Objective
Embryo-derived PreImplantation Factor (PIF) is essential for pregnancy immune modulation and synthetic PIF (sPIF), reverses neuroinflammation, and prevents diabetes mellitus through its immune modulatory properties. Herein, we explore sPIF’s systemic effects on peripheral blood mononuclear cells (PBMCs).
Study Design
sPIF’s effects on PBMCs and subset populations from nonpregnant patients (n = 7) and male patients were evaluated by the assessment of binding characteristics, mixed lymphocyte reaction, proliferation, cytokine secretion, and associated gene expression. Data analysis was by analysis of variance ( P < .05).
Results
Fluorescein isothiocyanate–sPIF bound all myelomonocytic cells; binding was 30-fold up-regulated in mitogen-activated T and B cells ( P < .05). sPIF decreased mixed lymphocyte reaction by 70% and blocked anti-CD3 antibody stimulated-PBMC proliferation by approximately 80% ( P < .05). In naïve PBMCs, sPIF reduced interleukin (IL)-10 and -2; in activated PBMCs, sPIF increased IL-4, -5, -10, and -2, tumor necrosis factor–α, interferon-γ, and granulocyte-macrophage colony-stimulating factor ( P < .05).
Conclusion
Physiologic concentrations of PIF exert potent systemic antiinflammatory effects on nonpregnant activated immune cells.
Pregnancy is an immune and engraftment paradox. The mother/host “accepts” the embryo/allograft, enabling engraftment while preserving maternal immune balance. Nonviable embryos fail to achieve maternal acceptance ; likewise, impaired tolerance towards the conceptus leads to pregnancy failure. However, the successful implantation rate for allogenic embryos is unexpectedly high. Despite evidence that supports a maternal “permissive” environment, which includes the observation that autoimmune disorders often improve only to frequently flare-up during the postpartum period, maternal defense mechanisms are generally well-maintained during pregnancy. We therefore postulate that embryo-specific compounds that modulate both tolerant T-suppressor (T H 2) and inflammatory T-helper 1 (T H 1) cytokine profiles are essential for successful mammalian reproduction, and their imbalance is associated with pregnancy loss.
Maternal recognition of pregnancy occurs before implantation, which suggests operative embryo-specific signaling. Identification and characterization of the putative agent have important implications for therapies that address immune-related disorders in pregnancy and autoimmunity.
PreImplantation Factor (PIF) is a peptide that is secreted by the preimplantation embryo and is specific to pregnancy. PIF is secreted only by viable embryos, starting at the 2-cell stage, and is absent in nonviable embryos. PIF is expressed by the fetus and the placenta, and is detectable in maternal circulation. PIF levels correlate with embryonic development by exerting direct, prosurvival autotrophic effects in the setting of an embryo-toxic environment in vitro. PIF plays an essential role in promoting implantation by directly acting on the decidua, modulating local immunity, enhancing embryo-decidual adhesion, and controlling apoptosis. Moreover, PIF facilitates trophoblast invasion in vitro .
We demonstrated PIF’s immunomodulatory effects in nonpregnant models. In a juvenile diabetes mellitus model, synthetic PIF (sPIF) modulates systemic T H 1/T H 2 cytokines and prevents diabetes mellitus development long-term. In an autoimmune encephalitis model, sPIF reverses advanced paralysis, down-regulates neural proinflammatory T H 1-type genes and proteins, and inhibits interleukin (IL)-6 and -17 secretion through direct action on activated splenocytes.
Because systemic sPIF administration has tissue-specific effects in autoimmune models, we aimed to determine whether it also affects human immune cell lineages. Such documentation would have profound implications in the explanation of systemic immune changes that are associated with pregnancy and autoimmunity in general. We demonstrated that PIF is secreted from viable embryos and detectable shortly after fertilization. Now we seek to examine its effects in the context of embryo-maternal cross-talk before implantation, which would expand beyond the previously described proreceptive effect of sPIF on embryo implantation. In this study, we use chromatographic methods to isolate and further characterize PIF. We use sPIF as a single embryo-derived agent to elucidate its specific effects on nonpregnant, male naïve, and stimulated peripheral blood mononuclear cells (PBMCs) by assessing binding characteristics, mixed lymphocyte reaction (MLR), proliferation, cytokine secretion, and associated gene expression.
