The placenta utilizes both active and passive mechanisms to evade rejection by the maternal immune system. Recently, the mRNA for two newly cloned members of the B7 family of immunomodulatory cell-associated proteins have been identified in the human term placenta. In this article, we review the current knowledge of the B7 family member B7-H1, and discuss how it may participate in modulation of the maternal immune system at the maternal-fetal interface. B7-H1 has been found to possess immunostimulatory or immunoinhibitory properties, and immunohistological examination of first trimester and term placenta has revealed that this protein is abundant in the placenta. B7-H1 is highly expressed by both the syncytiotrophoblast and extravillous cytotrophoblast, both of which lie in direct contact with maternal blood and tissue. Further, treatment of the choriocarcinoma cell line, JEG-3, with recombinant human interferon (IFN)-gamma resulted in a dose-dependent increase in the abundance of the message for B7-H1, suggesting that IFN-gamma could regulate expression of B7-H1 by the trophoblast. These studies document that the positioning of B7-H1 at the maternal-fetal interface is such that it could participate in suppression of activated maternal leukocytes.

The human placenta utilizes both active and passive mechanisms to evade rejection by the maternal immune system. We investigated the pattern of expression of the B7 family of immunomodulatory molecules B7-H1 (PD-L1), B7-2 (CD86), and B7-1 (CD80) at the term maternal-fetal interface. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that B7-H1 mRNA is abundant in term placenta and that cytotrophoblasts are sources of this message. Immunohistochemistry demonstrated that B7-H1 is constitutively expressed by the syncytiotrophoblast and by extravillous cytotrophoblasts, both of which are juxtaposed to maternal blood and tissue. By contrast, placental stromal cells, including macrophages, lacked the protein. Expression of B7-H1 protein was low in first-trimester placenta compared to second- and third-trimester tissue (P < 0.05) and was enhanced in cultured cytotrophoblasts by treatment with either interferon-gamma or epidermal growth factor (P < 0.05), suggesting that one or both of these mediators regulates B7-H1 expression in the placenta. RT-PCR and immunofluorescence analysis of term placental tissue revealed different patterns of expression of the immunostimulatory protein, B7-2. In contrast to B7-H1, B7-2 mRNA and protein were absent in cytotrophoblast cells but present in maternal macrophages and some fetal macrophages. The B7-1 mRNA and protein were absent at the maternal-fetal interface. These studies document expression of the B7 family proteins at the maternal-fetal interface and demonstrate that B7-H1 is positioned such that it could facilitate protection of fetal cells against activated maternal leukocytes. Conversely, B7-2 was absent on trophoblasts and was appropriately localized to fetal and maternal macrophages, which may participate in antigen presentation.

The HLA-G message is alternatively spliced into multiple transcripts, two of which encode soluble isoforms. To initiate studies on the specific functions of the soluble isoforms, we produced soluble rHLA-G1 (rsG1) and rsG2 in human embryonic kidney 293 cells and characterized the proteins. Both isoforms were glycosylated and formed disulfide-bonded oligomers. Recombinant sG1 associated with beta(2)-microglobulin, whereas rsG2 did not. Mouse mAb generated to rsG1 (1-2C3), which identified exclusively sG1, and mAb generated to rsG2 (26-2H11), which identified both soluble and membrane G2 (m/sG2), were used for inummohistochemical isoform mapping studies on placental tissue sections. Soluble G1 protein was abundant in many subpopulations of trophoblast cells, whereas m/sG2 protein was present exclusively in extravillous cytotrophoblast cells. Although both isolated placental villous cytotrophoblast cells and chorion membrane extravillous cytotrophoblast cells contained mRNAs encoding sG1 and sG2, protein expression was as predicted from the immunostains with m/sG2 present only in the invasive trophoblast subpopulation. Analysis of function by Northern and Western blotting demonstrated that both rsG1 and rsG2 inhibit CD8alpha expression on PBMC without changing CD3delta expression or causing apoptotic cell death. Collectively, the studies indicate that: 1) both sG1 and m/sG2 are produced in placentas; 2) transcription and translation are linked for sG1, but not G2; 3) expression of G2 is exclusively associated with the invasive phenotype; and 4) the two isoforms of sG may promote semiallogeneic pregnancy by reducing expression of CD8, a molecule required for functional activation of CTL.

