Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.0108067 on August 6, 2008

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(Journal of Leukocyte Biology. 2008;84:1346-1352.)
© 2008 by Society for Leukocyte Biology

Chorionic gonadotropin up-regulates long pentraxin 3 expression in myeloid cells

Hui Wan*,1, Cornelia G. van Helden-Meeuwsen*, Cecilia Garlanda{dagger}, Lonneke M. E. Leijten*, Virginia Maina{dagger}, Nisar A. Khan*, Hemmo A. Drexhage*, Alberto Mantovani{dagger}, Robbert Benner* and Marjan A. Versnel*

* Department of Immunology, Erasmus MC, Rotterdam, The Netherlands; and
{dagger} Istituto Clinico Humanitas, Milan, Italy

1 Correspondence: Department of Immunology, Erasmus MC, Dr. Molewaterplein 50, NL-3015 GE Rotterdam, The Netherlands. E-mail: h.wan{at}erasmusmc.nl


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ABSTRACT
 
Pentraxin 3 (PTX3) is an acute-phase response protein that initiates innate immunity against diverse microorganisms. It is produced in response to proinflammatory stimuli by many cell types including myeloid cells. Increased serum levels of PTX3 are found in pregnancy, a condition characterized by increased serum levels of the pregnancy hormone human chorionic gonadotropin (hCG). As myeloid cells bear the receptor for hCG, we hypothesized that hCG can promote innate immunity by affecting the PTX3 production by myeloid cells. In this paper, we demonstrate that hCG increases PTX3 expression by human monocytes, mouse dendritic cells, and mouse macrophages in vitro. This increased PTX3 expression by hCG is mediated via the protein kinase A signaling pathway. hCG injection in mice also increases the PTX3 serum levels. This serum PTX3 is produced mainly by blood monocytes, which from pregnant women, express more PTX3 compared with nonpregnant controls. The hCG-induced hormones progesterone and estrogen also increase the PTX3 production by human monocytes. In conclusion, hCG increases innate immunity via induction of PTX3 in myeloid cells.

Key Words: hCG • PTX3 • monocytes • M{varphi} • DC • pregnancy • innate immunity


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INTRODUCTION
 
Pentraxins are a superfamily of proteins with cyclic pentameric structure, including the short pentraxins C-reactive protein and serum amyloid P component and prototypic long PTX3, which is also called TNF-stimulated gene 14 and is highly conserved in evolution. PTX3 is produced mainly by monocytes, macrophages (M{varphi}), and dendritic cells (DC), as well as epithelial cells and fibroblasts, in response to inflammatory signals such as LPS, TNF-{alpha}, and IL-1β [1 2 3 ].

The function of PTX3 has not been fully unraveled, but there are suggestions that PTX3 has a dual role: the protection against pathogens and the control of autoimmunity [4 ]. First, PTX3 behaves as an acute-phase response protein that initiates innate immunity against some specific microorganisms. PTX3 levels increased rapidly and dramatically during endotoxic shock, sepsis, and other inflammatory and infectious conditions and correlated with the severity of the disease [4 , 5 ]. PTX3 activates the classical complement pathway by binding to immobilized C1q and facilitates extracellular pathogen recognition by M{varphi} and DC [4 ]. Moreover, overexpression of PTX3 results in increased resistance, and PTX3 deficiency causes increased susceptibility to specific microorganisms [4 5 6 ]. In PTX3-deficient mice, no generalized impairment of host resistance to microbial pathogens is observed.

Second, PTX3 binds specifically to apoptotic cells and inhibits self-antigen recognition by DC. PTX3, however, does not influence the presentation of exogenous, soluble antigens, an event required for immunity against extracellular pathogens [7 , 8 ].

