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*
Department of Experimental Medicine and Biochemical Sciences and
Division of Obstetrics and Gynecology, University of Rome Tor Vergata, Rome, Italy; and
Fatebenefratelli Association for Research, San Giovanni Calibita Hospital, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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reduced the
activity and the protein expression of FAAH. Human chorionic
gonadotropin or cortisol had no effect on FAAH activity. At variance
with FAAH, the lymphocyte anandamide transporter and cannabinoid
receptors were not affected by treatment with progesterone or
cytokines. Good FAAH substrates such as anandamide and
2-arachidonoylglycerol inhibited the release of leukemia-inhibitory
factor from human lymphocytes, but
N-palmitoylethanolamine, a poor substrate, did not. A
clinical study performed on 100 healthy women showed that a low FAAH
activity in lymphocytes correlates with spontaneous abortion, whereas
anandamide transporter and cannabinoid receptors in these cells remain
unchanged. These results add the endocannabinoids to the
hormone-cytokine array involved in the control of human
pregnancy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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9-tetrahydrocannabinol, the psychoactive
principle of hashish and marijuana (1). AEA and 2-AG have
cardiovascular activity, by inducing vasorelaxation (4, 5), and act as immune modulators (6, 7), much alike
the exogenous cannabinoids (8, 9). Also,
N-palmitoylethanolamine (PEA) is a biologically active
endocannabinoid, reported to have antiinflammatory activity
(10). However, its ability to bind to CB receptors is
still controversial (2). Recently, we reported on the
association between decreased concentrations of the AEA-degrading
enzyme, fatty acid amide hydrolase (FAAH), in peripheral lymphocytes
and early pregnancy failure in humans (11). This finding
seemed of interest, because the cellular and molecular mechanisms of
spontaneous abortion remain largely unknown, yet about one-half of all
conceptuses are lost before the expected menses (12).
Progesterone (P), a hormone essential for the maintenance of
pregnancy, is also known to modulate immune function (13)
and to elicit an immunological response critical for normal gestation
(14). Indeed, P has been shown to favor the development of
human T lymphocytes producing Th2 cytokines (ILs 4 and 10), which
inhibit Th1-type cytokines (IL-12 and IFN-), thus allowing the
survival of fetal allograft and therefore a successful pregnancy
(15, 16). More recently, the P-induced Th2 bias has been
found to stimulate the release of LIF from T lymphocytes, mediated by
IL-4 (17). Clinical data showing that women with
unexplained recurrent abortions have a reduced LIF production suggest
that the latter is indeed critical for implantation and maintenance of
fetus in humans (17, 18, 19). In this study, we sought to
investigate whether P stimulates FAAH activity and expression in human
lymphocytes. This would provide a biochemical ground to our previous
observation that low FAAH activity correlates with pregnancy loss in
humans (11). Since AEA and related compounds bind to CB
receptors, and their degradation by intracellular FAAH requires
cellular uptake through a specific transporter (20, 21),
the effect of P on CB receptors and AEA transporter in lymphocytes was
also investigated. Moreover, we studied the role of Th1- and Th2-type
cytokines in the regulation of FAAH, to ascertain a possible
correlation among the effects of P. We also investigated the effect of
human chorionic gonadotropin (hCG) on FAAH activity, because of its
role in pregnancy (22), and its use as a marker to monitor
human gestation (23). Finally, we investigated whether AEA
and its congeners might reduce LIF release from lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemicals were of the purest analytical grade. AEA, P, P
3-(O-carboxymethyl)oxime:BSA conjugate (P-BSA),
mifepristone (RU486), hCG, cortisol (17-hydroxycorticosterone), and
human rIL-2 were purchased from Sigma (St. Louis, MO).
N-(4-hydroxyphenyl)arachidonoylamide (AM404),
arachidonoyltrifluoromethyl ketone (AACOCF3), and
2-arachidonoylglycerol (2-AG) were from Research Biochemicals
International (Natick, MA). Human rIL-4, rIL-10, rIL-12, human
rIFN-, PMA, tetanus toxoid (Clostridium tetani), and
capsazepine
(N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide,
CAPS) were from Calbiochem (La Jolla, CA).