Human placentae and two of the cell types in placentae (cytotrophoblasts and macrophages) were examined by RT-PCR for transcripts of the eight TNF superfamily ligands known to induce death of activated immune cells, tumour cells, and virus-infected cells (TNFα, LTα, LTβ, FasL, TRAIL, TWEAK, LIGHT, 4-1BBL). Transcripts for all ligands were detected in term placenta but LTα and 4-1BBL were not detected in first trimester placenta. Although term cytotrophoblasts contained mRNAs specific for TNFα, LTα, TWEAK, and 4-1BBL, messages encoding LTβ, FasL, TRAIL, and LIGHT were absent. In term placental macrophages, messages for all ligands except 4-1BBL were present. Transcripts for the 14 receptors to which the ligands bind, six of which contain death-domains (TNFR1, Fas, DR3, DR4, DR5, DR6), were also identified using RT-PCR. Term and first trimester placentae contained transcripts for all receptors except 4-1BB. Although term cytotrophoblasts lacked receptor mRNA encoding 4-1BB and OPG, term placental macrophages lacked DcR1 and OPG. Detection of nearly all the death-inducing TNF superfamily ligands and their receptors in human placentae implies that these powerful cytokines contribute to programmed or activated cell death in this organ.

Mechanisms accounting for protection of the fetal semiallograft from maternal immune cells remain incompletely understood. In other contexts, interactions between TRAIL (TNF-related apoptosis-inducing ligand/Apo-2L) and its receptors kill activated lymphocytes. The purpose of this study was therefore to investigate the potential of the TRAIL/TRAIL-R system to protect the placenta against immune cell attack. Analysis by Northern blotting demonstrated mRNAs encoding TRAIL as well as the four TRAIL receptors (DR4, DR5, DcR1/TRID, DcR2/TRUNDD) in human placentas. Immunohistochemical experiments demonstrated that TRAIL protein is prominent in syncytiotrophoblast, an uninterrupted placental cell layer that is continuously exposed to maternal blood, as well as in macrophage-like placental mesenchymal cells (Hofbauer cells). Studies on cell lines representing trophoblasts (Jar, JEG-3 cells) and macrophages (U937, THP-1 cells) showed that both lineages contained TRAIL mRNA and that steady state levels of transcripts were increased 2- to 11-fold by IFN-gamma. By contrast, cell lineage-specific differences were observed in expression of the TRAIL-R genes. Although all four lines contained mRNA encoding the apoptosis-inducing DR5 receptor, only trophoblast cells contained mRNA encoding the DcR1 decoy receptor and only macrophages contained DcR2 decoy receptor transcripts. DR4 mRNA was present only in THP-1 cells and was the only TRAIL-R transcript increased by IFN-gamma. Cytotoxicity assays revealed that the two trophoblast cell lines were resistant, whereas the two macrophage lines were partially susceptible to killing by rTRAIL. Collectively, the results are consistent with a role for the TRAIL/TRAIL-R system in the establishment of placental immune privilege.