These functions of PTX3 are of great importance during pregnancy, where PTX3 is expressed in the placenta in amniotic epithelium, chorionic mesoderm, trophoblast terminal villi, and perivascular stroma. PTX3 serum levels were found to rise steadily during normal gestation and peaked during labor [9 ]. Implantation and trophoblast invasion are characterized by a progressive and continuous induction of apoptosis in the maternal tissue surrounding the fetus [10 , 11 ]. The removal of apoptotic cells is crucial to the resolution of local inflammation and prevents exposure of self-antigens. The above data suggest that PTX3 is an important enhancer of the innate immune system, which is essential in the host protection against infections and maintenance of a successful pregnancy [12 ].

During pregnancy, there are important changes in the production of hormones such as human chorionic gonadotropin hormone (hCG), which is a placental glycoprotein mainly secreted by trophoblasts that peaks at approximately 300 U/ml in blood with wide variations among pregnant individuals [13 ]. The most well-known function of hCG is to induce the production of progesterone (P4) and estrogen (estradiol, E2). The hCG level peaks in the first trimester of pregnancy and then declines to a stable level in the remaining second and third trimesters, while progesterone and estrogen continue to increase until the end of the pregnancy [14 15 16 17 ]. Luteinizing hormone (LH)/hCG receptors have been found in several tissues and cell types, including ovary, fallopian tube, endometrium, and resident tissue M{varphi} [12 , 18 ]. The effect of hCG on cells of the immune system has hardly been studied.

Earlier, we showed that hCG enhances innate immunity by stimulating M{varphi} functions, among others, the phagocytic capacity [19 ]. Interestingly, an improved phagocytic activity is observed in M{varphi} from PTX3-overexpressing mice. Furthermore, we recently found an altered PTX3 mRNA expression in hCG-exposed DC (unpublished results). Therefore, we hypothesized that by induction of PTX3 production by myeloid phagocytic cells, hCG contributes to the increased phagocytosis and the protection against infection during pregnancy. Here, we studied the effect of hCG on PTX3 production by monocytes, M{varphi}, and DC in vitro and in vivo.


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MATERIALS AND METHODS
 
Isolation of human monocytes, cell culture, and stimulation
Human monocytes were isolated from peripheral blood. Briefly, blood was diluted 1.5 times with PBS containing 0.1% BSA (BioWhittaker, Verviers, Belgium), layered on a Ficoll gradient (1.077 g/ml; Pharmacia, Uppsala, Sweden), and centrifuged (2280 rpm, 15 min, room temperature). The cells at the interface were collected, washed, layered on a Percoll gradient (1.063 g/ml, Pharmacia), and centrifuged (1900 rpm, 40 min, room temperature). The monocytes were collected from the interface and washed, and a final purity was reached of 85–95%. Monocytes were cultured in 12-well plates (Nunc A/S, Roskilde, Denmark) in a concentration of 2 x 106 cells/well in 2 ml RPMI 1640 (BioWhittaker), supplemented with 10% FCS (Hyclone Perbio, Brevieres, France) overnight. For stimulation, control PBS, LPS 50 ng/ml (Sigma Chemical Co., St. Louis, MO, USA), or human (h)IL-1β 20 ng/ml (R&D Systems, Minneapolis, MN, USA) was added, with or without hCG 300 U/ml (Pregnyl®; Organon, Oss, The Netherlands) for 4 h. Then, the cells were collected, counted, and used for RNA isolation. From pregnant females (average age: 22.67±3.14), the monocytes were isolated at first and third trimesters and used directly for RNA isolation.

Two subsets of human monocytes CD16+ and CD16– populations were isolated by using magnetic beads coated with anti-CD16 mAb (Miltenyi Biotec, Auburn, CA, USA). Then, the cells were incubated with PBS or hCG (300 U/ml) for 48 h, followed by supernatant collection for PTX3 ELISA.