N-Piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide
(SR141716) and
N-[1(S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide
(SR144528) were a kind gift of Sanofi Recherche (Montpellier,
France). PEA was synthesized and characterized (purity >96% by
gas-liquid chromatography), as reported (24).
[3H]AEA (223 Ci/mmol) and
[3H]CP55.940
(5-(1,1'-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)cyclohexyl]phenol,
126 Ci/mmol) were from NEN DuPont de Nemours (Cologne, Germany).
2-[3H]AG was synthesized from
1,3-dibenzyloxy-2-propanol and [3H]arachidonic
acid (200 Ci/mmol; ARC, St. Louis, MO), as reported
(25). Anti-FAAH polyclonal Abs were elicited in rabbits
against the conserved FAAH sequence VGYYETDNYTMPSPAMR (26)
conjugated to OVA, and were prepared by Primm (Milan, Italy).
Anti-human CD3 mAbs were from Calbiochem. Soluble IL-4R (sIL-4R), human
rLIF, anti-human IL-4R (anti-IL-4R), anti-human
insulin-like growth factor I receptor (anti-IGF-IR) mAbs, and
Quantikine human LIF colorimetric sandwich ELISA were from R&D Systems
(Minneapolis, MN). Goat anti-rabbit Abs conjugated with alkaline
phosphatase (GAR-AP) were from Bio-Rad (Richmond, CA).
Isolation and treatment of lymphocytes
Blood samples (20 ml/donor) were drawn from the antecubital vein of healthy nonpregnant women (age range 28–35 years), who gave informed consent to the study, and were collected into heparinized sterile tubes. All subjects had regular cycles, and sampling was perfomed between the seventh and the tenth day from the last menstrual period. Peripheral lymphocytes were purified by gradient centrifugation, using the density separation medium Lymphoprep (Nycomed Pharma, Oslo, Norway), as reported (11). Purified lymphocytes were resuspended in RPMI 1640 medium (Life Technologies, Paisley, U.K.), supplemented with 25 mM HEPES, 2.5 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (24), at a density of 1.5 x 106 cells/ml, in ventilated 25-ml flasks. Incubation of lymphocytes with P, alone or in the presence of different compounds, with cytokines, with hCG, with cortisol, or with endocannabinoids was performed at 37°C in humidified 5% CO2 atmosphere, at the indicated concentrations and for the indicated periods of time. Controls were incubated with vehicles alone. In all cases, cells were treated in serum-free medium for 1 h; then heat-inactivated FBS (Life Technologies) was added at a final concentration of 10% (24). Cell viability after each treatment was tested by trypan blue dye exclusion, and was found to be higher than 90% in all cases.
AEA hydrolase activity and expression
FAAH (EC 3.5.1.4) activity was assayed at pH 9 with 10 µM
[3H]AEA as substrate, by the reversed-phase
HPLC method previously described (24). In a preliminary
series of experiments, T cells were isolated from the other PBMCs by
means of the Dynal CD2 CELLection kit (Dynal, Olso, Norway), according
to the manufacturer’s instructions. It was found that FAAH activity in
T lymphocytes accounted for 85% of the activity measured in the whole
cell population; the remaining 15% was found in B cells (6%) and
monocytes (9%). Cell homogenates (20 µg/lane) were prepared as
described (24) and were subjected to SDS-PAGE (12%),
under reducing conditions. Rainbow m.w. markers (Amersham Pharmacia
Biotech, Little Chalfont, U.K.) were phosphorylase
b (97.4 kDa), BSA (66 kDa), and OVA (46.0 kDa). For
immunochemical analysis, gels were electroblotted onto 0.45-µm
nitrocellulose filters (Bio-Rad), and FAAH was visualized with
anti-FAAH polyclonal Abs (1/200), using GAR-AP, diluted 1/2000, as
second Ab (24). Densitometric analysis of filters was
performed by means of a Floor-S MultiImager, equipped with a Quantity
One software (Bio-Rad). The same anti-FAAH Abs were used to
determine FAAH protein content also by ELISA. Wells were coated with
human lymphocyte homogenates (20 µg/well), which were then reacted
with anti-FAAH polyclonal Abs (diluted 1/300), as first Ab, and
with GAR-AP, diluted 1/2000, as second Ab (24). Color
development of the alkaline phosphatase reaction was measured at 405
nm, using p-nitrophenyl phosphate as substrate. The
OD405 values could not be converted into FAAH
