Reproductive BioMedicine Online (2013) 26, 79– 87
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ARTICLE
Preimplantation factor inhibits circulating
natural killer cell cytotoxicity and reduces
CD69 expression: implications for recurrent
pregnancy loss therapy
Roumen G Roussev a, Boris V Dons’koi b, Christopher Stamatkin a,
Sivakumar Ramu a, Viktor P Chernyshov b, Carolyn B Coulam a,
Eytan R Barnea c,d,e,*
a
CARI Reproductive Institute/BioIncept LLC, Chicago, IL, United States; b Laboratory of Immunology, Institute of Pediatrics,
Obstetrics and Gynecology, Academy of Medical Sciences, Kiev, Ukraine; c SIEP – Society for the Investigation of Early
Pregnancy, Cherry Hill, NJ, United States; d BioIncept LLC, Cherry Hill, NJ, United States; e Department
of Obstetrics, Gynecology and Reproduction, UMDNJ – Robert Wood Johnson Medical School, Camden, NJ, United States
* Corresponding author. E-mail address: [email protected] (ER Barnea).
Dr Eytan R Barnea, MD, FACOG is double board certified in obstetrics, gynaecology and reproductive
endocrinology. He investigates embryo-derived signalling in pregnancy, translating such observations into
clinical applications in pregnancy and immune disorders. He is the founder of the Society for the Investigation of
Early Pregnancy, director of obstetrics and gynaecology at CAMcare and associate clinical professor of obstetrics
and gynaecology and reproduction at University of Medicine and Dentistry of New Jersey/Robert Wood Johnson
Medical School. In 1989, he received the Elkeles Prize ‘Scientist of the Year in Medicine’ from the Israel Health
Ministry/Jewish National Fund.
Abstract Embryo-secreted preimplantation factor (PIF) is necessary for, and its concentration correlates with, embryo develop-
ment in humans by promoting implantation and trophoblast invasion. Synthetic PIF (sPIF) modulates systemic immunity and is effective in autoimmune disease models. sPIF binds monocytes and activated T and B cells, leading to immune tolerance without
suppression. This study examined the effect of sPIF on natural killer (NK) cell cytotoxicity in 107 consecutive nonselected, nonpregnant patients with recurrent pregnancy loss (RPL) and 26 infertile IVF patients (controls). The effects of sPIF, intravenous gamma
immunoglobulin (Ig), Intralipid and scrambled PIF (PIFscr; negative control) on NK cell cytotoxicity to peripheral-blood cells were
compared by flow cytometry of labelled-K562 cell cytolysis. The effects of sPIF and PIFscr on whole-blood NKCD69+ expression were
also compared. In patients with RPL, sPIF inhibited NK cell cytotoxicity at doses of 2.5 and 25 ng/ml (37% and 42%) compared with
PIFscr (18%; P < 0.001), regardless of the proportion of peripheral-blood NKCD56+ cells to lymphocytes. Pre-incubation of blood
from infertile patients with sPIF for 24 h decreased NKCD69+ expression versus incubatino with PIFscr (P < 0.05). In conclusion, sPIF
inhibits NK cell cytotoxicity by reducing NKCD69 expression, suggesting a significant role in RPL patients. RBMOnline
ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
KEYWORDS: NK cell cytotoxicity, preimplantation factor (PIF), recurrent pregnancy loss (RPL), CD69, therapy
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.rbmo.2012.09.017
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Introduction
The aetiologies for isolated and/or recurrent pregnancy loss
(RPL) have not yet been fully elucidated and often remain
undefined (Simpson, 2007). The main factor leading to RPL
is genetic, being associated in 60% of cases with chromosomally abnormal embryos. Other recognized aetiologies
arise from mechanical and endocrine disorders (Christiansen
et al., 1995; Regan, 1988).
Immune disorders are also recognized as significant
contributors to RPL (Varla-Leftherioti, 2007). Peripheral
natural killer (NK) cells are an important part of the altered
immune repertoire found in RPL (Emmer et al., 2000).