Human monocytic cell line MonoMac6 (MM6) was a kind gift from Prof. Silvano Sozzani (University of Brescia, Italy). MM6 cells were cultured in RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin (BioWhittaker), 1% nonessential amino acids (Gibco, Eggenstein, Germany), 1% sodium pyruvate (Gibco), and 1% sodium chloride (solvent for Pregnyl®). The cells were collected into 12-well plates in concentrations of 2 x 106 cells/well in 2 ml culture medium and stimulated with LPS 50 ng/ml or PBS, with or without different concentrations of hCG (10, 50, 150, 300, and 600 U/ml). Polymyxin B 50 µg/ml (Sigma Chemical Co., Milan, Italy) was added to study a possible LPS contamination. To inhibit the intracellular protein kinase A (PKA) signaling pathway, 10 µM H-89 (Biomol, Plymouth Meeting, PA, USA) was applied. Stimulation experiments with progesterone (P4) 100 ng/ml (Sigma Chemical Co., St. Louis, MO, USA) or estrogen (E2) 100 ng/ml (Sigma Chemical Co.) were performed in the absence or prescence of hIL-1β 20 ng/ml. After 48 h incubation, the culture supernatants were collected and used for further cytokine measurement by ELISA.

Mice
Specific pathogen-free C57BL/6, female mice were purchased from Harlan (Horst, The Netherlands). The mice were 8 weeks of age when killed. All experiments were performed with the approval of Erasmus University Animal Welfare Committee (Rotterdam, The Netherlands).

Murine cell culture and stimulation
Murine bone marrow-derived DC (BMDC) and bone marrow-derived M{varphi} (BMDM) were generated by culturing BM cells in Petri dishes (Becton Dickinson, Le Pont De Claix, France) at a concentration of 2 x 106 live cells per Petri dish in 10 ml RPMI 1640 containing 10% FCS, 1% penicillin/streptomycin, and 20 ng/ml recombinant mouse (rm)GM-CSF (Biosource International, Camarillo, CA, USA) or 10 ng/ml rmM-CSF (PeproTech, Rocky Hill, NJ, USA) in a 37°C, 5% CO2 incubator. At Day 3, another 10 ml medium with rmGM-CSF or rmM-CSF was added to the cultures.

On Day 5 or 6, BMDM or BMDC were collected by using a cell scraper (Corning Inc., Corning, NY, USA) and stimulated with LPS 50 ng/ml in the absence or presence of hCG 300 U/ml. After incubation, the cells were collected, counted, and used for RNA isolation.

Similar as described above for human monocytes, murine monocytes and lymphocytes were isolated and cultured with or without hCG 300 U/ml for 5 h at a concentration of 1 x 106 cells/well in 2 ml culture medium in 12-well plates, followed by cell collection for RNA isolation.

Mouse serum collection
C57BL/6 mice were injected i.p. with hCG (500 U/20 g body weight) or PBS in a volume of 200 µl as used before [20 , 21 ]. After 5 h, the mice were killed, and blood was pooled for serum collection.

PTX3 ELISA
ELISA kits for the detection of hPTX3 and mPTX3 were used according to the protocols described earlier [22 ]. For detection of hPTX3, ELISA plates (96-well, MaxiSorp, Nunc A/S) were coated with 100 µl rat anti-hPTX3 mAb MNB4 in coating buffer (PBS plus 0.05% Tween 20), followed by overnight incubation at 4°C. After washing, the plates were incubated for 2 h at room temperature with 5% milk powder diluted in coating buffer to block the nonspecific binding. After three washes, 100 µl rhPTX3 standards (diluted in PBS+2% BSA) or cell supernatant were added for 2 h at 37°C. Plates were washed, and 100 µl biotin-labeled polyclonal rabbit anti-PTX3 antibody was added for 2 h at 37°C. After three washes, streptavidin-HRP was added for 1 h at room temperature. Plates were washed and then incubated with 100 µl chromogen tetramethylbenzidine for 30 min, followed by the addition of 2 M H2SO4 (50 µl/well). Absorbance values were read at 450 nm by an ELISA reader (Thermo Labsystems, Vantaa, Finland).