In mice and humans, expression of the tumour necrosis factor receptor-1 (TNF-R1) gene in placental trophoblast cells is constitutive whereas expression of the TNF-R2 gene is developmentally programmed. In order to study the individual functions of TNF-R1 and -R2 in this lineage, cell lines were generated from placental explants of homozygous matings of gestation day 10 outbred mice (Swiss–Webster), TNF-R1-deficient (TNF-R1−/−) and TNF-R2−/−transgenic mice as well as the background strain for the TNF-R2−/−mice (WT, C57BL/6×129). All of the cells exhibited trophoblast markers; they contained cytokeratin intermediate filaments, expressed alkaline phosphatase activity and displayed transferrin receptors, but were negative for vimentin filaments and the macrophage marker, F4/80. Analysis of DNA by polymerase chain reaction demonstrated the expected TNF-R genotype in each line. In experiments testing the effects of recombinant mouse TNF-α (rmTNF-α) on viability and proliferation of the cell lines, rmTNF-α modestly but dose-dependently inhibited the growth of WT and TNF-R2−/−cells while having no effect on TNF-R1−/−cells. Actinomycin D-treated WT and, to a lesser extent, TNF-R2−/−cells, were more sensitive to growth inhibition than untreated cells whereas TNF-R1−/−cell responses remained unchanged. These data indicated that rmTNF-α inhibits growth of trophoblastic cells through TNF-R1 and that newly synthesized protein(s) provide partial protection against toxicity. In contrast to the receptor species-specific effects on cell growth exerted by rmTNF-α, both TNF-R mediated inhibition of alkaline phosphatase activity. Collectively, the observations support the postulate that receptor expression is the key factor which determines the nature and extent of TNF-α effects on trophoblast cell growth and function.

The reactive nitrogen intermediate, nitric oxide (NO) is important in host defense against both NO-sensitive microorganisms and tumor cells. Macrophages are one of the chief inflammatory sources, especially when stimulated with the combination of LPS and interferonγ (IFNγ). It is not known, however, whether IFNγ-mediated augmentation of LPS-induced production of NO is the result of greater production by all cells or to the recruitment of more producer macrophages within a given population. This question was addressed, first, by stimulating mouse macrophages (either bone marrow culture-derived, inflammatory peritoneal or those of the cell line, RAW 264.7) with up to 10 U/ml IFNγ for as long as 24 h. Under these conditions, there was little or no production of NO and rare or no cells were immunocytochemically positive for the inducible form of nitric oxide synthase (iNOS), which catalyzes the production of NO. Populations similarly exposed to 1 ng/ml LPS were low producers of NO and contained somewhat more, but still only a few (< 15%), iNOS-positive cells. In contrast, as the concentration of IFNγ was increased (≥ 1 U/ml) in the presence of a constant amount of LPS (1 ng/ml), the principal effect was to increase both the production of NO and the number of iNOS-positive macrophages. The amount of iNOS expressed by some cells also appeared to be increased. Two important conclusions can be drawn from these findings: (1) there is heterogeneity in mouse macrophage populations with respect to the production of iNOS; and (2) increasing concentrations of IFNγ appear to augment LPS-induced secretion of NO by recruiting increasingly greater numbers of macrophages into the production of iNOS. Such results potentially provide important clues as to how IFNγ may be acting at the subcellular level to enhance iNOS synthesis.

The inducible protein p71/73 marks the response of mouse macrophages to one of several stimuli (e.g., bacterial lipopolysaccharide or poly I:C) that trigger the expression of cytolytic activity when these cells have previously been primed for tumor cell killing by interferon-gamma (IFN-gamma). The results reported here identify this marker protein as the inducible prostaglandin endoperoxide synthase (PES), TIS10/PES-2. Identification was based on four findings: (1) p71/73, like the TIS10/PES-2 protein, was associated with cellular membranes; (2) the sequence of amino acids in the NH2 terminus of both p71 and p73 was 96% identical to the predicted NH2-terminal sequence of the TIS10/PES-2 protein; (3) a polyclonal antiscrum raised against the COOH-terminal region of the TIS10/PES-2 gene product recognized p71/73 in immunoblots; and (4) dexamethasone, which blocks induction of TIS10/PES-2 expression, inhibited the induction of both p71/73 synthesis and tumoricidal activity in macrophage. Several regulatory roles for this protein in the activation process are possible.