Materials and Methods
PIF peptide isolation and synthesis
Partial characterization and PIF assay information was published previously. Briefly, 1 L of mouse embryo that had been conditioned in culture media or control media was filtered through a 3 kd YM membrane (Millipore Corporation, Bedford, MA) and was passed through an affinity column (Anti-CD2 T11-1-antibody; BD Pharmingen, San Diego, CA) with Affi-Gel Hz Immunoaffinity (Bio-Rad Laboratories, Hercules, CA). Eluted fractions with PIF activity passed through C18 (Clipeus) high-performance liquid chromatography (HPLC) column and rechromatographed on Vydac C18 HPLC column (Grace Davison, Deerfield, IL). PIF-active fractions’ size was determined by MALDI-TOF mass spectrometry (Perseptive Biosystems, Cambridge, MA). Purified PIF peptides were sequenced by Edman degradation (Pulsed Liquid Sequencer; Applied Biosystems, Foster City, CA). Released amino acids were derivatized with phenylisothiocyanate and detected by a reversed-phase HPLC system in line with the sequencer. Synthetic MVRIKPGSANKPSDD (sPIF), PIFscr (GRVDPSNKSMPKDIA), and irrelevant peptide (GMRELQRSANK) of mouse origin and fluorescein isothiocyanate (FITC)–labeled ligands of >95% purity were also generated. Media from human embryo–conditioned media and control were analyzed with anti-PIF monoclonal antibody (mAb) by mass spectrometry (Ciphergen Systems, Freemont, CA).
Flow cytometry
Nonpregnant patients who underwent infertility treatment signed standard informed consent. Studies were approved (Repromedix Corp, Bedford, MA; CARI Institute, Chicago, IL). Blood was drawn as part of the work-up process with the use of excess specimen without identifiers (n = 7). Additional samples were obtained from 18 male volunteers after consent (Boston Biomedical Research Institute, Boston, MA; MDS Pharma Services, Bothell, WA). Twelve nonpregnant and 13 pregnant women in the first trimester were also studied (CARI Institute). PBMCs were isolated from peripheral blood (Ficoll Hypaque density gradient method). PBMCs were incubated with FITC-PIF, FITC-PIFscr, and size-matched irrelevant peptide at (0.19-48 μmol/L) concentrations along with antibody cocktail (anti-CD14,CD3, CD4,CD8,CD19,CD56; BD Pharmingen). In certain experiments, PBMCs were stimulated (1 μg phytohemagglutinin) at 24-72 hours, and FITC-PIF binding was examined. Isotype antibodies were used as negative controls. Two- and 3-color staining was performed. Fluorescence measurements (20,000-50,000 gated events/sample) by Coulter Epics XL Flow Cytometer were analyzed with System II software (BeckmanCoulter, Inc, Miami, FL).
PBMC proliferation
Cells (200,000/well) were stimulated with plate-bound anti-CD3 antibody or anti-CD3/anti-CD28 mAb 10 μg/mL or by heterologous MLR that was cultured 2-4 days in serum-free medium (GIBCO AIM-V Medium; Invitrogen Corporation, Carlsbad, CA) that contained 0-500 nmol/L sPIF or PIFscr. In some experiments, IL-2 recombinant (30 U/mL) ± sPIF was added. Proliferation was determined by [ 3 H]-thymidine incorporation. In other experiments, sPIF 0-1000 nmol/L was tested on isolated T cells (CD3+) on both MLR and anti-CD3/anti-CD28 MAb10 μg/mL–stimulated PBMCs.
