Published online before print May 22, 2003
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Departments of
* Anatomy and Cell Biology and
Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City; and
Human Genome Sciences, Rockville, Maryland
Correspondence: Joan S. Hunt, Ph.D., Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7400. E-mail: jhunt{at}kumc.edu
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B, osteoprotegerin, CD27, OX40/CD134, AITR). Analysis by reverse transcriptase-polymerase chain reaction revealed mRNAs encoding only three of the ligands (APRIL, BLyS, CD30L/CD153). Immunoblots demonstrated all three proteins in first-trimester and term placentas, and immunohistochemical experiments showed that expression was cell-specific and gestation-related. Although mRNAs encoding receptors for the three expressed ligands were absent, those encoding receptors for all of the unexpressed ligands were detectable. Collectively, the results are consistent with the postulate that nonapoptosis-inducing, placenta-derived TNF superfamily cytokines contribute to the T helper cell type 2 bias required for successful pregnancy. Patterns of placental expression of receptors suggest bidirectional maternal–fetal cytokine communication.
Key Words: maternal–fetal immunology • humoral response • immune privilege
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With the exception of implantation and parturition, pregnancy is characterized by a bias toward antibody production and against cytotoxic T lymphocyte production [8 ]. It is not certain how this deviation is achieved, although the major hormone of pregnancy—progesterone—undoubtedly has a role [9 ]. TNF superfamily members that do not mediate apoptosis could easily contribute, as many are key regulators of immune cell development (reviewed in ref. [10 ]). With the exception of CD30L/CD153 [11 ], it has not been determined whether any of these powerful cytokines or their receptors are expressed at the maternal–fetal interface.
In this study, we analyzed first-trimester and late-gestation human placentas as well as trophoblast cells for transcripts encoding eight nondeath-dealing ligands and nine of their receptors. Immunoblotting was used to verify translation of the specific ligand mRNAs that were detected in placentas, and immunohistochemistry was used to determine cellular locations of the proteins.
These experiments revealed that two ligands that contribute to B lymphocyte maturation and development, a proliferation-inducing ligand (APRIL) and B lymphocyte stimulator [BLyS; also known as B cell-activating factor belonging to the TNF family (BAFF), TALL-1, TNF homologue that activates apoptosis, nuclear factor (NF)-B, and c-Jun NH2-terminal kinase (THANK), and zTNF4] [12
13
14
], and one that promotes T helper cell type 2 (Th2) development [15
], CD30L/CD153, are readily detected in placentas by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry. By contrast, mRNAs encoding five ligands that support T cells and antigen-presenting cells (APC), CD40L/CD154, TNF-related activation-induced cytokine [TRANCE; also known as receptor activator of NF-B ligand (RANKL) and osteoprotegerin ligand (OPGL)], CD27L/CD70, OX40L, and activation-inducible TNF receptor ligand [AITRL; human glucocorticoid-induced TNF receptor ligand (hGITRL)] [16
17
18
19
20
21
], are absent. Thus, placenta-derived, nonapoptosis-inducing TNF superfamily members are in position to contribute to the Th2 bias of pregnancy.
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Primers and RT-PCR analysis
Oligonucleotide primers were designed using the PrimerSelect program (DNASTAR, Inc., Madison, WI) and cDNA sequences available from the National Center for Biotechnology Information (Bethesda, MD) databases. GenoMechanix (Alachua, FL) or Integrated DNA Technologies (Coralville, IA) synthesized primers. All primer pairs are known to span at least one exon–intron boundary each. RT-PCR analysis of total RNA was performed as described previously [5
]. Briefly, 2 µg each RNA sample was treated with DNase I to remove genomic DNA and then was reverse-transcribed in a 40-µl reaction. This first-strand cDNA was then diluted fivefold with sterile water, and 5 µl each diluted sample in a 50-µl reaction volume was subjected to 30 cycles of PCR. The primers used, with optimal annealing temperatures and positions of the nucleotides within the protein encoding region of the cDNA sequence, are listed in Table 1
. OPG primers were as described previously [5
]. Correct sequences of products were confirmed by dRhodamine terminator cycle sequencing using the ABI 310 DNA sequencer (Applied Biosystems, Foster City, CA) in the Center for Reproductive Sciences at the University of Kansas Medical Center.