For detection of mPTX3, the protocol was similar as for hPTX3 but with the following antibodies and standards: rat anti-mPTX3-coating mAb 2C3, rmPTX3 standards, and biotin-labeled, anti-mPTX3 antibody 6B11.

Quantitative real-time PCR (qRT-PCR)
Collected cells were lysed in RLT buffer, followed by total RNA extraction using an RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the protocol supplied by the manufacturer. cDNA was synthesized using the Superscript First Strand synthesis system (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Mouse qRT-PCR was performed using an ABI 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA) and Taqman probe-based chemistry. Primers for hPTX3 (Hs00173615_m1) and mPTX3 (Mm00477267_g1) and ABL were supplied by Primer ExpressTM (Applied Biosystems). Each PCR sample was run in duplicate. The mean value of the two reactions was defined as representative for the sample. The resulting PTX3 comparative threshold (CT) values were corrected relative to an ABL CT. For interpretation of results, we have used the following equation for the figure: 2 (ABL–PTX3) x 100%. Therefore, an increase is proportional to an increase in expression of the particular target gene.

Statistical analyses
Data are expressed as mean values ± SEM in all figures. Statistical analyses were performed using Student’s paired t-test or Mann-Whitney test. P values <0.05 were considered significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.


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RESULTS
 
hCG increases PTX3 expression by human monocytes
Human monocytes were incubated in vitro with different stimuli for different time periods to determine the kinetics of the induction of PTX3, and PTX3 mRNA expression rose from 2 h and peaked at 6 h after LPS stimulation and 4 h after IL-1β stimulation (Fig. 1A ). These results are in line with data described earlier for monocytes and DC [1 , 23 , 24 ]. The addition of hCG significantly increased PTX3 mRNA expression by nonstimulated human monocytes. No effect on PTX3 expression was observed in LPS or IL-1β-stimulated cells (Fig. 1B) .


Figure 1
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  		Figure 1. PTX3 expression by human monocytes upon hCG treatment. (A) Human monocytes were isolated from peripheral blood and stimulated with LPS or IL-1β for 0, 2, 4, 6, and 8 h. Then, the cells were collected for RNA isolation. A representative of three individual experiments is shown. (B) Human monocytes were stimulated with the above stimulation ± hCG for 4 h, and then PTX3 qRT-PCR was performed (n=4). The equation is: 2 (ABL–PTX3) x 100%. (C) Human monocytes and the human monocytic cell line MM6 were stimulated with LPS, with or without hCG for 48 h. Then, the culture supernatants were collected to measure PTX3 protein production by ELISA. hCG increased the PTX3 expression by unstimulated monocytes but not by LPS-stimulated monocytes. The data shown were relative to non-hCG treatment (human monocytes: n=6; MM6: n=3).

To study the PTX3 protein production, primary human monocytes and the human monocytic cell line MM6 were stimulated with hCG for 2 days. hCG significantly enhanced PTX3 production by unstimulated human monocytes and MM6 cells. No effects on PTX3 protein production by LPS-stimulated cells incubated with hCG were observed (Fig. 1C) . As PTX3 was described originally as a TNF-{alpha}-inducible gene, the expression of TNF-{alpha} was studied. hCG addition did not influence TNF-{alpha} production by monocytes. Therefore, the hCG-induced PTX3 expression in human monocytes appeared not to be mediated by TNF-{alpha} (data not shown).

The increased PTX3 expression by hCG is dose-dependent and PKA pathway-mediated
During normal pregnancy, the hCG levels vary from 20 to 300 U/ml but can be much higher than 300 U/ml in some individuals [13 ]. Therefore, we tested the effect of different concentrations of hCG (ranging from 0 to 600 U/ml) on PTX3 expression by MM6 cells. The PTX3 protein expression by MM6 cells upon hCG treatment increased dose-dependently (Fig. 2A ). In humans, two major monocyte subsets are distinguished: the "immature" CD14+CD16– monocytes and "mature" CD14lowCD16+ monocytes. The latter subset is considered to give rise to different APC subsets [25 ]. CD16+ and CD16– monocytes were isolated using CD16 beads and analyzed separately for the induction of PTX3 by hCG. Both subsets increased the PTX3 protein expression to a similar level after 48 h incubation with hCG (Fig. 2B) .


