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Original Article |
Department of Gynecology and Obstetrics, Faculty of Medicine (K.K., H.F., K.T., S.Y., Y.S., T.H., S.F.), Institute for Virus Research (M.U.), and Institute for Frontier Medical Science (M.M.), Kyoto University, Sakyo-ku, Kyoto 606-8507; and Department of Obstetrics and Gynecology (H.E., T.N.), Japan Baptist Hospital, Sakyo-ku, Kyoto 606-8273, Japan
Address all correspondence and requests for reprints to: Hiroshi Fujiwara, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail: fuji{at}kuhp.kyoto-u.ac.jp.
Abstract
To investigate immune-endocrine interactions between the embryo and the mother early in pregnancy, we examined the effects of human chorionic gonadotropin (HCG) on IL-8 production by peripheral blood mononuclear cells (PBMC). Recombinant HCG promoted IL-8 secretion by PBMC derived from nonpregnant women. The induction of IL-8 mRNA expression was observed after 30 min of HCG stimulation. Adsorption of the HCG with anti-HCG antibodies confirmed the specificity of this effect. The translocation of nuclear factor 
B into the nucleus and subsequent IL-8 production were observed mainly in monocytes, and IL-8 production was reduced when a proteasome inhibitor was added to inactivate nuclear factor B. Although fluorescein isothiocyanate-labeled HCG was bound to the majority of monocytes, cell surface expression of HCG receptor was hardly detected. IL-8 production by HCG was not affected by inhibitors of protein kinases A and C. In contrast, this stimulation was attenuated by D-mannose, which inhibits binding to C-type lectins. The basal IL-8 production by PBMC from women early in pregnancy was significantly elevated, compared with that from nonpregnant women.
This study showed that human monocytes respond to HCG and secrete IL-8 through a pathway different from the HCG receptor system, suggesting that this glycoprotein hormone can react with not only endocrine cells but also immune cells early in pregnancy, probably via primitive systems such as C-type lectins.
HUMAN CHORIONIC GONADOTROPIN (HCG) is a placental glycoprotein hormone that is mainly produced by the syncytiotrophoblasts in the chorionic villi. In a normal pregnancy, HCG is detectable in maternal serum as early as 1 d after the initiation of embryo implantation. Then HCG rapidly rises to a peak value of more than 100,000 mIU/ml around 60–90 d gestation and gradually decreases to levels under 50,000 mIU/ml within the second trimester (1). The main role of HCG is considered to stimulate the corpus luteum in the ovary to produce progesterone and maintain embryo implantation (2). Recently, we showed that not only HCG but also peripheral blood mononuclear cells (PBMC) derived from women early in pregnancy promote progesterone production by luteal cells, suggesting that PBMC play an important role in the endocrine system during early pregnancy (3, 4). On the other hand, on the basis of immunological considerations, it had been suspected that HCG induces immunological tolerance of the maternal immune system against trophoblasts. In 1973, crude HCG purified from urine was reported to suppress immune reactions (5). Subsequent studies demonstrated that contaminating factors other than HCG were responsible for these effects, and highly purified HCG had no effect on lymphocyte function (6, 7, 8). Thereafter, the secretion of several cytokines from PBMC in vitro was shown to be modified by HCG stimulation (9, 10, 11). In those studies, however, the authors did not examine the specificity of HCG or the effects of contaminating factors in the HCG preparation. In addition, the cell surface expression of LH/HCG receptor on PBMC has not yet been demonstrated, and it is still unclear whether or not HCG can affect PBMC function.
Recently, we found that PBMC derived from women early in pregnancy promoted spreading and invasion of murine embryos in vitro. In addition, culturing of PBMC derived from nonpregnant women in the presence of HCG, which is one of the most important embryonal hormonal signals, increased the ability of the PBMC to promote murine embryo invasion. This finding suggests that HCG modulates the secretion by PBMC of soluble factor(s) that promote embryo implantation (12). To identify the HCG-induced factors responsible for promoting embryo invasion, biologically active molecules known to be secreted from PBMC, including various growth factors and cytokines, were screened using RT-PCR. Then, IL-8 mRNA expression in PBMC was revealed to be remarkably increased by HCG. IL-8 is a neutrophil chemoattractant/activating factor that was first reported to be produced by lipopolysaccharide-stimulated monocytes and is now well known to be expressed in a wide variety of cells, including lymphocytes (13, 14). IL-8 has been shown to stimulate the proliferation of several cell types and the migration of endothelial cells (15, 16, 17). Because it is important to clarify the functional alterations of PBMC by HCG stimulation with respect to both endocrine and immune functions, we further examined the precise mechanism by which recombinant HCG (r-HCG) promoted IL-8 production in PBMC. The specificity of the effects of HCG on PBMC function was also analyzed by specifically adsorbing HCG with anti-HCG antibodies (Abs). In addition, we investigated the cell populations responsible for IL-8 production and their expression of HCG receptor to elucidate whether or not HCG stimulation of PBMC is mediated by the LH/HCG receptor.