These large, peripheral, granular lymphocytes mostly
express the CD16 and always CD56, but not the CD3,
markers (Kwak-Kim and Gilman-Sachs, 2008). Thus,
CD3 CD16+CD56+ cells expressing NK cell-associated
molecules include CD3 CD16++CD56+ cells, which are cytotoxic and express the killer immunoglobulin-like receptor
(KIR) family, as do CD3 CD16+CD56++ cells with lower
CD16+ expression, which have a lower cytotoxicity (Cooper
et al., 2001). KIR receptors are expressed on all NK cell
subsets. However, CD56++ cells express a high-affinity
interleukin 2 receptor, have higher concentrations of interferon c, tumour necrosis factor a and interleukins 10 and 13
and have important immune modulatory roles (Fukui et al.,
2008, 2011; Shi et al., 2007; Vivier, 2011). Based on recent
studies, there may be an excess of activating KIR or a
slightly lowered ratio of inhibitory to activating KIR in RPL
patients. Also, specific ‘less inhibiting’ combinations of
maternal inhibitory KIR and fetal human leukocyte C
combinations are found at higher proportions in subfertile
couples (Pantazi et al., 2010; Varla-Leftherioti, 2005).
Circulating NK cell numbers and cytotoxic activity have
been extensively investigated in patients with RPL. Patients
with a history of primary spontaneous abortions have a
higher percent circulating NK cells compared to lymphocytes than those with secondary recurrent abortion (King
et al., 2000; Shakhar et al., 2003). This is especially true
in patients who have lost chromosomally normal embryos
(Yamada et al., 2003). In general, 10% peripheral NK cells
is considered to be elevated. It has been shown that >12%
NK cells 5 days after miscarriage in RPL patients is associated with an immune aetiology of fetal loss (Paparistidis
et al., 2008). Further, with >18% NK cells, pregnancy loss
was reported to be inevitable (Beer et al., 1996).
For patients with a history of RPL and an elevated
proportion of peripheral NK cells, several therapies have
been advocated. They include intravenous gamma immunoglobulin (Ig) (Coulam and Goodman, 2000; Coulam and
Roussev, 2003; Kwak et al., 2000; Perricone et al., 2006),
intravenously administered Intralipid, which is reported to
increase implantation rates (Roussev et al., 2007, 2008),
sildenafil, which increases blood flow to the uterus and
increases lining thickness (Jerzak et al., 2008), and prednisolone, which was shown to effectively suppress NK cell
cytotoxicity in vitro (Thum et al., 2008). However, a
recent large placebo-controlled study found limited efficacy of intravenous gamma Ig in treating RPL patients
(Stephenson et al., 2010).
RG Roussev et al.
The testing method for NK cell activation has been
re-evaluated, demonstrating that incubation of peripheralblood mononuclear cells (PBMC) with different targets
results in activation and increased CD69 expression on NK
lymphocytes (Giavedoni et al., 2000; Korbel et al., 2005;
Ntrivalas et al., 2001. It was reported that incubation of
freshly isolated PBMC or whole blood with K562 cells
stimulates NK cells and results in a significant increase of
CD69 expression on NK cells, but less so on NK T cells
(CD3+CD56+; Dons’koi et al., 2011a). The same authors
further showed that CD69 expression on NK cells, and
more significantly up-regulation of the CD3 CD56+CD69+
subset, correlates with NK cell cytotoxicity and could be
valuable for examining in NK cell activity (Dons’koi et
al., 2011b).
Viable embryos secrete preimplantation factor (PIF), a
peptide essential for embryo development and absent in
nonviable embryos. PIF exerts a targeted autotrophic
effect on the embryo (Barnea and Coulam, 1997; Barnea
et al., 1999, 2007; Barnea, 2004, 2007a; Barnea and
Sharma, 2006; Stamatkin et al., 2011a). PIF is detected
in the maternal circulation and is expressed by the placenta (Barnea, 2007b). A synthetic PIF analogue (sPIF) in
a physiological dose range displays multitargeted effects,
namely regulating local immunity, promoting embryo adhesion and controlling apoptosis in decidual cell cultures
(Barnea et al., 2012a; Paidas et al., 2010). In parallel, sPIF
promotes trophoblast invasion thereby supporting placental development (Duzyj et al., 2010). In nonpregnant
models, sPIF modulates peripheral immunity, leading to
tolerance without immune suppression (Barnea, 2007a;
Barnea and Kirk, 2009; Barnea et al., 2012b). For example,
in juvenile diabetes investigated in the non-obese diabetic
mouse model, short-term administration of sPIF prevents
diabetes development in the longer term by preserving
pancreatic islet function (Weiss et al., 2011a). In a
separate nonpregnant model, sPIF reverses chronic neuroinflammation, while promoting neural repair (Weiss
et al., 2011b).
