeled anti-human CD3 as described above and fixed in 1 ml cold 70% ethanol at 4°C. After being extensively washed, cell pellets were gently resuspended in 1 ml hypotonic fluorochrome solution [50 µg propidium iodide (PI)/ml diluted in 4 mM sodium citrate, 0.3% NP-40] and were kept at 4°C for 18 h in the dark. The PI fluorescence emission of individual nuclei was filtered through a 585/42 nm band pass filter. The number of apoptotic cells was determined by evaluating the percentage of hypo-diploid nuclei in the <2 N DNA peak and the cells in G0/G1 and S/G2/M cell-cycle stage by the amount of DNA per cell. Ten thousand events were acquired in a flow cytometer (Ortho Diagnostic System) using the Research program (Ortho Cytoronabsolute) to measure apoptosis or with the Cell Cycle program (Ortho Cytoronabsolute) to measure cell-cycle phases. The results were analyzed with WinMDI Software (The Scripps Institute, La Jolla, CA).

Statistical analysis
Data were analyzed using the Sigma Plot statistical package (Jandel Scientific Software, San Rafael, CA). Comparisons between two groups were made using the unpaired t-test. Values were considered statistically significant if P < 0.05. All experiments were repeated at least three times with similar results.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
rec-PSG1a synthesized in mammalian culture cells has similar features to placental PSG
The PSG1a biosynthesis in Vac-PSG-infected HeLa and J774 cells was evaluated in whole cell extracts and culture supernatant by Western blot analysis using a polyclonal rabbit antibody against human PSG1a. The rec-PSG1a from culture supernatant is detected as a single, thick, 72-kDa migrating band, whereas two fast migrating bands are observed in whole cell extracts (Fig. 1 A , lanes 7 and 8 and 5 and 6, respectively). In contrast, no specific protein is detected in the same cell lines infected with the wt-Vac virus used as a negative control (Fig. 1A , lanes 3 and 4 and 1 and 2, respectively). The rec-PSG1a molecule produced in cell culture supernatants shows the same 72-kDa molecular weight as the major placental PSG fraction, suggesting that it is secreted in its glycosylated form (Fig. 1 , compare lanes 7 and 9). To analyze the carbohydrate content of the rec-PSG1a protein, it was digested with PNGase F. The results confirmed that the rec-PSG1a underwent N-glycosylation as a result of its susceptibility to this enzyme, rendering a faster migrating 48-kDa protein that is roughly the same molecular weight as the PSG1a amino acid sequence deduced from its cDNA (Fig. 1B , lanes 10 and 11). Thus, like the placental PSG, the rec molecule is synthesized and processed by human and mouse culture cells to be secreted as a glycoprotein containing roughly 30% of its molecular mass of carbohydrates as was already reported [18 , 37 ]. In addition, rec-PSG1a interacted with the Con A lectin, and the binding was inhibited by mannose analogs ({alpha}-methyl d-mannoside), a feature that is shared with the placental PSG [18 , 38 ] (not shown).



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  		Figure 1. Analysis of PSG1a synthesized in mammalian cells by a Vac-based expression vector. (A) rec-PSG1a production in HeLa (H) and J774 (J) cells. Whole-cell extracts (W.C.; 100 µg, lanes 1 and 2 and 5 and 6) or culture supernatants (Sup.; 10 µl, lanes 3 and 4 and 7 and 8) from infected cells by a Vac virus bearing or not the PSG1a cDNA were analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with the anti-PSG antibody. A human placental extract (100 µg, P) was used as positive control. (B) rec-PSG1a SDS-PAGE pattern upon digestion with PNGase F. rec-PSG1a from culture supernatant of HeLa-infected cells was incubated in the absence (lane 10) or in the presence (lane 11) of PNGase F (1 U) during 1 h and was then analyzed by SDS-PAGE and Western blot. Arrows indicate the molecular weights (MW) of major detected bands.