concentrations, because the purified enzyme is not available to make
calibration curves. However, the ELISA test was linear in the range
0–50 µg/well of cell homogenate, and its specificity for FAAH was
validated by Ag competition experiments (24). RT-PCR was
performed using total RNA isolated from human lymphocytes (10 x
106 cells) by means of the simple nucleic acid
preparation. Total RNA Isolation Kit (Invitrogen, Carlsbad, CA),
as described (24). RT-PCR reactions were performed using
the EZ rTth RNA PCR kit (Perkin-Elmer, Norwalk, CT), according to the
manufacturer’s instructions. The reaction conditions were carefully
examined to stop the reaction during the exponential phase of
amplification of each gene. Briefly, 100 ng total RNA, for the
amplification of FAAH, or 0.4 ng, for 18S rRNA, were reversibly
transcribed and amplified in the same tube in a total reaction volume
of 10 µl, in the presence of 3 mCi
[
-32P]dCTP (3000 Ci/mmol; Amersham Pharmacia
Biotech). The amplification parameters were as follows: 2 min at
95°C, 45 s at 95°C, 30 s at 55°C, and 30 s at
60°C. Linear amplification was observed after 20 cycles. The primers
were as follows: (+), 5'-TGGAAGTCCTCCAAAAGCCCAG, and (-),
5'-TGTCCATAGACACAGCCCTTCAG, for FAAH; (+),
5'-AGTTGCTGCAGTTAAAAAGC, and (-), 5'-CCTCAGTTCCGAAAACCAAC, for
18S rRNA.
Five microliters of the reaction mixture were electrophoresed on a 6%
polyacrylamide gel, which was then dried and subjected to
autoradiography (24). The autoradiographic films were
subjected to densitometric analysis, by means of a Floor-S MultiImager
equipped with a Quantity One software (Bio-Rad). In some experiments,
the RT-PCR products were excised from the gel and counted in a LKB1214
Rackbeta scintillation counter (Amersham Pharmacia Biotech). Products
were validated by size determination and sequencing. Linear
amplification sequencing was performed by using Cyclist DNA Sequencing
Kit (Stratagene, La Jolla, CA), according to the manufacturer’s
instructions. RT-PCR products for sequencing were prepared without the
[-32P]dCTP and sequenced with the same
primers used for amplification, after labeling them with
[-32P]dATP (3000 Ci/mmol; Amersham Pharmacia
Biotech).
Analysis of AEA uptake and cannabinoid receptors
The uptake of [3H]AEA by intact lymphocytes (2 x 106/test) was studied essentially as described (21). To discriminate non-carrier-mediated from carrier-mediated transport of AEA through cell membranes, control experiments were conducted at 4°C. Q10 value was calculated as the ratio of AEA uptake at 30°C and 20°C (27), and apparent Km and Vmax of the uptake were determined by Lineweaver-Burk analysis (21). The effect of different compounds on AEA uptake (15 min) was determined by adding each substance directly to the incubation medium, at the indicated concentrations. Cell viability after each treatment was higher than 90% in all cases.
For cannabinoid receptor studies, membrane fractions were prepared from lymphocytes (10 x 106/test) as reported (28), were quickly frozen in liquid nitrogen, and stored at -80°C for no longer than 1 wk. These membrane fractions were used in rapid filtration assays with the synthetic cannabinoid [3H]CP55.940 at 400 pM, as described previously (28). Unspecific binding was determined in the presence of 10 µM AEA (29).
Release of LIF
To induce LIF production, short-term tetanus toxoid-specific T cell cultures were generated from human lymphocytes, as described (15). Peripheral lymphocytes (3 x 106/test) were stimulated for 5 days with tetanus toxoid (0.2 µg/ml), in the absence or presence of AEA, 2-AG, PEA, SR141716, SR144528, CAPS, AM404, AACOCF3, or different combinations of them, at the indicated concentrations. Human IL-2 (20 U/ml) was then added to all cultures, which were continued for additional 9 days. On day 14, T cell blasts were stimulated with PMA (20 ng/ml) plus anti-CD3 mAbs (100 ng/ml), and 36 h later culture supernatants were collected for LIF assay (17). LIF was quantitated by the Quantikine human LIF immunoassay, according to the manufacturer’s instructions. OD450 nm values of unknown samples were converted into LIF concentrations by calibration curves with human LIF (linearity range 10–1000 pg/ml).