In patients with a history of RPL, putative (Stamatkin
et al., 2011b) and specific circulating antiphosopholipid and
antinuclear antibodies (Kaider et al., 1999; Roussev et al.,
1996) and/or oxygen radicals (Ornoy, 2007) can have harmful effects on the embryo, causing their demise. It has also
been reported that sPIF acts as a rescue factor and negates
the embryotoxicity of serum from RPL patients when added
to mouse embryo cultures, increasing the proportion of
embryos that reaches the blastocyst stage, while reducing
embryo demise rates (Stamatkin et al., 2011b). Having
shown that sPIF protects against RPL serum toxicity to
embryos, it is further postulated that documenting a
sPIF-induced mitigating effect on peripheral NK cell cytotoxicity might help to substantiate PIF’s role in protecting
the embryo against maternally induced hostility.
The present work tests the effects of sPIF in vitro on NK
cell cytotoxicity and compares its effect with currently used
treatment regimens, namely intravenous gamma Ig and
Intralipid, in parallel in RPL patients. Scrambled PIF (PIFscr)
is used as a negative control. To determine the specificity of
sPIF action, its effect on the NK cell activity marker
CD3 CD56+CD69+ was examined.
PIF inhibits NK cell cytotoxicity in RPL patients
Materials and methods
PIF peptide synthesis
The production of sPIF (MVRIKPGSANKPSDD) and PIFscr
(GRVDPSNKSMPKDIA) has been reported previously (Duzyj
et al., 2010; Paidas et al., 2010). Briefly peptides were synthesized using solid-phase peptide synthesis (Peptide Synthesizer; Applied Biosystems, Foster City, CA, USA)
employing 9-fluorenylmethoxycarbonyl chemistry. Final
purification was carried out by reversed-phase high-performance liquid chromatography (HPLC) and peptide identity
>95% purity was verified by mass spectrometry (BioSynthesis, Lewisville, TX, USA).
Patient population
Patients (n = 107) experiencing repeated primary or secondary pregnancy loss were referred to CARI’s immunology laboratories for NK cell cytotoxicity evaluation and were
included in the IRB-approved study as previously reported
(Roussev et al., 2007), initiating the study on 19 August
2008. Patients were not on any treatment at the time of
blood collection. Blood samples were collected and the proportion of NKCD56+ cells to lymphocytes was determined.
Subsequently, blood samples were placed on a Ficoll
Hypaque column and isolated PBMC were further analysed
by using a standard NK cell cytotoxicity assay (Roussev
et al., 2008).
Infertile women (n = 26) undergoing IVF–embryo transfer
at the Institute of Reproductive Medicine (Kiev, Ukraine)
were selected as controls for this investigation. Except for
two of the infertile women, all had no previous IVF attempts
or obstetric complications. The women were aged
28 ± 1 years and in all cases the cause of infertility was peritoneal-tubal adhesions.
Immunological investigations, performed for the IVF
patients, were carried out at the Laboratory of Immunology,
Institute of Pediatrics, Obstetrics and Gynecology, Academy
of Medical Sciences, Kiev, Ukraine. Patients were not under
treatment with immunoglobulins, corticosteroids or heparin. Also, no patients had active or a history of autoimmune
disease.
All study subjects signed an informed consent prior to
entering the study. The study was carried out in accordance
with the Bio-ethic Convention EU Council 1997 and the
World Medical Association Declaration of Helsinki 1996.
NK cell cytotoxicity assay
NK cell cytotoxicity was determined by flow cytometry using
a previously described technique (Roussev et al., 2008).