Cytokine secretion
IL-4, IL-10, interferon-γ (IFN-γ), tumor necrosis factor–α (TNF-α) levels were determined in PBMC culture supernatants at 72 hours by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Concentrations of IL-2, IL-5, IL-8, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-4, IL-10, TNF-α, IFN-γ, IL-1β, and IL-6 were tested with sPIF (0-500 nmol/L) 4-96 hours ± plate-bound anti-CD3 mAb (10 μg/mL) or anti-CD3/anti-CD28 mAb (10/μg/mL). Effects compared with PIFscr, irrelevant peptide, or vehicle (0.005% dimethyl sulfoxide) alone. Culture media were collected and analyzed (Fluorokine MAP MultiAnalyte Profiling, Luminex; R&D Systems).
Real-time polymerase chain reaction analysis of sPIF effect on PBMC cytokine expression
PBMCs were cultured with plate-bound anti-CD3 mAb/anti-CD28 mAb (10 μg) per 24 hours ± 50 nmol/L sPIF. sPIF 50 nmol/L was compared with vehicle-only–treated controls. Briefly, after the culture procedure, cells were washed with phosphate-buffered saline solution, and messenger RNA (mRNA) was extracted, which was followed by hybridization to Oligo GEArray Human T H 1-T H 2-T H 3 Microarray (evaluates 84 immunity-related gene expression, 5 housekeeping genes GADPH, B2M, HPRT1, ACTB, HGDC and controls; SABiosciences, Frederick, MD). Delta delta Ct method was used for data analysis. Fold-changes were calculated between sPIF compared with coactivated or vehicle-only–treated PBMCs where a ≥2-fold change was considered significant.
sPIF effect on PBMC cytokine mRNA expression
sPIF50 nmol/L–treated PBMCs compared with vehicle-only and sPIF 50 nmol/L added to plate-bound anti-CD3 mAb/anti-CD28 mAb (10 μg) at 24 hours stimulated PBMCs compared with costimulated cells without added sPIF were studied. Postculture cells were washed with phosphate-buffered saline solution and chemically fragmented complementary RNA that was hybridized on Affymetrix HG_U133 Plus 2.0 human chips, GeneChip Hybridization Oven 640 (Affymetrix, Santa Clara, CA), screened for 18,400 human genes and expressed-sequence tags, followed by fluorescence labeling Fluidics Station 450 (Affymetrix), and optical scanning with an Affymetrix GeneChip Scanner 3000 (Codon Biosciences, LP, Houston, TX). A P < .05 value and a >2-fold change were considered significant.
Statistical analysis
Data were analyzed by analysis of variance with Dunnett error protection and 95% CI (Analyse-it for Microsoft Excel; Microsoft Corporation, Redmond, WA; Analyse-it Software, Ltd, Leeds, UK); a probability value of < .05 was considered statistically significant. Regression analysis was used to determine FITC-PIF binding to PBMC lineages.
Results
Predicted 3-dimensional structure; homology to malaria protein
Isolation of PIF from conditioned mouse embryo culture media vs media alone, control was carried out ( Figure 1 ). PIF is a 15 aa peptide; where the first 9 aa are common for all 4 peptides ( Figure 2 , A and B). Figure 1 , C and D, shows a predicted 3-dimension PIF (15 aa) image. We found that the 15 aa peptide (PIF) is by far the most abundant peptide in the embryo culture media, based on mass spectrometry. Therefore, the sPIF 15 aa (sPIF) was used for all experiments that assessed its interaction with nonpregnant or male PBMCs. An examination of the human embryo culture media demonstrated the presence of a mass spectrometry peak that corresponds to the 15 aa PIF peptide ( Figure 2 , E).
Low-dose sPIF binds all naïve monocytes
sPIF interaction with PBMCs was examined with FITC-PIF as ligand. FITC-sPIF binds all naïve myelomonocytic (CD14 + ) cells, although only minimally targets T cells at low concentrations ( Figure 3 , A). FITC-PIF binding specificity was demonstrated because irrelevant peptide GMRELQRSANK (control) binds <5% of CD14+ cells, even at high concentration ( Figure 3 , B). FITC-PIF also binds to all naïve CD14 + cells in pregnant patients ( Figure 3 , C).