Clinical study
A cohort of 100 white Italian women (age range 28–35 years) was enrolled after obtaining informed consent, and was selected from those who applied for antenatal booking at the outpatient clinic of San Giovanni Calibita Hospital. Women had normal singleton pregnancies, and embryos with crown-rump lengths of >10 mm and a heart rate of >110 beats/min. We excluded from enrollment patients with chronic diseases (diabetes, hypertension, lupus erythematosus systemicus), those with a known history of recurrent spontaneous abortions, those with uterine anomalies, those under chronic ongoing treatments (corticosteroids, low dose aspirin), and those who smoked >20 cigarettes per day. We also excluded women with any sign of vaginal bleeding, abdominal pain, or any other abnormal sign. Women had repeated ultrasonographs and a single blood test at gestational ages between 7 and 8 wk, and were all observed until 13 wk of gestation. Pregnant women were then examined at 22 and 32 wk of gestation. Gestational age was assessed on the basis of the date of the last normal menstrual period, confirmed through the first crown-rump length measurement. Spontaneous (missed) abortion was defined as the absence of cardiac activity in the embryo, assessed by ultrasonography. All the ultrasound examinations were performed using a 6.5-MHz transvaginal probe and a 3.5-MHz transabdominal probe (AU4 IDEA Esaote, Ansaldo, Milan, Italy). Lymphocyte isolation and assays of FAAH activity, of AEA uptake, and of CP55.940 binding were performed as described above for the in vitro studies. There were no differences in phenotype among lymphocyte preparations used in this study, especially between normal and aborted pregnancies.
Statistical analysis
Data reported in this work are the mean (±SD) of at least three independent determinations, each in duplicate. Statistical analysis was performed by the nonparametric Mann-Whitney test, elaborating experimental data by means of the InStat program (GraphPad, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro treatment of human lymphocytes with P enhanced their FAAH
activity in a dose-dependent manner, while P conjugated with BSA was
ineffective in the same concentration range (Fig. 1
A). FAAH activation reached
statistical significance (p < 0.05) at 250 nM
P and a maximum at 1 µM. Therefore, the last concentration was chosen
to further investigate the effect of P on FAAH. Time-course experiments
showed that P-induced activation of FAAH was significant
(p < 0.05) 12 h after lymphocyte
treatment and reached a maximum at 24 h (Fig. 1B).
Western blot analysis of lymphocyte extracts showed that specific
anti-FAAH Abs recognized a single immunoreactive band of the
molecular size expected for FAAH, the intensity of which increased dose
dependently in P-treated cells (Fig. 2A). Densitometric analysis of
the filter shown in Fig. 2A (representative of triplicate
experiments) indicated that FAAH protein increased to 130, 160, or
220% of the control (100% = 10,000 ± 1,000
U/mm2), in cells treated for 24 h with 250,
750, or 1,000 nM P, respectively. The same anti-FAAH Abs were used
to quantify FAAH content by ELISA, showing that P increased time
dependently FAAH protein in human lymphocytes in parallel to the
increase of enzymic activity (Fig. 1B). Kinetic analysis
showed that FAAH activity toward AEA had apparent maximum velocity
(Vmax) values of 190 ± 20 and
380 ± 35 pmol/min/mg protein, in homogenates of lymphocytes
treated for 24 h with either vehicle or 1 µM P, whereas the
apparent Michaelis-Menten constant
(Km) was unchanged (8 ± 1 µM
in both cases). Moreover, FAAH in untreated lymphocytes was also able
to hydrolyze 2-AG with apparent Km =
7 ± 1 µM and Vmax = 230
± 25 pmol/min/mg protein, whereas PEA was hydrolyzed with apparent
Km = 15 ± 2 µM and
Vmax = 50 ± 5 pmol/min/mg
protein. RT-PCR amplification of cDNA of human lymphocytes showed a
single band of the expected molecular size for FAAH gene, which
increased dose dependently in P-treated cells (Fig. 2B).