Briefly, K562 leukaemia cells were cultured as stationary
cultures at 37C in 5% CO2. To be certain they were in the
log growth phase, cells were subcultured for 3 days before
the assay. Before use in the assay, cells were incubated with
10 ll of 30 mmol/l dioctadecyl oxacarbocyanine perchlorate
per ml for 20 min at 37C with 5% CO2. To evaluate NK cell
response to suppression, sPIF 2.5–2500 ng/ml, intravenous
gamma Ig 12.5 mg/dl (Baxter, Glendale, CA, USA), Intralipid
81
18 mg/ml (Frezenius, Clayton, NC, USA) or PIFscr
25 ng/ml–50 lg/ml (control) was used. A total of five
12 · 75 mm Becton-Dickinson tubes per RPL patient was
used (one technical replicate per test): target cells at standard concentration (10 ll of 1 · 106/ml) were mixed with
100 ll of 5 · 106/ml PBMC (effector cells) to create a target/effector ratio of 1:50. A separate tube with target cells
only without PBMC was also used for background control
(spontaneous cytolysis). The mixture was centrifuged for
30 s at 1000g to pellet the cells. The mixture was incubated
with test agents for 2.5 h at 37C with 5% CO2, and 15 min
before flow-cytometry acquisition, 100 ll propidium iodide
was added to the tubes to label the dead cells. Data were
collected for analysis using a Becton-Dickinson fluorescent-activated cell sorter using the CellQuest program and
cytolysis software (Becton-Dickinson, Brea, CA, USA). Spontaneous cytolysis was subtracted from the actual cytolysis
obtained for each sample.
FACS analysis
The forward- and side-scatter parameters were adjusted to
accommodate the inclusion of both target and effector cells
within the acquisition gate and the 10,000 cells required for
counting. Ten thousand cells were analysed in order to
increase the accuracy of the FACS-based measurement.
Quadrant markers are drawn to distinguish dioctadecyl oxacarbocyanine perchlorate-labelled cells (quadrant 4) from
cells with incorporated propidium iodide (dead cells; quadrant 2). The proportion of cytolysis was calculated as [quadrant 2 events/(quadrant 2 + quadrant 4 events)] · 100.
Data analysis was carried out in the following manner.
Readings from tubes with K562 cells only were considered
as background and subtracted from all readings (dead cells).
Natural NK cell cytolysis was assessed using the tube containing K562 and PBMC and used as a basis to compare with
the test agents (PIF, intravenous gamma Ig, Intralipid) or
control (PIFscr). Therefore, the following formula was used
to report the data: percentage change in NK cell cytolysis = [(natural NK cell cytolysis NK cell cytolysis after
treatment)/natural NK cell cytolysis] · 100.
Measurement of NK cell activation
NK cell activation was determined as described recently
(Dons’koi et al., 2011b). Briefly, 0.1 ml blood sample from
infertile patients undergoing IVF were diluted in 0.4 ml RPMI
1640 penicillin/streptomycin (Sigma, St Louis, MO, USA) and
pre-incubated for 6 h, 12 h or 24 h at 37C with sPIF or PIFscr
(as control) at a final concentration of 100 ng/ml. Preincubated samples were incubated for another 16 h at 37C
with or without freshly washed K562 cells (2.5 · 105/ml).
Tubes were gently shaken after 2 and 3 h of incubation.
Spontaneous and K562-stimulated CD69 expression on NK
cells was determined. Cells were stained by FITC-, PE- and
Cy5-conjugated monoclonal antibodies to CD3, CD56 and
CD69, respectively (BD Bioscience, San Jose, USA). Lysed
and washed samples were analysed on a FACScan cytometer
using CellQuest software (Becton-Dickinson, Brea, CA, USA).
The method has been recently published (Dons’koi et al.,
2011b). Briefly, CD69 stimulation (CD69stim) for PIF and
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RG Roussev et al.
Statistical analysis
Statistical analysis of NK cell cytotoxicity was performed
using ANOVA followed by two-tailed t-test, with P < 0.05
being considered statistically significant. Analysis of CD69
activation results were performed using Fisher’s Exact test
(unpaired, nonparametric, two-sided P-value) and two-way
contingency table or chi-squared analysis (Stat version 3.0
for Windows; Graph Pad Software, San Diego, CA, USA).