Materials and Methods
Reagents
r-HCG (10,000 IU/mg), which is produced from mouse fibroblast cell line LM(TK-), was obtained from Rhoto Pharmaceutical Co. Ltd. (Osaka, Japan). A rabbit anti-HCG polyclonal Ab and control rabbit Ig were purchased from DAKO Corp. (Glostrup, Denmark). A mouse anti-HCG monoclonal Ab (mAb; clone 094-10627, IgG1 class) was purchased from OEM Concepts Inc. (Toms River, NJ). Phycoerythrin (PE)-labeled or nonlabeled antihuman CD2, CD14, and CD20 mouse mAbs and isotype-matched mouse negative control mAbs were purchased from PharMingen (San Diego, CA). Fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse Ig (DAKO Corp.) was used as secondary Ab for the immunohistochemistry. Mouse antihuman LH/HCG receptor mAb (3B5) was a generous gift from Dr. J. Wimalasena (Abramson Family Cancer Research Institute, Philadelphia, PA; Ref. 18). The murine anti-trinitrophenyl mAb (IgG1; Ref. 19) was used as negative control.
PBMC culture
PBMC were prepared as described previously (3). Volunteers were recruited from healthy nonpregnant women (secretory phase, cycle d 16–24; n = 35) with a regular menstrual cycle and pregnant women (4 or 5 wk gestation, n = 7; 6–11 wk gestation, n = 8; second trimester, n = 6; third trimester, n = 6). Informed consent for the use of PBMC including gene expression analysis was obtained from all participants. The analysis of PBMC function was approved by the Ethical Committee of Kyoto University Hospital. PBMC were isolated from 20 ml of venous blood by centrifugation using Ficoll-Hypaque and were suspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Dainippon Pharmaceutical Co., Osaka, Japan), 100 U/ml penicillin, and 100 µg/ml streptomycin. PBMC (1 x 106 cells/ml) were cultured in a 24-well tissue culture plate (Becton Dickinson and Co.Labware, Franklin Lakes, NJ) in triplicate at 37 C in a humidified atmosphere of 5% CO2 in air for 48 h in the presence or absence of r-HCG (0, 10, 100, 1,000, 10,000, or 100,000 mIU/ml). In some experiments, PBMC were cultured with r-HCG at concentrations of 0, 10, 50, 100, 500, or 1000 mIU/ml. In other experiments, a proteasome inhibitor, MG-132 (5 µM, Calbiochem-Novabiochem Co. La Jolla, CA) was added to the PBMC culture in the presence or absence of r-HCG (10,000 mIU/ml). A protein kinase A inhibitor, adenosine 3', 5'-cyclic monophosphothioate, Rp-isomer (0, 5 x 10-5 M, or 5 x 10-4 M; Wako Pure Chemical Industries Ltd., Osaka, Japan), and/or a protein kinase C inhibitor, bisindolylmaleimide 1 (0, 10-6 M, or 10-5 M; Calbiochem-Novabiochem Co.), was also added to the PBMC culture in the presence or absence of r-HCG (10,000 mIU/ml). After a 48-h incubation, the culture medium was collected for IL-8 ELISA analysis.
To investigate the effects of longer continuous stimulation on PBMC by HCG, PBMC were cultured for 4 or 6 d under one of the following three conditions: 1) PBMC were cultured without HCG for 6 d; 2) PBMC were cultured without HCG for an initial 2 or 4 d and were then incubated in the presence of HCG (10,000 mIU/ml) during the following 2 d; or 3) PBMC were cultured in the presence of HCG (10,000 mIU/ml) for 6 d. The culture medium was changed and collected every 2 d for IL-8 ELISA analysis.