Densitometric analysis of the autoradiographic film shown in Fig. 2B (representative of triplicate experiments) indicated that
FAAH mRNA increased to 120, 155, or 190% of the control (100% =
2700 ± 300 U/mm2), in cells treated for
24 h with 250, 750, or 1000 nM P, respectively. Under the same
experimental conditions, the expression of the 18S rRNA gene was
unaffected (Fig. 2B). Liquid scintillation counting of
RT-PCR products showed that P increased time dependently FAAH mRNA in
human lymphocytes, in a way parallel to that of enzymic activity and
protein content (Fig. 1B).
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The mechanism of FAAH up-regulation by P was investigated by using
different substances. In pilot experiments, various doses of each
compound were used, and the results obtained with the most effective
concentrations are shown in this study. Treatment of human lymphocytes
(3 x 106/test) with Th2-type cytokines IL-4
(5 ng/ml) or IL-10 (1 ng/ml) for 24 h increased FAAH activity up
to 230% or 180% of the control, respectively, whereas treatment with
Th1-type cytokines IL-12 (5 ng/ml) or IFN- (0.2 ng/ml) reduced FAAH
activity to approximately 50% of the control (Fig. 3A). FAAH expression at the
protein level, measured by ELISA, paralleled the enzyme activity (Fig. 3A).
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B). Finally, RU486, a synthetic antiprogestinic
compound (30), fully reverted the effect of 1 µM P on
FAAH activity and expression (Fig. 3B), when used at a
concentration of 14 µM (=6 µg/ml) (Fig. 3B). Instead,
hCG did not significantly (p > 0.05) affect
FAAH activity in human lymphocytes, at concentrations up to 400 IU/ml;
neither did 2 µM cortisol. P and cytokines do not affect AEA uptake and cannabinoid receptor binding
Intact lymphocytes were able to accumulate
[3H]AEA in a temperature
(Q10 = 1.6)-, time
(t1/2 = 5 min)-, and
concentration-dependent manner (data not shown), according to a
saturable process with apparent Km =
130 ± 15 nM and Vmax = 75
± 8 pmol/min/mg protein. The uptake of 200 nM
[3H]AEA was almost completely inhibited by 10
µM AM404 (Table I), a specific AEA
transport inhibitor (31). However, treatment of
lymphocytes (3 x 106/test) for 24 h
with P, IL-4, IL-10, IL-12, IFN-, hCG, or cortisol did not quite
affect [3H]AEA uptake (Table I).
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). Treatment of lymphocytes (20 x
106/test) for 24 h with P, IL-4, IL-10,
IL-12, IFN-, hCG, or cortisol did not affect
[3H]CP55.940 binding (Table II).
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Treatment of human lymphocytes (3 x
106/test) with 0.5 µM AEA reduced the
production of LIF to 33% of the untreated control (Table III). This effect of AEA was counteracted
by 0.1 µM SR141716, but not by 0.1 µM SR144528 or by 1 µM CAPS, a
selective antagonist of vanilloid receptors (33),
suggesting that it was mediated by CB1 receptors only (Table III). On
the other hand, coincubation of lymphocytes with 0.5 µM AEA and 10
µM AM404 or 10 µM AACOCF3, a FAAH inhibitor
(34), further reduced the LIF release to 17% of the
untreated controls (Table III). 2-AG also reduced LIF production by
lymphocytes to 42% of the untreated control, when used at the same
concentration as AEA, whereas twice concentrated PEA was ineffective
(Table III).