 
Hence, the rec-PSG1a produced in these cells was used to analyze its activity over macrophage activation pathways and T cell proliferation in the absence of other PSG isoforms, related PSG family members, or potential interference that normally co-purifies with these molecules when isolated from pregnant sera.

rec-PSG1a induces alternative activation of human and mouse monocytes
To investigate the potential role of PSG1a in the modulation of the arginine metabolism in human Mo, we incubated PBMo or the human monocyte cell line U937 for 48 h in the presence of rec-PSG1a. Then, the cells were lysed, and the arginase activity was measured. As it is shown in Figure 2 , rec-PSG1a induces strong activation of the arginase activity in these cells, up to three- to fourfold. It is well-known that LPS, an amphiphilic, structural component of the gram-negative bacteria outer membrane, is a potent activator of Mo, which is able to induce arginase and/or iNOS activity in a variety of cell types [9 , 39 , 40 ]. A combination of LPS and rec-PSG1a treatment increased the arginase activity by fourfold in U937 cells, whereas LPS alone does not modify the activity of this enzyme.



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  		Figure 2. rec-PSG1a induces arginase activity in human monocytes. PBMo and U937 cells were cultured with LPS (10 µg/ml), IL-4 (25 ng/ml), or medium alone in the presence or the absence of rec-PSG1a (50% v/v) or control supernatant (50% v/v) for 48 h. Then, the cells were lysed, and arginase activity was determined. Results shown are mean ± SD of duplicates from one representative of three independent experiments. The experiments were done using different fresh preparations of rec-PSG1a and control supernatant. *, P < 0.001 versus control.

 
It is well-established that murine Mo produce NO following cytokine or LPS activation, but human macrophages produce low levels or no NO at all under similar conditions [40 ]. Therefore, to determine whether PSG1a is also involved in the classical pathway of Mo activation, NO production was determined in murine J774 and RAW 264.7 cell lines. The results indicate that rec-PSG1a induces a weak but significant up-regulation of the arginase activity with a concomitant down-regulation of iNOS activity, and these effects were more marked in the presence of LPS (Fig. 3 ).



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  		Figure 3. rec-PSG1a induces arginase activity and down-regulates the iNOS activity in murine cell lines. (A) J774 or RAW 264.7 cells were cultured with IL-4 (25 ng/ml), LPS (10 µg/ml), or medium alone in the presence or absence of rec-PSG1a (50% v/v) or control supernatant (50% v/v) for 48 h. Then, the cells were lysed, and arginase activity was determined. (B) J774 or RAW 264.7 cells (1x106) were treated with IFN-{gamma} (500 pg/ml), LPS (10 µg/ml), or medium alone in the presence or absence of rec-PSG1a (50% v/v) or control supernatant (50% v/v) for 48 h in a final volume of 1 ml, and then supernatant media samples were assessed for content of nitrite. Results shown are mean ± SD of duplicates from one representative of three independent experiments. The experiments were done using different fresh preparations of rec-PSG-1a and control supernatant. *, P < 0.001 versus control.

 
As shown, arginase was coinduced together with iNOS activity in the J774 cells upon LPS stimulation; however, only arginase activity was enhanced after incubation with rec-PSG1a. It is interesting that RAW 264.7 cells, which were shown to be refractory to arginase induction mediated by IL-4 or LPS, increased the arginase activity threefold upon treatment with a combination of LPS and rec-PSG1a. In these experiments, appropriate concentration of IL-4 was used as a positive control for arginase activity induction (Figs. 2 and 3A) . Moreover, a pronounced synergism between rec-PSG1a and IL-4 was observed (not shown).

Taken together, these results are consistent with a biological role of human PSG1a in the regulation of Mo activation pathways through the up-regulation and down-regulation of the arginase and iNOS activities, respectively. Furthermore, human PSG1a function exhibits cross-species activity in the regulation of human and murine Mo.