Results
Physiologically low sPIF concentrations block NK
cell cytoxicity
The sPIF effect was tested with NK cell cytotoxicity on a
consecutive, unselected group of nonpregnant RPL patients
(n = 21). Co-cultured PBMC and K562 cells were incubated
with sPIF for 2.5 h to determine cytolysis using a standard
protocol. The results in Figure 1 show that sPIF consistently
inhibited toxicity: 2.5 and 25 ng/ml (37 ± 4% and 42 ± 4%)
versus PIFscr 18 ± 18% (mean ± SD, both P < 0.001). The natural NK cell cytolysis (K562 and PBMC) was 6 ± 2.1%. The
higher sPIF concentration tested was found only mildly more
effective than the lower tested peptide dose. At even
higher sPIF concentrations (up to 2500 ng/ml), the suppression of NK cell cytotoxicity did not further improve (data not
shown). Thus, sPIF is effective in inhibiting NK cell cytotoxicity at low doses, comparable to the physiological PIF concentrations seen in pregnancy.
100
80
NK suppression (%)
for PIFscr was analysed by subtracting the value for CD69
spontaneous expression (CD69sp) from that for CD69 expression following stimulation with K562 cells (CD69exp): proportion of CD69stim = [(CD69exp CD69sp)/CD69sp] · 100.
The proportion of sPIF-induced inhibition was calculated
as [(CD69stim with sPIF/CD69stim with PIFscr)] · 100.
60
40
20
0
i.v. gamma Ig Intralipid
25 ng/ml sPIF
PIFscr
Figure 2 Synthetic preimplantation factor (sPIF) inhibits NK
cell cytotoxicity in recurrent pregnancy loss patients with
elevated (10%) NKCD56+ cells. The effect of low-dose sPIF
25 ng/ml was compared with intravenous gamma Ig
(12.5 mg/ml) and Intralipid (18 mg/ml; n = 25). sPIF efficacy
was similar to the two other agents (all P < 0.001) when
compared with the scrambled PIF control (PIFscr) data obtained
in a different group of patients (same as in Figure 1).
Low-dose sPIF inhibits NK cell cytotoxicity both in elevated and in normal proportions of NKCD56+ cells, as shown
by comparison with intravenous gamma Ig or Intralipid.
The effect of 25 ng/ml sPIF on NK cell cytotoxicity in 86
RPL unselected patients was tested comparing with intravenous gamma Ig (12.5 mg/ml) or Intralipid (18 mg/ml) in parallel. The results obtained were divided into samples with
<10% or 10% NKCD56+ cells.
In 25/86 (29%) of patients, an elevated proportion
(10%) of circulating NKCD56+ cells was found. The remaining patients had a normal NK cell proportion. In those with
elevated (10%) NKCD56+ cells (n = 25), sPIF significantly
inhibited NK cell cytotoxicity (41 ± 1%), similarly to intravenous gamma Ig (40 ± 5%) or Intralipid (39 ± 6%) versus PIFscr
(18 ± 18%; all P < 0.001; Figure 2). The natural NK cell
cytolysis (K562 and PBMC) was 14 ± 2%. In the patients
with normal (<10%) NKCD56+ cells (61/86, 71%), sPIF
100
80
60
60
NK suppression (%)
NK suppression (%)
70
40
20
0
25 ng/ml sPIF
2.5 ng/ml sPIF
PIFscr
50
40
30
20
10
0
i.v. gamma Ig
Figure 1 Low doses of synthetic preimplantation factor (sPIF)
inhibit NK cell cytotoxicity. The effect of sPIF at two different
concentrations (2.5 and 25 ng/ml) was tested with a NK cell
cytotoxicity assay using blood samples from an unselected
group of nonpregnant recurrent pregnancy loss patients
(n = 21). sPIF at a physiological range was effective in both
doses tested, as compared with scrambled PIF control (PIFscr)
tested at 25 and 50 lg/ml (neither with significant effect) in 16
of the same patients (P < 0.001).
Intralipid
25 ng/ml sPIF
PIFscr
Figure 3 Synthetic preimplantation factor (sPIF) inhibits NK
cell cytotoxicity in recurrent pregnancy loss patients with
normal (<10%) NKCD56+ cells. The effect of low-dose sPIF
(25 ng/ml) was compared with intravenous gamma Ig
(12.5 mg/ml) and Intralipid (18 mg/ml; n = 61). sPIF efficacy
was similar to the two other agents (all P < 0.001) when
compared with the scrambled PIF control (PIFscr) data obtained
in a different group of patients (same as in Figure 1).