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In a clinical study, we measured FAAH activity,
[3H]AEA uptake, and
[3H]CP55.940 binding in lymphocytes isolated
from 100 healthy women at 7–8 wk of gestation. This is the earliest
time point in gestation in which the difference between FAAH content in
women who miscarried and those who did not was found to be significant
(11). Ultrasonography showed that approximately 10 days
after the blood tests, 15 subjects miscarried and 85 did not. The a
posteriori association between the gestation outcome and FAAH activity,
AEA uptake, or CP55.940 binding showed that FAAH activity (Fig. 4A) was lower in all the 15
women who miscarried than in the 85 who did not (48 ± 5 vs
133 ± 9 pmol/min/mg protein; p < 0.0001),
whereas AEA uptake (49 ± 4 vs 50 ± 4 pmol/min/mg protein;
p > 0.05) and CP55.940 binding (20,400 ± 1,795
vs 20,380 ± 1,930 cpm/mg protein; p > 0.05) were
similar in both groups (Fig. 4, B and C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A),
suggesting that P had to reach its intracellular receptor (PR) to be
active (35). P stimulates FAAH activity by up-regulating
gene expression at the transcriptional and translational level (Fig. 2, A and B). Therefore, it can be proposed that,
once bound to its receptor, P forms a P/PR complex that enhances the
trancription of FAAH gene. This effect can be either direct, i.e.,
through binding of the P/PR complex to the FAAH gene promoter, or
indirect, i.e., through P/PR-dependent production of a transcription
factor that binds to the promoter. In this context, it seems noteworthy
that a specific steroid/steroid receptor motif has been recently
described in the promoter region of FAAH gene (36), which
may suggest a direct effect of the P/PR complexes on FAAH
transcription. On the other hand, the effect of RU486 (Fig. 3B), a PR antagonist that stabilizes PR in a form unable to
bind DNA (30), is compatible with both hypotheses.
Remarkably, RU486 was effective at a concentration known to modulate
immunoendocrine interactions in early pregnancy of humans
(37). At any rate, the up-regulation of FAAH expression by
P is a major finding of this investigation, associated with higher FAAH
activity.
During pregnancy, P stimulates T lymphocytes to produce Th2-type
cytokines (IL-4 and IL-10), but lowers the production of Th1-type
cytokines (IL-12 and IFN-) (13, 15, 16). In this study,
we show that IL-4 and IL-10 stimulated the activity and protein
expression of lymphocyte FAAH, which were instead inhibited by IL-12
and IFN- (Fig. 3A). Moreover, IL-4, which is known to
mediate the favorable effects of P on pregnancy (14, 15, 16, 17),
also mediated the effect of P on FAAH. In fact, the FAAH up-regulation
was significantly lower when IL-4 was sequestered by sIL-4R or by
anti-IL-4R Abs (Fig. 3B). However, FAAH activity and
protein content remained significantly higher in lymphocytes treated
with P in the presence of sIL-4R or anti-IL-4R than in the
untreated controls, suggesting that IL-4 enhanced, but was only in part
responsible for the effects of P. Physiological concentrations of hCG
were ineffective on FAAH activity, which is in keeping with the
observation that the levels of hCG do not always correlate with the
outcome of pregnancy (22, 23). Consistently, the same
concentrations of hCG used in this study failed to modulate
Th1/Th2-type cytokine production by human lymphocytes
(15). Also the glucocorticoid cortisol failed to induce
FAAH, ruling out that P might act through glucocorticoid receptors
(38).
Human lymphocytes have a specific AEA transporter, and its affinity for
AEA is very close to that of human platelets (39), and
identical to that of human lymphoma (24) and endothelial
cells (21). Therefore, it can be proposed that the same
carrier is present on the surface of these different human blood cells.
Interestingly, P, IL-4, IL-10, IL-12, IFN-, hCG, or cortisol under
the same conditions tested for FAAH activity did not affect AEA
transport (Table I). This finding suggests that the transport was not a
"checkpoint" for AEA degradation during pregnancy. This concept is
in keeping with the notion that AEA is taken up through a facilitated
diffusion mechanism (20, 21, 24, 27) that is driven by the
AEA gradient between the outer and inner side of the plasma membrane,
maintained also by FAAH activity. Lymphocytes express CB1 receptors,
which, however, are not involved in the up-regulation of FAAH by P
(Table II). Indeed, P, IL-4, IL-10, IL-12, IFN-, hCG, or cortisol
were ineffective on CB receptors of lymphocytes, supporting the concept
that degradation by FAAH is the only critical event in controlling the
endocannabinoid level during gestation. The clinical study, showing
that FAAH activity was defective in lymphocytes of women who miscarried
compared with those who did not (Fig. 4A), whereas AEA
transporter and CB receptors were the same in both groups (Fig. 4, B and C), is a further evidence for the critical
role of this enzyme. Moreover, data reported in Fig. 4A
extend to enzyme activity our recent observation on FAAH protein
content in women who miscarried compared with those who did not
(11).