Figure 2
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  		Figure 2. Increased PTX3 expression by hCG is dose-dependent and mediated by the PKA signaling pathway. (A) MM6 cells were stimulated with different concentrations of hCG (0, 10, 50, 150, 300, and 600 U/ml) for 48 h. hCG increased their PTX3 production in the culture supernatants in a dose-dependent manner. (B) CD16+ or CD16– human monocytes were stimulated with PBS or hCG (300 U/ml) for 48 h. These two monocyte subsets produced similar amount of PTX3 upon hCG treatment. (C) MM6 cells were stimulated with LPS, with or without hCG (300 U/ml), or hCG, with or without polymyxin B (Poly-B)/H-89, for 48 h. Then, the PTX3 protein production in the culture supernatants was determined by ELISA. LPS increased PTX3 expression, which was inhibited by polymyxin B. The increased PTX3 expression by hCG can only be inhibited by H-89 but not by polymyxin B (n=3).

hCG binding to its receptor activates the intracellular cAMP/PKA signaling pathway [26 , 27 ]. We have shown that the increased ROS production induced by hCG in M{varphi} is mediated via the PKA signaling pathway [28 ]. To study whether the induction of PTX3 by hCG was also medicated by PKA signaling, the PKA inhibitor H-89 was added together with hCG. H-89 significantly reduced the increased PTX3 production by MM6 cells upon hCG treatment (Fig. 2C) . The cell counts after different culture conditions were the same, suggesting the viability of cells remained unchanged (data not shown).

Low concentrations of LPS can induce PTX3 expression by monocytic cells [29 ]. To exclude that the observed effect was a result of LPS contamination within the hCG batch used, polymyxin B, an antibiotic known to inhibit the biological activity of LPS, was used. Polymyxin B inhibited the PTX3 production induced by LPS but did not change the PTX3 production induced by hCG (Fig. 2C) . Furthermore, recombinant hCG induced PTX3 expression by the monocytic cell line MM6 to a similar level as purified hCG, and the addition of H-89 also blocked this increased PTX3 production (data not shown).

Progesterone and estrogen increase PTX3 expression by IL-1β-stimulated human monocytes
To study the effect of hCG-inducible hormones on PTX3 expression, human monocytes were stimulated with P4, E2, or P4 or E2 in combination with IL-1β. E2 and P4 significantly increased PTX3 mRNA expression by IL-1β-stimulated cells (data not shown). To further study the PTX3 protein production, MM6 cells were stimulated with LPS, hCG, P4, E2, or P4 or E2 in combination with IL-1β. As shown above, LPS, hCG, and IL-1β increased the PTX3 production by MM6 cells. Stimulation with E2 alone did not change PTX3 production. P4 slightly increased PTX3 production, but the result was not statistically significant. E2 and P4 addition to IL-1β increased the PTX3 production by MM6 cells compared with cells stimulated with IL-1β only (Fig. 3 ).