PIF inhibits NK cell cytotoxicity in RPL patients
significantly inhibited NK cell cytotoxicity (41 ± 3%), similarly to intravenous gamma Ig (45 ± 7%) and Intralipid
(39 ± 6%) versus PIFscr (18 ± 18%; all P < 0.001; Figure 3).
The natural NK cell cytolysis (K562 and PBMC) was 6.9 ± 2%.
Thus it was shown that low-dose sPIF is effective in both
patients exhibiting elevated or normal NKCD56+ cells. The
results were similar to those obtained using intravenous
gamma Ig or Intralipid.
sPIF blocks NK cell cytotoxicity, as shown by
reduced CD69+ activation
To determine whether sPIF-induced inhibition of cytotoxicity was exerted by modulating NK cell activity, the sPIF
effect was tested using a specific NK cell-activation marker,
namely CD69+ expression on NKCD3 CD56+ cells. Pre-incubation with sPIF or PIFscr (control) in parallel for 6–24 h
was followed by activation with K562 cells for 16 h. sPIF
100 ng/ml significantly reduced K562 cell-induced stimulation of NK cells expressing the CD69+ marker following
24 h pre-incubation versus PIFscr 100 ng/ml (P < 0.05;
Figure 4). Figure 5 shows the significant reduction in NK
cell activation following 24 h pre-incubation of individual
patient blood samples with sPIF as a proportion of activation
in the presence of PIFscr (P < 0.05). Examination of the
shorter incubation times, 12 h and 6 h, revealed a progressive increase in the sPIF-induced effect, as compared with
24 h pre-incubation (two-way contingency table chi-squared
analysis, P < 0.01, DF = 7.9). Interestingly, a mild, albeit
significant, increase in spontaneous NKCD69+ expression
was noted following pre-incubation with sPIF for 12 h but
not 24 h (5.2 versus 2.8%; P < 0.05; data not shown). This
confirms that sPIF specifically inhibits NK cell cytotoxicity
as suggested by the reduction seen in the CD69+ activation
marker.
83
Discussion
There is a continuous quest to develop targeted and safe
treatments for patients with a history of immune-based
RPL, specifically for those patients who lose chromosomally
normal embryos. Overactive circulating NK cells with elevated cytotoxicity may lead to RPL (Roussev et al., 2007,
2008). sPIF acts as a rescue factor and blocks RPL serum
toxicity to embryos by increasing blastocyst rates and
lowering embryo demise (Stamatkin et al., 2011b).
This study, examining other contributing elements to
recurring pregnancy loss, tested the effect of sPIF on NK cell
toxicity by using two complementary methods. sPIF blocked
peripheral NK cell cytotoxicity in patients with RPL history:
low-dose sPIF blocked PBMC samples having normal or elevated proportions of NKCD56+ cells. The effect of nontoxic
sPIF was comparable to that obtained using intravenous
gamma Ig or Intralipid. sPIF-induced reduction in NK cell
cytotoxicity was associated with decreased activation of
the specific NK cell marker CD69+.
The finding that sPIF was highly effective in inhibiting NK
cell cytotoxicity at low concentrations (similar to that present in maternal circulation in early pregnancy) indicates
that the embryo-secreted peptide may protect the embryo
against maternally induced hostility (Barnea, 2007a; Duzyj
et al., 2010). Also, at higher concentrations (supra-physiological), the peptide did not further improve NK cell cytotoxicity inhibition. The higher sPIF concentration
(25 ng/ml) was used to compare with intravenous gamma
Ig or Intralipid since the 2.5 ng/ml concentration was less
effective. The scrambled PIF used as the control had no significant effect, confirming sPIF’s specificity of action. At
very high concentrations, PIFscr had a mild agonist activity
which was not seen within the physiological range of
concentrations.
Figure 4 CD69+ expression on NK cells following 24 h pre-incubation with synthetic preimplantation factor (sPIF) or scrambled PIF
(PIFscr) with and without stimulation by K562 cells. Pre-incubation with sPIF 100 ng/ml significantly inhibited CD69stim compared
with PIFscr 100 ng/ml. Data are mean ± standard error for each treatment for blood samples from 26 infertile patients undergoing
IVF. CD69sp = CD69 spontaneous expression after incubation without K562; CD69exp = CD69 expression after incubation with K562;
CDstim = [(CD69exp CD69sp)/CD69sp] · 100.