To examine the effects of monosaccharides, 1 h before they were cultured, PBMC were incubated with D-mannose, D-glucose, D-galactose, L-fucose, N-acetylglucosamine (GlcNAc), or N-acetylgalactosamine (GalNAc; 0, 1 x 10-6 M, 1 x 10-5 M, or 1 x 10-4 M; Sigma Chemical Co., St. Louis, MO), and then they were cultured for 48 h in the presence or absence of HCG (10,000 mIU/ml). At the end of this culture period, the medium was collected and subjected to IL-8 ELISA.
Monocyte-macrophage preparation from PBMC
Separation of monocytes from PBMC was performed using macrophage-separating plates (Japan Immunoresearch Laboratories Co. Ltd., Takasaki, Japan) according to the instruction manual. Flow cytometric analysis of the cell fractions obtained by this method showed that more than 85% of the monocyte-rich population consisted of CD14-positive cells, and the contamination of CD14-positive cells in the lymphocyte-rich population was less than 1%. The monocyte-rich (1 x 105 cells/ml) and the lymphocyte-rich (1 x 106 cells/ml) populations were cultured in the presence or absence of r-HCG (10,000 mIU/ml) for 48 h. In addition, the concentration of IL-8 in the culture medium was measured by ELISA.
Human granulosa cell culture
To assess the biological activity of r-HCG, human granulosa cells were isolated from eight patients undergoing in vitro fertilization treatment as described previously (4). The separated granulosa cells were cultured in 96-well plates in RPMI supplemented with 10% FCS. The next day, unattached cells were removed by gentle washing, and the attached granulosa cells were cultured for 6 d in the presence or absence of r-HCG (0, 0.5, 1, 5, 10, or 100 mIU/ml). Culture media were changed every 2 d, and the final media after 6-d culture were collected and frozen at -20 C until they were subjected to RIA to measure the progesterone concentration.
RNA isolation
To analyze IL-8 mRNA expression, PBMC were first cultured for 24 h without HCG and were then incubated in the presence or absence of HCG (10,000 mIU/ml) for 0, 15, or 30 min or 1, 2, 3, 6, 12, 24, or 48 h. The PBMC were collected, immediately frozen in liquid nitrogen, and stored at -80 C until RNA extraction. The total RNAs in these cells were isolated by the TRIzol method using a commercial kit (Life Technologies, Inc., Rockville, MD). The culture medium was also collected and subjected to ELISA for IL-8. RNA was similarly extracted from freshly isolated human granulosa cells and PBMC.
RT-PCR analysis
Five micrograms of total RNAs from PBMC were reverse-transcribed with random primers using a commercial kit (First Strand cDNA Synthesis kit; Pharmacia, Inc., Piscataway, NJ). The resulting cDNA mixtures were subjected to 25 cycles of PCR amplification with oligonucleotides from the human IL-8 cDNA as primers (Ref. 13 ; sense primer, 5'-AACTTTCAGAGACAGCAGAG-3': position 15–34; antisense primer, 5'-TACAACAGACCCACACAATA-3': position 482–501) or with human S26 primers (Ref. 20 ; sense primer 5'-GGTCCGTGCCTCCAAGATGA-3': position 8–27; antisense primer, 5'-TAAATCGGGGTGGGGGTGTT-3': position 308–327). After PCR amplification, 10 µl of each PCR product were electrophoresed on a 1% agarose gel and stained by ethidium bromide. For detection of LH/HCG receptor, the cDNA mixture derived from freshly isolated PBMC and granulosa cells was also subjected to RT-nested PCR according to the method of Lin et al. (21) with some modifications. Briefly, first-round PCR was carried out for 30 cycles, and one tenth of the product of the first-round PCR was then subjected to 30 cycles for the second-round PCR. The nucleotide sequences of the primers used for PCR detection of LH/HCG receptor were as follows: the sense and antisense primers for first-round PCR were 5'-GCATCTGTAACACAGGCATC-3': position 394–413, and 5'-CATCTGGTTCAGGAGCACAT-3': position 1034–1053, respectively; and the sense and antisense primers for second-round PCR were 5'-GCAGAAGATGCACAATGGAG-3': position 638–657, and 5'-CTCTCAGCAAGCATGGAAGA-3': position 960–979, respectively (22).