rec-PSG1a inhibits the accessory cell-dependent pathway of T cell proliferation, promoting T cell cell-cycle arrest on the S/G2/M phase
Accessory cells participate in the growth of T cells initiated by anti-CD3 mAb or mitogens such as PHA by providing a matrix that favors the cross-linking of the CD3 complex and by secreting cytokines [41 ]. T cell activation and proliferation using these mitogens require Mo-lymphocyte contact that can only be achieved with viable Mo [42 ]. Previous studies have shown that alternatively activated Mo are able to inhibit the lymphocytes and tumor cell line proliferation [43 , 44 ]. Taking into account the results of PSG1a regarding the modulation of Mo function, the effect of rec-PSG1a on accessory cell-dependent T cell proliferation pathways was analyzed. The PBMC proliferation induced by optimal concentrations of soluble anti-CD3 or PHA was inhibited in a dose-dependent manner by rec-PSG1a in comparison with the negative control (Fig. 4 A ). In addition, the inhibitory effect was abolished when culture supernatant was depleted of rec-PSG1a by immunoprecipitation (Fig. 4B) .



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  		Figure 4. rec-PSG1a inhibits the accessory cell-dependent pathways of T cell proliferation. (A) PBMC were cultured with anti-human CD3 mAb (2.5 µg/ml) or PHA (5 µg/ml) in the absence or the presence of increasing volumes of rec-PSG1a or control supernatant for 3 days and were pulsed with [3H]TdR for the final 18 h of culture. (B) PBMC were cultured with PHA (5 µg/ml) in the absence or the presence of rec-PSG1a (50% v/v), control supernatant (50% v/v), or rec-PSG1a-depleted supernatant for 3 days and were pulsed with [3H]TdR for the final 18 h of culture. Results shown are mean ± SD of triplicate wells from one representative of three independent experiments. The rec-PSG1a protein level before (lane 2) and after PSG1a immunodepletion (lane 3) is shown at the bottom. Lane 1 refers to the control supernatant. The experiments were done using different fresh preparations of rec-PSG1a and control supernatant. *, P < 0.001; **, P < 0.0002 versus control.

 
To exclude the possibility that rec-PSG1a could be inherently toxic to the cells, their viability was checked at different times using a colorimetric test based on the activity of dehydrogenase enzymes found in metabolically active cells. The number of living cells cultured with medium alone or with different volumes of rec-PSG1a or control supernatant was not significantly different at any time studied. Similar results are obtained with the trypan blue exclusion test. Moreover, using this assay, we observed that the number of living cells was increased when anti-CD3 or PHA was added to the culture but it was significantly reduced in the presence of rec-PSG1a, showing again that rec-PSG1a was able to inhibit the mitogen-induced proliferation (data not shown).

These findings show that PSG1a, secreted to the culture supernatant of Vac-PSG-infected HeLa cells, inhibits the accessory cell-dependent, mitogen-induced proliferation of PBMC in a dose-dependent manner and that the immunosuppressive effect is not a result of an inherent toxic effect to the cells.

To discern if the inhibitory effect of rec-PSG1a on the mitogenic response elicited by PHA was affecting T cell activation, we evaluated the percentage of CD3+ CD25+-activated T cells by flow cytometry. T cells incubated with rec-PSG1a cells are able to express CD25 (the IL-2R or Tac antigen) to a similar extent as the negative control (Fig. 5 ), although 50–80% reduction in proliferation was observed. Thus, T cells can still become activated in response to mitogenic stimulation, but cell proliferation is blocked by rec-PSG1a treatment. This observation was further investigated by the cell-cycle analysis of T cells by flow cytometry. A significant accumulation of cells at the S/G2/M phase of the cell cycle is observed following incubation with rec-PSG1a, and the population of hypodiploid cells was not affected, indicating that apoptosis is not involved in the suppressive effects mediated by this molecule (Fig. 5) .