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Table 1. RT-PCR Primer Pairs and Annealing Temperatures
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isotype control (BD Biosciences PharMingen) was used at the same concentration as the matching primary antibody. Incubation with primary antibody was overnight at 4°C (APRIL, CD30L/CD153) or for 1 h at room temperature (BAFF/BLyS). Species-appropriate, secondary peroxidase-conjugated antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA) and were used at 0.01 µg/ml for 1 h at room temperature.
Immunohistochemistry
Immunohistochemical procedures for paraffin-embedded and frozen tissues were performed essentially as described previously [22
]. Paraffin-embedded tissues after deparaffinization were heated for 20 min at 100°C in citrate buffer (Reveal, Biocare Medical, Walnut Creek, CA). The same primary antibodies used in immunoblots were used for immunohistochemistry. Goat anti-APRIL, rabbit anti-BAFF (BLyS), and their respective control IgGs were used at 20 µg/ml for 1 h at 37°C on paraffin-embedded tissues. Mouse anti-CD153 (CD30L) mAb or mouse IgG1 isotype control was used at 10 µg/ml at 4°C overnight on frozen tissue sections.
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Table 2. TNF Superfamily Ligands and Receptors and Reported Cellular Locations of the Ligands
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Figure 1. Ligand-receptor interactions for nonapoptosis-inducing TNF superfamily members. Receptor–ligand pairs for which bidirectional signaling has been reported are connected by double-headed arrows.
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Figure 2. Analysis of mRNAs encoding nonapoptosis-inducing TNF superfamily ligands by RT-PCR in first-trimester and term human placentas and in villous CTB cells purified from term placentas. Negative-control (NEG; no added cDNA) and positive-control (POS; primer-specific cDNA) lanes are indicated. For APRIL, BLyS, CD30L/CD153, CD40L/CD154, TRANCE, CD27L/CD70, and OX40L, no fewer than two term placentas were examined; for AITRL, one term placenta was examined. For BLyS, two first-trimester placentas were examined; for APRIL, CD30L/CD153, CD40L/CD154, TRANCE, CD27L/CD70, OX40L, and AITRL, one first-trimester placenta was examined. For BLyS, four term CTB preparations were examined; for APRIL, CD30L/CD153, CD40L/CD154, TRANCE, CD27L/CD70, OX40L, and AITRL, one term CTB preparation was examined.
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Parallel PCR analysis of RT-negative controls for all samples and primer pairs failed to yield any products, confirming lack of genomic DNA contamination (data not shown). All amplicons were sequenced and found to be authentic.
Detection of APRIL, BLyS, and CD30L/CD153 proteins in human placentas by immunoblotting
Having determined that transcripts encoding three TNF superfamily ligands, APRIL, BLyS, and CD30L/CD153, were present in total RNA from early and late gestation placentas, immunoblotting was used to determine whether the messages were translated into protein.
Figure 3 shows that although the positive-control cell lines, HeLa and HL-60, yielded detectable signals for APRIL protein, little of this secreted cytokine [32 ] was identified in samples from placentas. Although the 8-week placenta was positive, signal in the 12-week placenta was barely detectable, and no APRIL protein was detected in term placentas by this method. By contrast, BLyS was present in both positive-control cell lines, HeLa and HL-60, as well as in all samples of early and late gestation placentas. CD30L/CD153 was detected in the two positive-control cell lines, and weak signals were detected in some but not all placentas. The molecular weights observed for BLyS and CD30L/CD153 were consistent with those expected for glycosylated monomers; the observed molecular weight for APRIL was higher than expected and more consistent with that of a trimer.
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Figure 3. Detection of APRIL, BLyS, and CD30L/CD153 proteins in human placentas and control cell lines (HeLa, HL-60) by immunoblotting. Three late-gestation (Term) and three first-trimester (6-, 8-, and 12-week) placentas were analyzed. Molecular weight of markers (x103) is indicated on the left.
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Localization of APRIL, BLyS, and CD30L/CD153 proteins to specific cells in human placentas by immunohistochemistry
To determine which placental cells were the source of the APRIL, BLyS, and CD30L/CD153 proteins, we performed immunohistochemistry on paraffin-fixed (APRIL, BLyS) and frozen (CD30L/CD153) tissue sections taken from first-trimester and term placentas (n=at least 3 each). Staining patterns for the three ligands were essentially the same in all placentas of each stage, except as noted, and negative controls (insets in Fig. 4a
4b
4c
4d
4e
4f
) did not demonstrate positive staining.