Figure 3
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  		Figure 3. PTX3 expression by MM6 cells upon progesterone and estrogen treatment. MM6 cells were stimulated with PBS, LPS, hCG, progesterone, estrogen, and IL-1β, with or without progesterone/estrogen for 48 h. Then, the culture supernatants were collected for PTX3 protein determination. Progesterone and estrogen increased PTX3 expression by IL-1β-stimulated MM6 cells (n=3).

hCG increases PTX3 expression by murine BMDC and BMDM
DC and M{varphi} are generally believed to be derived from monocytes. DC and M{varphi} are capable of producing PTX3. The induction of PTX3 expression by murine DC and M{varphi} upon hCG treatment was investigated. BMDC and BMDM responded to LPS by PTX3 mRNA expression (Fig. 4 ). The PTX3 expression level varied among monocytes, DC, and M{varphi} (data not shown), which is in line with the previous findings that DC are better producers of PTX3 than monocytes and M{varphi} [23 ]. BMDC stimulated with LPS showed a PTX3 mRNA expression peak at 4 h (Fig. 4A) . hCG treatment for 4 h increased PTX3 mRNA expression by unstimulated BMDC but not significantly by LPS-stimulated BMDC (Fig. 4B) . A maximum induction of PTX3 mRNA by LPS was observed in BMDM at 2 h (Fig. 4C) . hCG treatment increased PTX3 mRNA expression in unstimulated BMDM. PTX3 expression in LPS-stimulated BMDM was not affected by hCG treatment (Fig. 4D) .


Figure 4
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  		Figure 4. PTX3 expression by murine BMDC and BMDM upon hCG treatment. BMDC (A) and BMDM (C) were stimulated with PBS or LPS for 0, 2, 4, 6, and 8 h. Then, the cells were collected for RNA isolation and PTX3 qRT-PCR. LPS significantly increased PTX3 mRNA expression by BMDC and BMDM, but BMDC expressed 100 times more PTX3 mRNA than BMDM. mRNA was calculated according to the equation: 2 (ABL–PTX3) x 100%; x100 was applied to shorten the zeros after the mentioned equation number. A representative of three individual experiments is shown. When hCG was added to the cells for 4 h, hCG increased PTX3 mRNA expression of unstimulated and LPS-stimulated BMDC (B), but only unstimulated BMDM and not the LPS-stimulated BMDM increased PTX3 mRNA expression (D; n=3).

Blood monocytes contribute to the increased serum PTX3 upon hCG injection and in pregnancy
It has been shown that pregnancy is accompanied with an increased PTX3 level in the peripheral blood [9 ]. To investigate the relationship between hCG and PTX3 production by monocytes in vivo, mice were injected with hCG, followed by collection of blood 5 h later and measurement of serum PTX3 protein levels. hCG injection significantly increased the PTX3 protein level in the serum (Fig. 5A ). To investigate whether blood monocytes contribute to the increased PTX3 production upon hCG administration, mouse monocytes were isolated after hCG injection. Monocytes from hCG-treated mice revealed a higher PTX3 mRNA expression than monocytes from nontreated mice (Fig. 5B) . To study if other cell types in the blood contribute to the observed, increased PTX3 serum level, murine monocytes and lymphocytes were isolated from PBMC and stimulated separately with hCG for 5 h in vitro. PTX3 mRNA measurement revealed that monocytes expressed PTX3 and increased their PTX3 expression upon hCG treatment in vitro. Lymphocytes did not express PTX3 and did not respond to hCG treatment in vitro (Fig. 5C) .


Figure 5
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  		Figure 5. Blood monocytes contribute to the increased serum PTX3 level upon hCG injection and in pregnancy. (A) C57BL/6 mice were injected i.p. with hCG or PBS. Five hours later, mice were killed, and peripheral blood was collected for serum PTX3 protein measurement. hCG injection increased the PTX3 protein level in the serum (n=20). (B) The blood monocytes from hCG i.p.-treated mice were isolated and analyzed for PTX3 mRNA expression by qRT-PCR. (C) Murine blood monocytes and lymphocytes were isolated from untreated mice and cultured with hCG in vitro for 5 h. Then, the PTX3 mRNA expression by these cells was measured. Monocytes showed increased PTX3 expression after hCG stimulation in vivo and in vitro. The data are pooled from two separate experiments; each experiment was performed with 20 mice. (D) Human blood monocytes were isolated from nonpregnant (control) and pregnant females of the first or third trimesters of pregnancy. Cells were then analyzed for PTX3 mRNA expression by qRT-PCR (n=11).