Adsorption of r-HCG by anti-HCG Ab-conjugated gel
To confirm the specificity of the effect of r-HCG on IL-8 production by PBMC, r-HCG was adsorbed from the working solution (where noted in Results) before use. Anti-HCG polyclonal Ab (DAKO Corp.) or unrelated rabbit Ig (negative control, DAKO Corp.) were conjugated to Affigel-10 (Bio-Rad Laboratories, Inc., Hercules, CA) as previously described (23). The r-HCG working solution was preincubated with anti-HCG Ab-conjugated or negative control Ab-conjugated gel for 2 h at 4 C. This procedure was repeated twice. Each solution that had been adsorbed with anti-HCG Ab or negative control Ab was sterilized by microfiltration and added to the PBMC culture at a dilution that would yield a concentration of 10,000 mIU/ml if the r-HCG were not removed. After the cultures were incubated for 48 h, the media were collected and subjected to ELISA analysis for IL-8. The r-HCG working solution was also adsorbed using anti-HCG mAb (clone 094-10627) or negative control mAb-conjugated Affigel-10, and the effects of the resultant solutions on IL-8 production by PBMC were examined as described above.
Preparation of FITC-labeled HCG
r-HCG (500 µg) was dissolved in H2O (100 µl) and buffered with 25 µl 1M Na2CO3/NaHCO3 (pH 9.5) at 4 C. This solution was incubated with 45 µg FITC isomer 1 (BBL Microbiology Systems, Cockeysville, MD) in dimethylsulfoxide (DMSO; 10 µg/µl) overnight. Additional incubation with 20 µl 0.2 M lysine for 2 h was then performed to abolish the binding activity of FITC. Then, the FITC-conjugated r-HCG was dialyzed and diluted with PBS to a concentration of 100 µg (1000 IU)/ml.
Flow cytometry
PBMC (1 x 106) were sedimented and incubated at 4 C for 30 min with 10 µl antihuman LH/HCG receptor mAb (3B5; 100 µg/ml) or negative control mAb (100 µg/ml). The cells were washed twice with Hanks’ balanced salt solution and incubated with 20 µl FITC-conjugated rabbit antimouse Ig (diluted 1:40) for 30 min at 4 C in the dark. After washing, PBMC were incubated with negative control mAb again for 30 min to neutralize the second antibody. Then, the washed PBMC were incubated with 10 µl PE-conjugated anti-CD2 mAb (clone RPA-2.10; 100 µg/ml), anti-CD14 mAb (clone M5E2; 100 µg/ml), anti-CD20 mAb (clone 2H7; 100 µg/ml), or PE-conjugated negative control mAb (100 µg/ml) for 30 min. Double-stained PBMC were then analyzed using a fluorescence-activated cell sorter (FACS; Becton Dickinson and Co.). The human granulosa cells isolated as described above were also stained with antihuman LH/HCG receptor mAb (3B5; 100 µg/ml) and analyzed using a FACS.
To examine whether HCG can bind to the cell surface of PBMC, 10 µl FITC-conjugated r-HCG (1000 IU/ml) was incubated with sedimented PBMC (1 x 106) for 30 min at 37 C in the dark. The PBMC were then incubated with PE-conjugated anti-CD14 mAb or negative control mAb. For inhibition assays, an excess of unlabeled r-HCG (10 µl of 1,000 IU/ml, 10,000 IU/ml, or 100,000 IU/ml) was added to the first incubation with FITC-conjugated r-HCG. In some cases, to examine the effects of D-mannose on the binding of r-HCG to CD14-positive monocytes, PBMC were incubated with FITC-conjugated r-HCG (1000 IU/ml) in the presence of D-mannose (0, 1 x 10-6 M, 1 x 10-5 M, or 1 x 10-4 M). FITC-conjugated swine antirabbit Ig (DAKO Corp.) was used as a negative control. After incubation with PE-conjugated anti-CD14 mAb or PE-conjugated negative control mAb, the binding of FITC-conjugated r-HCG to CD14-positive monocytes was analyzed using a FACS. Each of these inhibition assays was repeated three times.
Assays of IL-8, progesterone, and HCG concentrations
The concentration of IL-8 was measured using an ELISA kit (BioSource International, Inc., Camarillo, CA), and those of progesterone and HCG were determined using RIA kits obtained from Daiichi Radioisotope Research, Inc. (Tokyo, Japan; Ref. 24) and CIS Diagnostic Co. Ltd. (Sakura, Japan), respectively.