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  		Figure 5. rec-PSG1a induces partial growth arrest at S/G2/M phase of the cell cycle without significant changes in the percentage of CD3+ CD25+ cells. PBMC (106 in 2 ml) were cultured for 24 h (for anti-CD25 study), 48 h (for apoptosis and cell-cycle study), or 72 h (for [3H]TdR incorporation study) in the absence or the presence of PHA (5 µg/ml) with or without rec-PSG1a (25% v/v), control supernatant (25% v/v), or medium alone. For proliferation study, the cultures were pulsed with [3H]TdR for the final 18 h, and the results are shown as the mean of cpm ± SD of triplicate wells. For anti-CD25 study, cells were double-stained with FITC-labeled anti-CD25 and PE-labeled anti-CD3. CD3+ cells were gated, and the percentages of CD25+ cells are indicated. For apoptosis and cell cycle study, cells were double-stained with FITC-labeled anti-CD3 and PI. Ten thousand events were acquired in a flow cytometer (Ortho Diagnostic System) using Research program (Ortho Cytoronabsolute) to measure apoptosis or with Cell Cycle program (Ortho Cytoronabsolute) to measure cell cycle phases. T cells (CD3+) were gated, and the percentages of cells in sub Go/G1, Go/G1, or S/G2/M phase of the cell cycle were calculated. Significance of the differences (PBMC+PHA+rec-PSG1a vs. PBMC+PHA+control supernatant) was P < 0.01, calculated using the Student’s t-test.

 
rec-PSG1a inhibits the human mixed lymphocyte reaction
The PSG1a function on human T cell proliferation was further analyzed in the relative physiologic context provided by the allo-mixed lymphocyte reaction. In complete agreement with the results previously observed in mitogen-induced proliferation, rec-PSG1a also has the ability to significantly block the mixed lymphocyte reaction in a dose-dependent manner (Fig. 6 ).



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  		Figure 6. rec-PSG1a inhibits the human mixed lymphocyte reaction. Effectors PBMC and irradiated (30 Gy)-target PBMC from two healthy donors were cultured in the absence or the presence of increasing volumes of rec-PSG1a or control supernatant for 6 days. The cells were pulsed with [3H]TdR for the final 18 h of culture. Results shown are mean ± SD of triplicate wells from one representative of three independent experiments. The experiments were done using different fresh preparations of rec-PSG1a and control supernatant. *, P < 0.001; **, P < 0.002 versus control.

 
Effect of rec-PSG1a on accessory cell-independent pathways of T cell proliferation
The experiments shown above indicated that T cells underwent substantial growth inhibition in the presence of rec-PSG1a when they were stimulated through lymphocyte-proliferation, accessory cell-dependent pathways (allo-mixed lymphocyte reactions, PHA, and anti-CD3 stimulation). We further investigated the potential role of rec-PSG1a to interfere with accessory cell-independent pathways of human T cell proliferation. As shown in Figure 7 , the proliferative response of highly purified CD3+ cells to a mitogenic combination of ionomycin and PMA or a combination of anti-CD3 and anti-CD28 antibodies was identical in the presence or the absence of rec-PSG1a. Therefore, PSG1a immunosuppressive effect is not a result of a direct effect on T cells. These results further emphasize the central role of accessory cells in the immune regulation mediated by PSG1a.



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  		Figure 7. Effect of rec-PSG-1a on accessory cell-independent pathways of T cell proliferation. Highly enriched T cells were stimulated with plastic, immobilized anti-CD3 (2.5 µg/ml) in conjunction with anti-CD28 (1 µg/ml) or PMA plus calcium ionophore (ionomycin; Io) in the absence or the presence of rec-PSG1a (25% v/v), control supernatant (25% v/v), or medium alone for 3 days and were pulsed with [3H]TdR for the final 18 h of culture. Results shown are mean ± SD of triplicate wells from one representative of three independent experiments. The experiments were done using different fresh preparations of rec-PSG1a and control supernatant.

 
Vac-PSG1a expression "in vivo" impairs a T cell proliferative response induced by mitogens and specific antigen
Because PSG1a is a glycoprotein released to maternal circulation, according to our in vitro results it is interesting to hypothesize that this molecule could be involved in the induction of maternal T cell tolerance at the systemic level. To investigate this hypothesis, a Vac-PSG1a was injected into BALB/c mice four days before immunization with OVA in Frend’s adjuvant (OVA-CFA). The proliferative response against accessory cell-dependent stimulus such as OVA as well as mitogens such as Con A and anti-CD3 was measured eight days post-immunization. As observed in Figure 8 , the T cell proliferative response induced by mitogens and OVA-specific antigen was dramatically impaired in Vac-PSG1a-injected mice in comparison with Vac-wt-injected control mice. This defective response was observed in the experiments carried out with spleen mononuclear cells (from i.p.-immunized mice) as well as lymph node cells (from s.c.-immunized mice) cultures.