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RG Roussev et al.
Figure 5 Synthetic preimplantation factor (sPIF) inhibits NK cell activity by lowering CD69+ expression. The effect of
pre-incubation with sPIF 100 ng/ml for 6 h (n = 10), 12 h (n = 20) and 24 h (n = 26) in blood samples from infertile patients
undergoing IVF treatment compared with PIFscr 100 ng/ml as a control. P-values were calculated for mean activation of CD69stim
incubated with sPIF versus CD69stim incubated with PIFscr. A time-dependent sPIF-induced inhibition was significant following 24 h
pre-incubation (P < 0.05). Data are results from different patient blood samples.
The exact cut off above which the proportion of NKCD56+
cells is regarded as elevated is not fully established and may
vary within an individual and also multiple subtypes of NK
cells are known to be present (Kwak-Kim and Gilman-Sachs,
2008; Paparistidis et al., 2008; Shakhar et al., 2003). Therefore, for the present study it was important to examine sPIF
inhibitory effect on NK cell cytotoxicity in a diverse patient
population, having both normal and elevated proportions of
peripheral NK cells.
Owing to the multifactorial nature of RPL, and since the
lost embryos are chromosomally abnormal in 60% cases, it
has been difficult to devise clear and effective therapies for
RPL. Consequently, the NK cell cytotoxicity FACS assay is
commonly used to assess the efficacy of potential novel
therapies against RPL prior to their clinical use (Roussev
et al., 2007). Testing the sPIF inhibitory effect on NK cell
cytotoxicity in parallel with the other clinically used agents
has provided an opportunity to validate the observations
with respect to the assay performance. The similar inhibitory effects obtained with sPIF in both normal and elevated
NKCD56+ cell populations are in line with these agents’
inhibitory effect (Roussev et al., 2007).
Although not directly comparable, the significant effectiveness of low-dose sPIF compared with intravenous
gamma Ig and Intralipid may reflect a physiological effect
of PIF on these cell populations. sPIF is a single small-size
peptide synthetic analogue that has defined sites of intracellular action (Paidas et al., 2010). In comparison, intravenous gamma Ig is a complex antibody mixture and Intralipids
are fatty acids. Intravenous gamma Ig acts by targeting
CD94+ cells on NK cells (Shimada et al., 2009) on the
pro-tolerance ligand CD200 (Clark and Chaouat, 2005) and
binds to complement by the Fc fraction (Sewell and Jolles,
2002). Intralipid’s exact mechanism of action remains to be
defined. It acts on various nuclear receptors such as retinoid
X (Khan and Vanden-Heuvel, 2003) and its signalling is
involved in G-protein coupled receptors (Kostenis, 2004;
Roussev et al., 2007, 2008).
Having confirmed that sPIF was effective in the NK cell
cytotoxicity assay, this study examined whether the
observed effect was specific, namely whether the single
peptide acts on a critical marker of NK cell activation.
CD69+ expression is a specific NK cell activation marker
(Dons’koi et al., 2011b; Iizuka et al., 1999). The finding that
pre-exposure to sPIF significantly decreased this NK cell
activation marker expression clearly points to a specific
inhibitory action of the peptide on NK cell cytotoxicity.
Since the effect was time dependent and not replicated
by PIFscr tested as an internal control, sPIF’s specificity
was confirmed. Although in majority of patients NK cell
activity was reduced following exposure to sPIF, the variability of response to the peptide may be due to the use
of an unselected and known infertile patient group that
were undergoing IVF in order to achieve pregnancy. It is possible that individual NK cell populations may be differently
responsive to sPIF action. A slightly higher sPIF dose, within
the physiological range (100 ng/ml; Duzyj et al., 2010), may
have been required for testing in whole blood (not PBMC)
and for a prolonged experimental time. Nonetheless, given
that sPIF at low dose acts in targeted manner on CD69+ cells
and in whole blood (not only with PBMC) supports the view
that sPIF is a potent inhibitor of NK cell cytotoxicity in vitro.