FITC-PIF binding to PBMCs was further examined with increased ligand concentrations. Close-to-maximal binding to CD14 + cells was at approximately 3 μg/mL ( Figure 4 , A). In contrast, FITC-sPIF binding to naïve T (CD4 + , CD8 + ), B (CD19 + ), and NK (CD56 + ) cells was <10%. At high concentrations, a dose-dependent linear increase was observed in all phenotypes, however, which did not reach saturation (r 2 = 0.9) with multiple regression analysis. In contrast, scrambled FITC-PIF 25 μmol/L binding to NK (CD56 + ) cells was <10% ( Figure 4 , B). Binding to CD3 + cells significantly increased when compared with nonpregnant patients ( P < .001; Figure 4 , D and E). Therefore, naïve CD14+ cells are primary PIF targets.
sPIF binding to lymphocytes is markedly up-regulated by mitogen exposure
Response to immune challenge requires interaction with the adaptive immune system arm at low (physiologic) PIF levels that are present in maternal circulation. To mimic challenged immune environment, FITC-PIF binding to PBMCs after mitogen (phytohemagglutinin) stimulation was examined. After 24 hours, a substantially different binding profile was observed ( Figure 5 ). FITC-sPIF binding to T cells (CD4 + and CD8 + ) and B cells increased 30-fold up to approximately 90%. In contrast, no change in NK (CD56 + ) cells was noted (<10%), which remained low even at 72 hours. Thus, under challenge, sPIF interacts with the adaptive arm of immunity.
sPIF blocks MLR
After fertilization, the embryo through PIF promotes tolerance development. We aimed to determine whether sPIF affects MLR that reflects immune response to alloantigen presence. sPIF, but not PIFscr (control), significantly decreased MLR (approximately 70%) at 125 nmol/L ( P < .05; Figure 6 ). However, when sPIF (2-1000 nmol/L) was tested on isolated T cells (CD3+) and after activation with IL-2r (30 U/mL), the MLR decrease was minimal (10-20%). Thus, sPIF reduced MLR, but only in global PBMCs.
sPIF blocks anti-CD3 mAb-induced PBMCs proliferation
sPIF effect on (anti-CD3 mAb) activated PBMC proliferation was tested. Low sPIF concentrations significantly blocked activated proliferation although did not affect naïve PBMCs proliferation ( P < .05). In contrast, PIFscr had no effect ( Figure 7 ). Further, lymphocyte proliferation after stimulation with anti-CD3 mAb combined with IL-2 recombinant (potent inducer) or costimulation by (anti-CD3 mAb/anti-CD28 mAb) stimulated T (CD3+) cells was not affected by sPIF. Thus, sPIF inhibits only activated PBMC proliferation.
sPIF stimulates activated-PBMC cytokines secretion that creates a T H 2 bias
Because sPIF affected differently activated vs naïve PBMCs proliferation, effects on T H 1 and T H 2 cytokine secretion were assessed. After anti-CD3 mAb stimulation, sPIF increased T H 2/T H 1 cytokine ratio approximately 5-fold at 72 hours, mainly because of increased IL-10 secretion. In contrast, the effect on naïve cells was minimal ( Table ). We examined whether this response was due to only 1 time-point testing (72 hours) and examined time-dependent (4-96 hours) incubation. At 50 nmol/L concentration, sPIF increased both T H 1/T H 2 cytokines secretion in anti-CD3 mAb-stimulated PBMCs ( Figure 8 ) ; sequential changes were observed in cytokine secretion throughout the culture period. Remarkably, T H 2-type cytokines (IL-4, -5, and -10) increased, although a time-dependent (early decrease, followed by increase) in T H 1-type cytokines (IL-2, TNF-α, IFN-γ, and GM-CSF) secretion was noted. In contrast, in naïve PBMCs, none of 10 different cytokines that were tested increased during sequential culture. Actually, T H 1 cytokines (TNF-α, IL-1β, and GM-CSF) decreased (not shown; Figure 8 , A and C). In comparison, PIFscr increased only TNF-α secretion in both stimulated and naïve PBMCs ( Figure 8 , B and D); vehicle-only controls had no effect. At 96 hours of culture, all cytokine levels returned to baseline, except for IL-8. Thus, sPIF exerts divergent effects on naïve and activated PBMCs cytokines secretion.