Furthermore, in the first trimester when hCG serum levels are the highest during pregnancy, human blood monocytes express more PTX3 mRNA compared with nonpregnant controls (Fig. 5D) . This increased PTX3 mRNA expression by monocytes dropped in the third trimester but still stayed higher than control blood monocytes, although the difference is not statistically significant (Fig. 5D) .


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DISCUSSION
 
Pregnancy has been proposed to be a state of controlled inflammation [30 ] with increased innate immunity characterized by the activation of M{varphi} and granulocytes and the complement system [31 32 33 ]. PTX3 is produced by innate immune cells including monocytic cells in response to inflammatory mediators, such as IL-1β, TNF, and bacterial products [1 2 3 , 34 ]. Earlier, we have shown that hCG influences the innate immune effector functions of M{varphi} directly [28 ]. M{varphi} increased their oxygen radical production and phagocytosis of apoptotic cells upon hCG treatment. This effect of hCG on cells of the innate immune system led us to hypothesize that hCG also modulates PTX3 expression by myeloid cells and as such, further contributes to the increased innate immunity during pregnancy. Therefore, we studied the effect of hCG on PTX3 production by monocytes, DC, and M{varphi} in vitro and in vivo.

In the present study, we showed that hCG treatment induced increased PTX3 expression, at the mRNA and protein level, by unstimulated but not LPS-stimulated monocytes, DC, and M{varphi}. This is likely important during pregnancy, where PTX3 was found to be elevated locally as well as systemically [9 ]. LPS mimics an inflammatory situation; hCG did not increase PTX3 expression by LPS-stimulated cells, suggesting it does not play a harmful role to worsen the inflammation, whereas hCG increased PTX3 expression by unstimulated cells, suggesting an increased innate immunity during pregnancy.

PTX3 is generated in peripheral tissues under the control of inflammatory stimuli and in particular, is involved in the response against a selective set of microbes [34 ]. Binding of PTX3 to immobilized C1q activates the classical complement pathway and facilitates extracellular pathogen recognition by M{varphi} and DC [4 , 35 ]. This is evident from the observation that M{varphi} and DC from PTX3-deficient mice did not recognize conidia of pulmonary aspergillosis, resulting in increased susceptibility [5 ]. Furthermore, rPTX3 binds to zymosan and Paracoccidioides brasiliensis and functions as an opsonin, thereby increasing the phagocytic activity of peritoneal M{varphi} [35 ]. M{varphi} from PTX3-overexpressing mice have an improved phagocytic activity toward zymosan and P. brasiliensis [35 ]. Together with our findings, these data provide evidence that hCG-induced PTX3 expression in myeloid cells contributes to maintain a robust innate immune system against infections and thereby, contributes to successful pregnancy.

It should be noted that fluid-phase binding of PTX3 to C1q inhibits complement activation by blocking relevant interaction sites. C1q increases the phagocytosis of apoptotic cells by phagocytic cells. PTX3 binds in the fluid phase to C1q, decreasing C1q deposition and subsequent complement activation on apoptotic cells [36 ]. Implantation and trophoblast invasion are characterized by a progressive and continuous induction of apoptosis in the maternal tissue surrounding the fetus [10 , 11 , 37 , 38 ]. Extensive cell death/tissue necrosis represents a challenge for the immune system, as autoantigens are released in a context in which antigen presentation is favored. PTX3 binds selectively to late apoptotic and necrotic cells [8 ] and edits the cross-presentation of epitopes expressed by apoptotic cells to T lymphocytes. This leads to potential substrates for cross-presentation without enforcing a maternal immunization against the embryo [7 ]. PTX3 specifically concentrates at the phagocyte synapse between dying and DC, where it actively restricts the activation of T cells specific for apoptotic cell-associated antigens, including cytoskeleton components, tumor-associated antigens, and viral glycoproteins [7 ]. In the developing placenta, PTX3 possibly has a key role in quenching the cross-presentation of antigens of the embryo to maternal T lymphocytes, thus preventing alloimmunization.