This is the first report to show that sPIF is capable of
reducing CD69+ activation in context of this novel NK cell
PIF inhibits NK cell cytotoxicity in RPL patients
activity assay. CD69+ is one of the earliest surface sites that
is activated on NK cells (Iizuka et al., 1999). It was reported
that CD69+ is an important marker for NK cell activity which
reflects degranulation and cytotoxicity (Chernyshov et al.,
2010). Also, only a certain subset of NK cells and NK T cells
are activated, while T lymphocyte expression is not
affected (Dons’koi et al., 2011a). Elevated NK cell cytotoxicity has been previously shown to negatively affect implantation following IVF (Chernyshov et al., 2010; Coulam and
Roussev, 2003; Miko et al., 2010; Prado-Drayer et al., 2008).
Whether sPIF could additionally have a preventive role in
these patients having elevated NK cell cytotoxicity remains
to be demonstrated.
It has been previously reported that sPIF acts on immune
cells and binds all monocytes in naive PBMC, while in mitogen-activated PBMC it binds to most T and B cells, where
mRNA expression is greatly amplified in a time-dependent
manner (Barnea et al., 2012b; Barnea and Kirk, 2009). In
naı̈ve PBMC, sPIF down-regulated the NK cell triggering
receptor gene, which encodes cyclophylin B, a pro-tolerance molecule. Perhaps the transient increase in spontaneous CD69+ expression is related to a time-dependent effect
of the peptide. The possibility that NK cell inhibition is indirect and is exerted through monocytes has been previously
suggested (Higuchi et al., 1995), and possibly PIF acts in a
similar manner (Barnea, 2007a; Barnea et al., 2012b). Further studies are needed to fully elucidate the specific elements involved in sPIF’s mechanism of action on NK cells.
The demonstration of sPIF’s effectiveness in diverse
models of autoimmunity in vitro and in vivo supports and
strengthens the view that sPIF should equally be tested
for immune-based RPL therapy (Barnea, 2007b; Weiss
et al., 2011a,b). CD69+ inhibition has a major role in other
disorders beyond autoimmunity such as tumours and infections (Giavedoni et al., 2000; North et al., 2007) and therefore sPIF may prove to be relevant also for additional array
of clinical applications, which are currently being tested.
The current study is limited because the testing for NK
cell cytotoxicity use two different population cohorts
–patients with history of RPL and infertile patients undergoing IVF. The study’s strengths include using a large cohort of
unselected nonpregnant patients with a RPL history, examining inhibitory effect of low-dose sPIF on NK cell cytotoxicity versus other clinically used agents and comparing and
assessing K562 cell cytolysis. Further, the use of a novel
method to document sPIF-induced inhibition of a NK cell
activation marker, CD69 expression, supports its inhibitory
action. Additionally, documenting that PIF suppression of
NK cell cytotoxicity and activation is independent from circulating proportions of NKCD56+ cells.
Overall, the current and previous data place the embryo
– through PIF signalling – as having a critical co-ordinated
and beneficial effect on both systemic immunity and the
uterine environment (Barnea, 2007a; Barnea and Kirk, 2009;
Barnea et al.,2012a,b; Duzyj et al., 2010; Paidas et al.,
2010). This is shown by both by negating circulating
embryo-toxic factors in culture (Stamatkin et al., 2011b)
and in the current study, by blocking circulating NK cell
cytotoxicity in RPL patients. Thus, by counteracting potentially adverse circulating elements, sPIF protects embryos
against a hostile maternal environment in RPL patients.
85
Given the complex nature of RPL, sPIF’s clinical therapeutic
potential, although clearly suggestive, needs to be reconfirmed in additional studies.
In conclusion, nontoxic, low-dose sPIF blocks NK cell
cytotoxicity and inhibits NK cell activation marker CD69
expression. sPIF may represent a targeted therapy for
immune-based RPL prevention. Clinical testing of sPIF in
treating various immune disorders is warranted and is
planned shortly.
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Declaration: PIF is a patented compound owned by BioIncept. ERB is
the Chief Scientist (with no remuneration) of BioIncept. CBC is a
minority shareholder of BioIncept. CS, RGR and SR have received
funding from BioIncept. BVD and VPC declare no conflict of
interest.
Received 20 January 2012; refereed 13 September 2012; accepted
18 September 2012.