The intra-assay and interassay variations for IL-8, progesterone, and HCG are 2.6% and 5.5%, 5.1% and 3.3%, and 2.9% and 4.9%, respectively. The minimal detectable values for IL-8, progesterone, and HCG are 5 pg/ml, 0.3 ng/ml, and 0.5 mIU/ml, respectively. IL-8 ELISA kit has no cross-reactivity with IL-1, -2, -3, -4, -6, -7, -10, or other cytokines including IFN-
and -
, TNF-, and TGF-ß. Progesterone RIA kit has cross-reactivity with 11-OH-progesterone (72.9%) and corticosterone (6.6%), but not with other steroid hormones such as pregnenolone, 17-OH-progesterone, cortisol, estradiol, and testosterone. The cross-reactivity of the HCG RIA kit for TSH, LH, or FSH is less than 5%.
Immunocytochemistry
For detection of nuclear factor (NF) B localization in the cytoplasmic or nuclear region of monocytes, monocyte-rich cell fractions were cultured in an 8-well chamber slide (Lab-Tec, Nunc Inc., Naperville, IL) for 24 h without HCG, and then they were incubated in the presence or absence of HCG (10,000 mIU/ml) for 0, 5, 15, or 30 min, or 1 h. The cultured cells were fixed with methanol and indirectly stained using anti-NFB mAb (5 µg/ml, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or the negative control mAb (5 µg/ml) and then incubated with FITC-conjugated rabbit antimouse Ig second antibody (diluted 1:40). The slides were washed, mounted, and observed using a confocal laser scanning microscope (Carl Zeiss Inc., Jena, Germany). Four independent experiments were performed.
Statistics
The concentrations of progesterone and IL-8 and the mean fluorescence intensities detected by flow cytometry were expressed as means ± SEM, and their differences were analyzed by one-way ANOVA, followed by Scheffé’s F test. The differences were considered to be significant at P value less than 0.05.
Results
IL-8 production by PBMC in 2-d cultures
The IL-8 production by PBMC obtained from women at 4 and 5 wk gestation (n = 7) was significantly higher than that by PBMC obtained from nonpregnant women (n = 22), pregnant women at 6–11 wk gestation (n = 8), or pregnant women in the second trimester (n = 6) and third trimester (n = 6; Fig. 1
).
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Treatment of PBMC obtained from nonpregnant women with r-HCG for 48 h increased IL-8 production in a dose-dependent manner. A significant enhancement by r-HCG was observed at concentrations of at least 10,000 mIU/ml (Fig. 2A). No significant increase of IL-8 production was observed at concentrations of 500 mIU/ml or less. At the concentration of 1000 mIU/ml, a tendency to enhance IL-8 production was observed, but the effect was not significant (Fig. 2B).
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). On the other hand, a small amount of production of IL-8 was observed from 1 h after r-HCG administration (Fig. 2D). After 3 h, a clear increase of IL-8 production was detected, and the concentration continued to increase until at least 24 h, in agreement with the above profile of IL-8 mRNA expression.
Enhanced IL-8 production was observed in PBMC that had been cultured in the absence of r-HCG for 2 or 4 d and then treated with r-HCG (n = 4). The HCG-stimulated IL-8 production by these PBMC was significantly higher than the production by PBMC that were incubated with r-HCG from the beginning of the culture period (Fig. 2E).
Effects of r-HCG on progesterone production by human granulosa cells
r-HCG at concentrations of at least 5 mIU/ml significantly enhanced progesterone production by granulosa cells during 6-d cultures (n = 8), confirming that r-HCG has the previously reported biological activity (Fig. 3A).
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The r-HCG preparation that had been adsorbed with unrelated rabbit Ig or mouse negative control mAb significantly promoted IL-8 production by PBMC obtained from nonpregnant women as compared with the control (PBMC cultured with no reagent). On the other hand, when PBMC were incubated with r-HCG preparations that had been adsorbed with anti-HCG polyclonal (n = 3) or monoclonal (n = 4) Abs, the IL-8 production was significantly reduced to the same level as the control (Fig. 3, B and C). The concentrations of the working solutions adsorbed with negative control polyclonal and monoclonal Abs were 3600 mIU/ml and 2300 mIU/ml, respectively, as shown by evaluation with an RIA kit for HCG. On the other hand, the HCG concentrations of the working solutions that had been adsorbed with the two anti-HCG Abs were less than 2 mIU/ml.