High FAAH activity should lower the level of its substrates. Since it
has been shown that peripheral lymphocytes play a critical role in
human pregnancy by producing LIF (17, 18), we tested
whether the endocannabinoids would affect LIF release from peripheral T
cells. The results, summarized in Table III, show that indeed AEA
reduced LIF release from lymphocytes in a CB1 receptor-mediated manner,
and that increasing the extracellular concentration of AEA by blocking
its uptake or hydrolysis enhanced this effect. In this context, it
should be recalled that AACOCF3 is a powerful
inhibitor of FAAH, which can also inhibit phospholipase
A2 (34), leaving open the
possibility that this latter enzyme might be involved in the effect of
AEA on LIF release. However, the identity between the effect of
AACOCF3 and that of AM404, a specific inhibitor
of the AEA transporter (31), suggests that blockade of AEA
degradation was the critical step, making it unlikely that other
unrelated pathways might be involved. Moreover, the lack of effect of
CAPS rules out that AEA might reduce LIF release acting through
vanilloid receptors (32). Also, 2-AG inhibited LIF
production, while PEA did not (Table III). 2-AG, like AEA, binds to CB1
receptors (2, 3) and can be hydrolyzed by lymphocyte FAAH
as much as AEA (11). On the other hand, PEA was hydrolyzed
by lymphocyte FAAH 10-fold less efficiently than AEA, and its ability
to bind to CB receptors and to have antiinflammatory actions in humans
is still controversial (2, 28). Altogether, these data
suggest that a low FAAH activity, and hence higher AEA and 2-AG levels,
can lead to spontaneous abortion by reducing LIF production. This
unprecedented effect of AEA is consistent with its adverse effects on
embryo implantation and development in mouse (40, 41, 42, 43, 44).
Moreover, keeping in mind the role of LIF in regulating growth and
differentiation of neurons and endothelial cells (19), a
wider implication of the present findings can be anticipated. The
interplay among P, cytokines, FAAH, endocannabinoids, and LIF is
depicted in Fig. 5. It is shown that P,
by interacting with its receptor, increases the synthesis of FAAH,
which in turn reduces the extracellular concentration of AEA by driving
its import through the transporter. In this way, the effect of AEA on
LIF release by binding to type 1 cannabinoid receptors is reduced. FAAH
activation by P is further enhanced by IL-4. This cytokine can also
directly activate FAAH, as does IL-10, whereas IL-12 or IFN- inhibit
FAAH activity.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alessandro Finazzi-Agrò, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via di Tor Vergata 135, I-00133 Rome, Italy. E-mail address: Finazzi{at}uniroma2.it
3 Abbreviations used in this paper: AEA, anandamide (N-arachidonoylethanolamine); AACOCF3, arachidonoyltrifluoromethyl ketone; 2-AG, 2-arachidonoylglycerol; AM404, N-(4-hydroxyphenyl)arachidonoylamide; CAPS, capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide); CB1/2, type 1/2 cannabinoid; CP55.940, 5-(1,1'-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)cyclohexyl]phenol; FAAH, fatty acid amide hydrolase; GAR-AP, goat anti-rabbit Abs conjugated with alkaline phosphatase; hCG, human chorionic gonadotropin; IGF-IR, insulin-like growth factor I receptor; P, progesterone; P-BSA, P 3-(O-carboxymethyl)oxime:BSA conjugate; PEA, N-palmitoylethanolamine; RU486, mifepristone; sIL, soluble IL; SR141716, N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide; SR144528, N-[1(S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide.
Received for publication November 27, 2000. Accepted for publication April 10, 2001.
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) or P-BSA
(
) on FAAH
activity in human lymphocytes, after 24 h of incubation (100% =
140 ± 15 pmol/min//mg protein). B, Time course of
the effect of 1 µM P on FAAH activity (
),
and mRNA level (
) in human lymphocytes (100% = 140 ± 15
pmol/min/mg protein for the activity, or 0.220 ± 0.025 OD units
at 405 nm for the protein content, and 15,000 ± 1,500 cpm for the
mRNA level). In both panels, bars represent SD values. *,
p < 0.05 vs control; **, p
< 0.01 vs control.
,
p < 0.01 vs P-treated lymphocytes.
) or
spontaneous abortion (•). Biochemical data were associated a
posteriori with the outcome of pregnancy.