We further showed that the increased PTX3 expression by hCG is mediated by the PKA signaling pathway, which suggests hCG receptor involvement. This is consistent with our previous finding that the increased radical production and phagocytosis of M{varphi} upon hCG treatment were also mediated by the PKA signaling pathway [28 ]. In line with our findings are publications from Aggarwal and co-workers [39 , 40 ] showing that hCG binding to the hCG receptor increased the cAMP level and PKA activity. Meanwhile, former studies from Mantovani and co-workers [34 ] showed that there are several pathways involved in PTX3 induction, including TNF, IL-1β, TLR ligands, and others. Here, we addressed a new pathway for the induction of PTX3: the PKA signaling pathway. Taken together, these data suggest that PTX3 induction is mediated via multiple pathways by multiple cells, all of which contribute to an enhanced innate immunity.

To support our hypothesis that hCG affects PTX3 expression by monocytes in vivo, we injected mice i.p. with hCG, followed by the isolation of monocytes. Indeed, monocytes expressed more PTX3 mRNA upon hCG treatment. We further proved that only monocytes, not lymphocytes, exhibited an increase in PTX3 expression upon hCG treatment. This induction of PTX3 expression in monocytes was accompanied by an increase in PTX3 serum levels. It is further proved that in human pregnancy, blood monocytes express more PTX3 mRNA compared with nonpregnant controls. Moreover, blood monocytes from the first trimester express more PTX3 mRNA than blood monocytes from the third trimester, which parallels with hCG kinetics during pregnancy. However, we did not observe a direct correlation between PTX3 and hCG serum level from women in the first trimester of pregnancy (data not shown). Several possibilities may account for this: individual differences including inflammatory conditions, blood pressure, body weight, age of pregnancy, and pregnancy number; contribution by other cell types; and unidentified regulatory factors, apart from hCG, in the blood of pregnant women may also influence the PTX3 level, such as progesterone and estrogen.

hCG levels decrease in the second and third trimester of pregnancy, and other hormones induced by hCG, such as progesterone (P4) and estrogen (estradiol, E2), continue to increase and stay high until the end of the pregnancy [14 15 16 17 ]. Upon interaction of hCG with the LH/hCG receptor (or cross-interact with the follicle-stimulating hormone receptor), the corpus luteum starts to secrete progesterone and estrogen. Interestingly, P4 and E2 can increase PTX3 production by IL-1β-stimulated monocytes. Different from hCG, P4 or E2 does not increase PTX3 but only in collaboration with IL-1β, suggesting the possible interaction between those factors in the induction of PTX3 production. The above observation suggests that the whole period of pregnancy is characterized by increased PTX3 levels, first induced by hCG and later induced by hCG-induced hormones. These increased PTX3 levels contribute to the increased innate immunity in pregnancy.

In this study, we observed that hCG increases PTX3 expression by monocytic cells in vivo and in vitro. Furthermore, the hCG-induced hormones, such as progesterone and estrogen, can also increase the PTX3 production by inflammatory monocytes. In conclusion, hCG can enhance innate immunity via induction of PTX3 in myeloid cells, as well as via hCG-induced hormones such as progesterone and estrogen.


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ACKNOWLEDGEMENTS
 
This study was supported financially by Biotempt B.V. (Koekange, The Netherlands) and the European Network of Excellence on Embryo Implantation Control Project (LSHM-CT-2004-512040) from the European Commission. We thank Tar van Os for his support in preparation of the figures and Dr. Wim A. Dik and Professor Frank H. de Jong for their helpful discussion and suggestions.

Received January 25, 2008; revised June 8, 2008; accepted July 2, 2008.


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