PBMC population responsible for IL-8 production
IL-8 production was detected in the monocyte-rich population, and this production was enhanced by HCG stimulation. On the other hand, IL-8 production of the lymphocyte-rich population was very low, and no significant difference was observed between the cell populations with or without HCG stimulation (Fig. 4A). When monocyte-rich and lymphocyte-rich cells were cultured together, the IL-8 production was similar to that observed with the monocyte-rich population alone.
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, B–E).
Nuclear translocation of NFB by HCG stimulation
Immunocytochemical staining revealed that NFB was diffusely distributed in the cytoplasm of the monocytes in the absence of r-HCG. On the other hand, NFB could be detected in the nuclear region in some cells after 30 min of HCG stimulation. After 1 h, NFB was observed in the nuclear region in the majority of the cells, indicating that NFB was translocated into nucleus as a result of HCG stimulation (Fig. 5, A and B).
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In the presence of proteasome inhibitor MG-132, the HCG-stimulated IL-8 production by PBMC was significantly reduced (Fig. 6A). On the other hand, HCG-stimulated IL-8 production was not inhibited in the presence of the protein kinase A inhibitor adenosine 3', 5'-cyclic monophosphothioate, Rp-isomer, and/or the protein kinase C inhibitor bisindolylmaleimide 1 (Fig. 6B).
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The LH/HCG receptor was detected by flow cytometry at high levels on human granulosa cells, whereas it was hardly detected on CD14-positive cells (Fig. 7, A and B). Similarly, two fragments corresponding to the cDNA of LH/HCG receptor were detected in human granulosa cells by RT-PCR. The large fragment was verified to be identical to the full-length LH/HCG receptor cDNA by determining its sequence, whereas the smaller fragment corresponded to the exon 9-deleted variant (25, 26). In contrast, neither band was detected in RT-PCR analysis of PBMC derived from nonpregnant women (Fig. 7C).
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Although treatment with various monosaccharides did not affect the basal production of IL-8, the IL-8 production stimulated by HCG (10,000 mIU/ml) was significantly inhibited by D-mannose in a dose-dependent manner (Fig. 8A; n = 7). On the other hand, no significant inhibitory effects of HCG-stimulated IL-8 production were observed in the presence of D-glucose (n = 5), D-galactose (n = 5), L-fucose (n = 5), GlcNAc (n = 5), or GalNAc (n = 5; Fig. 8, B–F).
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By flow cytometry, the binding of FITC-conjugated r-HCG to CD14-positive PBMC was shown to be affected by D-mannose (Fig. 9). The partial inhibitory effects of D-mannose were observed at a concentration of 1 x 10-5 and 1 x 10-4 M (mean fluorescence intensities were 49.3 ± 9.5 and 44.0 ± 10.7, respectively) as compared with control incubation without D-mannose (111 ± 13.3; P < 0.05).
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This study demonstrated that high doses of r-HCG promoted IL-8 production by PBMC. The effective dose of HCG was more than 10,000 mIU/ml, which is almost 2,000-fold higher than the effective dose for LH/HCG receptor activity in the ovary. Previous studies showed that HCG inhibited IL-2 production and increased IL-1ß, IL-6, and TNF- production by PBMC (9), and that HCG significantly enhanced interferon production by PBMC when calcium ionophores were used as inducers (10). HCG was also reported to diminish IL-2 secretion and enhance the release of soluble IL-2 receptor from PBMC (11). In those reports, the effective dose of HCG was more than 10,000 mIU/ml, and the authors did not examine the biological activity or specificity of the HCG hormones that they used for the studies. Schmidt et al. (8) reported that crude HCG fractions with very low gonadotropic activity extensively inhibited mitogenic-induced or allogenic-induced lymphocyte transformation, but a highly purified fraction with strong biological activity had no inhibitory effect on lymphocyte reactions, suggesting that HCG had no specific effect on immune cell function. By dialysis treatment of purified HCG, it was also shown that other uncharacterized compounds that were partially copurified from pregnant urine along with HCG have immunoregulatory activity (6). In addition, Morse et al. (7) reported that some commercial batches of crude HCG that are purified from pregnant urine contain a mitogen for human peripheral blood lymphocytes.