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  		Figure 8. Vac-PGS1a-injected mice showed an impaired T cell-proliferative response against mitogens and specific antigen. SMC (1x106/ml; from i.p. OVA-immunized mice) or LNC (1 x 106/ml from s.c. OVA-immunized mice) from Vac-PSG1a or Vac-wt-injected mice were cultured in triplicate in 96-well microculture plates at 37°C in 5% CO2 for 72 h (mitogens) or 120 h (specific antigen) with Con A (5 µg/ml), soluble anti-CD3 (2.5 µg/ml), different concentrations of OVA, or medium alone, and proliferation was measured by [3H]TdR incorporation. Results shown are mean ± SD of triplicate wells from one representative of three independent experiments. *, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present work, the function of the human PSG1a, a major PSG isoform secreted into maternal circulation during pregnancy, on Mo activation pathways and T cell proliferation was investigated. For these purposes, a rec-PSG1a was expressed in mammalian cell cultures with a Vac-based expression vector. High PSG1a concentration was detected in the cell culture supernatant as an N-glycosylated 72-kDa protein, thus mimicking the natural PSG biosynthesis carried out in placenta.

We demonstrated that human rec-PSG1a induces alternative activation in human PBMo and human and murine Mo cell lines. We also demonstrated, using in vitro and in vivo experiments, that rec-PSG1a is able to modulate the maternal T cell immune response, acting as an important accessory cell-dependent T cell suppressor factor.

Recently, Snyder et al. [30 ] have proposed that some human PSG (PSG 1, PSG 6, and PSG 11) could modulate the immune response by inducing the secretion of anti-inflammatory cytokines such as IL-10, IL-6, and TGF-ß by human and murine cells. Our results not only agree with these findings (as alternatively activated Mo can secret TGF-ß and IL-10), but they also demonstrate that PSG1a is able to modify the Mo metabolism. Thus, rec-PSG1a induces iNOS down-regulation and arginase up-regulation in murine cell lines stimulated by LPS, suggesting that the presence of this protein at the site of Mo activation could induce a Mo metabolic pathway deviation toward the alternatively activated one with the consequent enhancement of its capacity for endocytosis and antigen presentation of a broad range of foreign antigens [7 ]. This activity of PSG1a on Mo could also be extrapolated to other cell populations such as dendritic cells (DC) in keeping with the knowledge that macrophages are a precursor of DC [45 ] and cells from fetal-maternal interface. In agreement, arginase activity has been detected in myometrium from pregnant guinea pigs, showing the maximal activity at the placental implantation site [46 ]. Moreover, it has been observed that arginase activity is increased in myometrium and some other organs such as liver, kidney, small intestine, and stomach in advanced pregnancy [47 ]. In addition, the significance of the arginase activity in the reproductive process was already demonstrated using some inhibitors of this enzyme before or during the pregnancy as a contraceptive agent [48 , 49 ]. In contrast, many researchers have argued that NOS activity and its product NO have a possible primary or secondary role in the development of pre-eclampsia [50 , 51 ]. The potential function of rec-PSG1a in the regulation of arginase activity in other tissues still remains to be investigated.