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Figure 4. Immunohistochemical detection of APRIL (a and d), BLyS (b and e), and CD30L/CD153 (c and f) in human first-trimester (a–c; n=3) placenta and term (d–f; n=3) placenta. The APRIL and BLyS antibodies were tested against sections taken from the same tissue blocks; first trimester was 9 weeks. (a, b, d, e) Staining of paraffin-embedded tissues by antibodies specific for APRIL and BLyS. (c and f) Staining of frozen tissues by an antibody to CD30L; first trimester was 13 weeks. Negative staining by control IgG for each antibody is shown in the inset for each panel. Large arrows point to syncytiotrophoblast; small arrows point to villous CTB cells; arrowheads point to placental villous endothelial cells. (f) Very small arrow points to positive cells in a fetal blood vessel. Original magnifications, x200.
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As illustrated in Figure 4b , BLyS in early placentas was localized to villous CTB cells, particularly the nuclei but also throughout the cells. As with APRIL, BLyS declined as gestation proceeded to termination. The protein was identified in only one of three term placentas, but the positive cells were of the same subpopulation (villous CTB cells) as the positive cells in early placentas, and BLyS protein was in the same location (predominantly cell nuclei). Figure 4e shows these positive cells. These observations are consistent with the results of the immunoblots, where BLyS was readily identified in two of three first-trimester placentas and yielded weaker positive signals in term placentas.
CD30L/CD153 was, as previously reported [11 ], identified in first-trimester as well as term placental villi. The antibody used to localize this ligand was useful only in frozen tissue sections where morphology is not well retained. Yet, it was possible to see that staining in early placentas (Fig. 4c) and term placentas (Fig. 4f) was mainly in villous endothelial cells. CD30L was also present in fetal blood cells. Unlike staining for APRIL and BLyS, staining with CD30L antibody was stronger in term placentas than in first-trimester tissues.
CTB cells synthesize BLyS
The immunohistochemical experiments reported above suggested that BLyS, a B lymphocyte stimulator, which has previously been identified in secondary lymphoid organs and cells as well as placenta (see Table 2
), might be a product of CTB cells. We purified villous CTB cells from four different term placentas and tested their RNAs for BLyS mRNA. Figure 5
shows that consistent with the immunohistochemical results described above, where only one in three term placentas contained immunoreactive BLyS in CTB cells, one of four CTB cell samples contained BLyS mRNA. Thus, villous CTB cells may produce BLyS, but the specific messages and the proteins are often lacking at term. Higher levels in first-trimester CTB cells could not be confirmed because of inadequate numbers of cells.
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Figure 5. Detection of BLyS mRNA in one of four samples of purified term villous CTB cells by RT-PCR. Negative-control (NEG; no added cDNA) and positive-control (POS; primer-specific cDNA) lanes are as indicated.
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Figure 6 shows that transcripts encoding the receptors for APRIL and BLyS (BCMA and TACI) were undetectable in first-trimester and term placentas. We also failed to detect specific messages in term CTB cells. By contrast, CD30, the receptor for CD30L/CD153, was present in early but not late gestation placentas (Fig. 6) . Consistent with this finding, purified, term CTB cells also lacked mRNA for this receptor.
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Figure 6. Detection by RT-PCR of transcripts encoding receptors for the nonapoptosis-inducing TNF superfamily ligands in first-trimester and term placentas and villous CTB cells purified from term placentas. Negative-control (NEG; no added cDNA) and positive-control (POS; primer-specific cDNA) lanes are as indicated. For CD30, CD40, OPG, CD27, and OX40/CD134, no fewer than two term placentas were examined; for BCMA, TACI, RANK, and AITR, one term placenta was examined. For all receptors, only one first-trimester placenta and one term CTB preparation were examined.