In this study, to reduce artifacts due to contamination, we used r-HCG and observed that HCG at the concentrations higher than 1000 mIU/ml was necessary to enhance IL-8 production. The effect of r-HCG on human luteinizing granulosa cells was also examined to assess the biological activity of r-HCG, and r-HCG was shown to significantly stimulate progesterone production by granulosa cells at a concentration as low as 5 mIU/ml. This indicates that r-HCG has biological activity that is compatible with the activity previously reported. Because high concentrations of r-HCG were required to stimulate IL-8 production, we performed experiments to further rule out the possibility that some contaminating factor(s) in the r-HCG preparation affected the IL-8 production by PBMC. After the r-HCG preparation was pretreated with anti-HCG polyclonal and monoclonal Abs, the promoting effect of the r-HCG preparation on IL-8 production disappeared. These findings demonstrated that the promoting effect on IL-8 production by PBMC was specifically caused by r-HCG and was not due to other contaminating factor(s). Thus, we conclude that HCG can affect PBMC function.
The concentrations of HCG that are necessary for IL-8 production are higher than the serum level of the midcycle LH surge. From the very early stage of embryo implantation, a high HCG concentration can be locally achieved at the implantation site. When the embryo is completely buried in the endometrium, the syncytiotrophoblasts develop the labyrinth around the embryo (27, 28, 29). In these sites, PBMC encounter a high concentration of HCG, and this may induce PBMC to produce IL-8. In support of this speculation, the basal production of IL-8 by PBMC was significantly increased in women at 4 and 5 wk gestation as compared with nonpregnant women. Except for these very early stages of pregnancy, the level of basal production of IL-8 by PBMC obtained from pregnant women is similar to that produced by PBMC of nonpregnant women. Although the precise mechanisms are not clear, this is compatible with the finding that the response of PBMC to HCG gradually decreased during in vitro culturing of PBMC.
To determine whether IL-8 production is directly or indirectly induced by HCG, the time course of IL-8 production by PBMC was analyzed by RT-PCR and ELISA in the presence of HCG. The adherence of PBMC to plastic or extracellular matrix-coated dishes was reported to induce the expression of IL-8 mRNA and production of IL-8 (30). In this study, IL-8 mRNA expression was demonstrated to be transiently increased when PBMC were separated from blood and transferred to in vitro culture. Therefore, the time course of stimulation by HCG was analyzed after a 24-h preincubation in the absence of HCG. Under these conditions, the expression of IL-8 mRNA could be detected after 30 min of HCG stimulation. An increase of IL-8 production was also detected from 3 h after HCG administration. At the same time, translocation of NFB from the cytoplasm into the nucleus was observed in monocytes/macrophages. Because NFB is well known to mediate transcription of IL-8 mRNA (31), it is reasonable to postulate that HCG induces the transcription of IL-8 mRNA at least partially through NFB activation, resulting in increased IL-8 production in PBMC. This speculation is supported by the finding that HCG-stimulated IL-8 production by PBMC was significantly suppressed by MG-132, which inhibits proteasome enzyme activity and thereby keeps NFB inactive and associated with IB.
HCG is considered to transmit its hormonal signal to target cells through the LH/HCG receptor, which was originally reported to be expressed on ovarian granulosa cells, theca interna cells, and luteal cells. After the nucleotide sequence of LH/HCG receptor cDNA was determined, various lines of evidence showed that the LH/HCG receptor is also expressed in the endothelium (32) and myometrium (33). In 1995, Lin et al. (21) reported that mRNA of the LH/HCG receptor was detected by RT-PCR in T lymphocytes derived from the peripheral blood of pregnant women. In the present study, the monocyte-rich cell population, which was separated from PBMC on the basis of its adhesive properties, was shown to respond to HCG by producing IL-8. We then examined the binding of HCG to monocytes using FITC- conjugated r-HCG. The labeled r-HCG was bound to more than 80% of CD14-positive monocytes, and this binding was blocked by unlabeled r-HCG in a dose-dependent manner. However, apparent expression of LH/HCG receptor was not observed on the cell surface of CD14-positive monocytes by flow cytometry. In addition, RT-PCR failed to detect expression of LH/HCG receptor mRNA in the PBMC obtained from nonpregnant women. These findings indicate a discrepancy between HCG-binding and LH/HCG receptor distribution on monocytes. In addition, HCG-stimulated IL-8 production by PBMC was not affected by the presence of inhibitors of protein kinase A and/or protein kinase C, which are well known to be involved in signal transmission via the LH/HCG receptor (34). Considering that the translocation of NFB into the nucleus was observed in the majority of monocytes after HCG stimulation, despite their lack of LH/HCG receptor expression, it can be concluded that some alternative pathway other than the LH/HCG receptor pathway is involved in the PBMC response to HCG stimulation.