Early reports had suggested that placental-derived PSGs, which contained a mixture of the PSG family members, suppress mixed lymphocyte reaction and T cell activation by mitogens [52 , 53 ]. In contrast, Arnold et al. [25 ] found no effects on T cell proliferation using purified rec-PSG1a or PSG11 molecules obtained from insect cells. In this sense, it is important to note that some of the differences in the post-translational processing, e.g., glycosylation, between mammalian and insect cells may explain these discrepancies. In agreement, we demonstrate with experimental designs in vivo and in vitro, that PSG1a synthesized in mammalian cells, glycosylated and secreted to natural PSG biosynthesis, can mediate T cell suppression. The rec-PSG1a-mediated T cell suppression activity seems to be mediated through pathways involving other cell populations rather than T cells, considering that the ability of highly purified T cells to respond to accessory cell-independent stimuli is not targeted by PSG1a. In contrast, when PBMC were stimulated with accessory cell-dependent mitogens, the addition of rec-PSG1a reproducibly inhibits the proliferation in a dose-dependent manner. Moreover, rec-PSG1a inhibits the human T cell allogenic response, a common parameter tested to study the immunosuppressive effect of a number of compounds. The possibility that the immunosuppressive effect mediated by rec-PSG1a could be a result of a toxic activity to the cells was excluded, as the cell viability was preserved during all the incubation times assayed. Accordingly, flow cytometry analysis of T cells stimulated with PHA revealed that rec-PSG1a produce a significant decrease in the percentage of cells in Go/G1 and a modest but significant increase in the percentage of cells in the S and G2/M phases. Furthermore, T cell activation is not affected by rec-PSG1a, as they are still able to express CD25. These findings are consistent with cells entering S phase and progressing into cell cycle slower than control cells [54 , 55 ].

Because PSG1a is a glycoprotein released to maternal circulation, according to our in vitro results, it was interesting to test the hypothesis that this molecule could be involved in the modulation of T cell response at the systemic level. In fact, we observed that in vivo injection of Vac-based expression vector coding PSG1a into BALB/c mice four days before immunization with OVA in Frend’s adjuvant markedly suppressed the T cell-proliferative response induced by mitogens and a specific antigen compared with control mice injected with Vac-wt.

Several studies performed with alternatively activated Mo or DC have demonstrated that they can inhibit the proliferation of lymphocytes and tumor cell lines [43 , 44 , 56 ]. Taking into account the results of PSG1a regarding the modulation of Mo function, this glycoprotein could be affecting the local and systemic T cell proliferation through the regulation of antigen-presenting cell (APC) metabolism. Because it has been demonstrated that these APC populations could modulate the T cell proliferation through a cytokine-mediated pathway (TGF-ß or IL-10) [57 , 58 ] involving direct cell contacts [43 ] or by the induction of regulatory T cells [59 ], the exact mechanism involved in PSG1a-mediated effects still remains to be elucidated.

Sacks et al. [6 ] have proposed that soluble placental products released directly into the maternal circulation can generate specific pregnancy signals through interaction with the innate immune system. Thus, the innate immunity might be able to distinguish the pregnant from the nonpregnant states, producing a unique signal that promotes or prevents the lymphocyte response to alloantigen stimulation. This signal, so-called "signal P" [6 ], might include up-regulation of some APC’s surface antigens and production of cytokines generating suppressor rather than costimulatory APC’s for lymphocyte function. In light of this hypothesis, our results acquire potential relevance, as the alternatively activated Mo targeted by PSG1a described in this report could have a suppressor role. In addition, it has been reported that alternatively activated Mo are able to induce differentiation of naïve T cells into Th2 cells [20 , 21 ]. In this context, experiments currently in progress are consistent with in vivo PSG1a function to mediate modulation of murine T cell response and differentiation into Th2 cells (unpublished results).

Finally, the results reported in this work suggest that PSG1a may have "signal P"-like role during pregnancy with opposite effects on maternal-innate and adaptative-immune systems. Thus, in addition to activating a noninflammatory pathway of the innate-immune system, PSG1a could actively contribute to the T cell suppression and/or shift of the maternal-cell immunity toward a Th2 phenotype to assure successful pregnancy.


   ACKNOWLEDGEMENTS
 
C. C. M. and F. L. D. are recipients of postdoctoral and graduate fellowships from the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) of Argentina. Grants from the Secretaría de Ciencia y Tecnología—Universidad Nacional de Córdoba (SECyT-UNC), Argentina, Agencia Córdoba Ciencia, and CONICET supported this work. We are grateful to Dr. Luis Patrito for his continuous encouragement and support and to Prof. Claude Kedinger for many helpful discussions. We thank the staff of the cell culture department of the IGBMC (Illkirch, France) for technical assistance during rec-Vac virus production. We also thank Eva Acosta for idiomatic corrections. A. G. and J. L. B. are career investigator members of CONICET.

Received November 9, 2001; revised April 8, 2002; accepted April 24, 2002.


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