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Our studies confirm and expand other reports. Messages encoding both of the TNF superfamily ligands that have been shown to promote B cells, APRIL and BLyS, were detected in first-trimester and term placenta (Fig. 2) , and their proteins were localized to specific types of cells. Our detection of APRIL mRNA in placenta was somewhat surprising as a result of its rarity in normal tissues and reported absence in placentas analyzed by Northern blotting [23 ]. This disparity in results could be a result of the relative sensitivity of RT-PCR and Northern blot hybridization techniques. APRIL protein has not previously been identified in human placentas. By using organ blots, others have detected BLyS mRNA in term placentas, peripheral blood leukocytes, lymphoid tissues, bone marrow, and several other organs and tissues [12 , 13 , 26 ]. We confirmed the observation for term placentas, added detection in early placentas, and demonstrated the presence of the protein at both stages. We here report mRNA encoding CD30L/CD153 in early and late gestation placentas and localization of the protein, thus confirming a previous report [11 ]. Our results are unique, however, in identifying placental endothelial cells as containing this protein.
A second major point to emerge from these experiments was that the three proteins, APRIL, BLyS, and CD30L/CD153, are found in a variety of placental cells. APRIL was mainly localized to the microvilli of syncytiotrophoblast, BLyS was present primarily in villous CTB cells, and CD30L/CD153 was detected in endothelial cells and fetal blood cells. Generally speaking, all of these ligands are believed to be produced mainly in leukocytes (Table 2) . As with previous experiments reported by our laboratory on apoptosis-inducing members of the TNF superfamily, expression of TNF superfamily members is common in unusual cell types such as trophoblasts and immature cells of many types that comprise the placenta [5 , 7 , 33 ].
A third major point emerged from our studies on receptor expression. We eliminated probable binding of APRIL, BLyS, and CD30L/CD153 to placenta cells, which supports the idea that these cytokines may target maternal tissues and/or embryonic tissues. Our results on BCMA confirm an earlier report on term placenta [34 ] and add information on early placenta. Our findings on TACI are entirely novel; this receptor has not been previously investigated in placentas [35 ]. It should be noted that we did not study a recently identified BLyS-specific receptor, BAFF-R, which has been detected only in secondary lymphoid organs and not in any other tissues, including placenta [36 ]. CD30 expression was detected in first-trimester placentas. Although this could be a result of contaminating decidua in the samples [11 , 28 ], our finding remains to be established experimentally.
Finally, our observation that the receptors for nonexpressed ligands are transcribed in placentas suggests a reciprocal pathway where the five ligands are synthesized in maternal and/or fetal tissues and target the placenta.
The consequences of interactions between the placental ligands and receptors on as-yet undetermined cells remain to be explored. APRIL is synthesized as a secreted protein, and BLyS can be processed by furin-like proteinases, so both of these ligands could circulate systemically as biologically active, soluble trimers [13 , 14 , 32 ]. It would be of considerable interest to know if these two ligands are present in maternal blood, where they could target peripheral lymphoid tissues and alter maternal immune responses. APRIL and BLyS are believed to act as costimulators of some T cells. APRIL stimulates proliferation of primary B and T cells purified from mouse spleen and may serve as a costimulatory molecule for T cells involved in the humoral response [14 , 25 ]. In vitro T cell stimulation assays have shown that BLyS acts as a costimulator of T cells but only when the recombinant ligand is immobilized and not in a soluble form [37 ]. The secreted form of BLyS is not believed to stimulate T cells. Neither our experiments nor those of other investigators have revealed whether placental BLyS is membrane-associated or secreted. It is interesting that APRIL and BLyS diminished as gestation progressed to term, suggesting that these cytokines are most useful in establishment of early events in placentation.
CD30L–CD30 interactions are believed to play a role in peripheral tolerance by inhibiting proliferation of cytotoxic T cells and increasing their susceptibility to apoptotic signals (reviewed in refs. [38 , 39 ]). This signaling has been reported to protect against autoimmunity and in some but not all instances, to correlate with Th2-type immune responses [40 ]. Decidua is known to produce the single receptor for CD30L, i.e., CD30 [28 ], but CD30L–CD30 signaling in decidua has not yet been established.
In summary, our detection in placentas of nonapoptosis-inducing TNF family members known to stimulate B cells or limit the action of cytotoxic T cells supports the overall postulate that members of this powerful gene family play important roles in the immunoprotection of the fetus.
Received January 21, 2003; revised March 18, 2003; accepted March 26, 2003.
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production in activated mouse macrophages by progesterone J. Immunol. 160,5098-5104 mRNA and protein are present in human placental and uterine cells at early and late stages of gestation Am. J. Pathol. 139,327-335[Abstract]B, and c-Jun NH2-terminal Kinase J. Biol. Chem. 274,15978-15981This article has been cited by other articles:
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