Recently, pituitary hormones such as LH and TSH were reported to interact with mannose receptor (35), which is a cell surface lectin and is well known as a phagocytosis-related receptor on macrophages (36). It was proposed that immune cells are involved in the clearance of these hormones (35). This receptor recognizes glycoconjugates terminating in mannose, fucose, or GlcNAc in a Ca2+-dependent manner via C-type carbohydrate-recognition domains (37). Therefore, we examined whether cell surface lectins play a role in the HCG-stimulated IL-8 production by PBMC. The addition of mannose did not affect the basal IL-8 production, but significantly suppressed HCG-stimulated IL-8 production. Furthermore, mannose interfered with the binding of HCG to CD14-positive monocytes. These findings suggest that cell surface lectins play a role in the HCG-signaling pathway. The so-called mannose receptor was reported to appear on monocyte/macrophage cell lineage during the differentiation process from monocytes to macrophages. In our preliminary experiments, mRNA of mannose receptor was not detected in freshly isolated PBMC or PBMC cultured for 2 d, but was clearly detected on PBMC cultured for more than 6 d (data not shown), suggesting that the so-called mannose receptor was not involved in the findings in this study. However, mannans that are complex polymers containing mannose also showed enhancement on IL-8 production (our unpublished data). Therefore, we speculate that undefined lectin receptors other than the so-called mannose receptor recognize mannose on HCG and play a key role in IL-8 production by PBMC.
HCG hormone has the following two characteristics. First, HCG possesses four N-linked glycosylation sites and four O-linked glycosylation sites (38). Second, a large amount of HCG is rapidly produced by chorionic villi in the first trimester. Taking these characteristics into account, it is possible that PBMC can respond to HCG hormone through a primitive pathway involving lectins in vivo. Very recently, Kovalevskaya et al. (39) reported that a hyperglycosylated form of HCG arises very early in pregnancy and is rapidly replaced by less glycosylated isoforms, showing that hCG glycoforms dynamically change as pregnancy progresses. This finding provides one possible explanation of why the basal production of IL-8 by PBMC is increased very early in pregnancy, then returns to a level similar to that produced by PBMC of nonpregnant women. The presence of an embryo in the maternal reproductive tract is the most important information for the mother, who must prepare herself for the subsequent events early in pregnancy. Among mammals, only primates and a few equines have this hormone (40). In this regard, it can be said that HCG is a highly evolved hormone that effectively elicits both endocrine and immune reactions in primates. The embryo may transmit the information about its presence to maternal immune systems using highly glycosylated HCG hormone, which mimics some product produced in bacterial infections. The involvement of glycosylation in the signaling pathway may explain the discrepancies among the previous reports concerning the effects of purified HCG on immune cells, because HCG purified from urine may have been considerably deglycosylated to various extents. Thus, the physiological roles of HCG hormone not only in the endocrine system, but also in the immune system should be investigated further with respect to aspects other than the so-called LH/HCG receptor.
In conclusion, the present study demonstrated that peripheral blood monocytes are able to respond to HCG at high concentrations by enhancing their production of IL-8. This study also showed that system(s) different from the LH/HCG receptor system are responsible for this effect of HCG on immune cells. In addition, IL-8 production was elevated in PBMC derived from women early in pregnancy. These findings suggest that an immune-endocrine network involving HCG and peripheral blood immune cells exists and plays an important role in early pregnancy.
Acknowledgments
We thank Ms. M. Oshima for technical assistance. We also thank Dr. T. Takahashi for valuable advice.
Footnotes
This work was supported in part by Grants-in-Aid for Scientific Research (no. 12470342, 13557140, 13671709, and 13671710).
Abbreviations: Ab, Antibody; DMSO, dimethylsulfoxide; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; HCG, human chorionic gonadotropin; mAb, monoclonal Ab; NF, nuclear factor; PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; r-HCG, recombinant HCG; RPMI, Roswell Park Memorial Institute.
Received March 4, 2002.
Accepted August 7, 2002.
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