Judith L. Pace

Placental Cell Expression of HLA-G2 Isoforms Is Limited to the Invasive Trophoblast Phenotype

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 β2-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 immunohistochemical 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 CD8α expression on PBMC without changing CD3δ 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.

Cellular localization and hormonal regulation of inducible nitric oxide synthase in cycling mouse uterus

Nitric oxide (NO), a potent and versatile free radical, is synthesized in macrophages and mast cells as well as in other types of cells by the inducible form of nitric oxide synthase (iNOS). In this study, cells containing iNOS were identified in the uteri of cycling mice by using a rabbit antibody generated to an iNOS‐specific peptide. Macrophages were identified in semiserial sections of the same tissues with the monoclonal antibody, F4/80, and mast cells were identified by toluidine blue staining. In tissue sections of uteri obtained from mice in the four stages of the estrous cycle (8 to 11 mice per stage), iNOS immunoreactivity was strongest in diestrus‐I uteri and weakest in diestrus‐II uteri. Myometrial mast cells and endometrial epithelial cells were prominent locations of iNOS, and specific protein was also present in myometrial smooth muscle and macrophage‐like cells in the endometrial stroma. Because cyclic variations suggested regulation of iNOS expression by ovarian steroid hormones, studies were done using ovariectomized mice. Seven days after ovariectomy, immunoreactive iNOS was low but detectable in mast cells and luminal epithelial cells. In the uteri of ovariectomized, estradiol‐17β (E2)–treated mice, mast cells were iNOS+ after 24 h whereas epithelial cells were negative; the reverse was observed in progesterone (P4)‐treated mice. Both mast cells and epithelial cells were iNOS+ in the uteri of mice that had received a combination of E2 + P4. These results indicate that several types of uterine cells produce iNOS and that expression of this enzyme in specific cell lineages is governed by ovarian steroid hormones. The data are consistent with the postulate that NO derived from uterine leukocytes and other types of cells plays a role in uterine cyclicity and preparation for pregnancy. J. Leukoc. Biol. 57: 27–35; 1995.

Synthesis of β2-microglobulin-free, disulphide-linked HLA-G5 homodimers in human placental villous cytotrophoblast cells

Human leucocyte antigen‐G (HLA‐G) is a natural immunosuppressant produced in human placentas that binds differently to the inhibitory leucocyte immunoglobulin‐like receptors LILRB1 (ILT2) and LILRB2 (ILT4) according to its biochemical structure. To predict the binding functions of the HLA‐G5 soluble isoform synthesized in placental villous cytotrophoblast (vCTB) cells, we investigated structural features of this protein. Biochemical and immunological studies showed that vCTB cell HLA‐G5 heavy (H)‐chain proteins are disulphide‐bonded homodimers unassociated with β2‐microglobulin (β2m) light‐chain proteins. Although comparatively low levels of β2m messenger RNA (mRNA) were identified by real‐time reverse transcription–polymerase chain reaction, immunoprecipitation studies failed to detect β2m protein even when specific mRNA was doubled by transduction of a lentivirus‐β2m complementary DNA into vCTB cells. No abnormalities were identified in the translational start codon of vCTB cell β2m mRNA and differentiation into syncytium did not promote β2m synthesis. The failure of vCTB cells to exhibit β2m in vitro was paralleled by a lack of detectable β2m in vCTB cells in vivo. Lack of the β2m protein could be the result of low levels of β2m transcripts or of as yet unidentified translational defects. Experiments with recombinant ectodomains of LILRB indicate that β2m‐free HLA‐G binds strongly to LILRB2, a receptor that is expressed by macrophages. This potentially immunosuppressive cell type is abundant in the pregnant uterus. Thus, our findings are consistent with the postulate that the natural β2m‐free homodimeric form of HLA‐G5 synthesized in primary vCTB cells could comprise a particularly effective tolerogenic molecule at the maternal–fetal interface.

Isolation and culture of term human trophoblast cells.

Experimentation with most human cell types is restricted to the use of cell lines, and this limits our ability to extrapolate interpretations to the in vivo condition. However, in studying human trophoblast cells, we have a unique opportunity to obtain large quantities of readily available human tissue. In this chapter, we outline the methodology for purification of human trophoblast cells from term placentas. The procedures are based on enzymatic dissociation of villous placental tissue, followed by gradient centrifugation and immunomagnetic bead purification. Purity may be assessed by immunocytochemistry or flow cytometry using a number of markers to identify both cytotrophoblast cells and cellular contaminants. The resulting cytotrophoblast cell populations have excellent viability and purity, and may be subjected to long-term culture.

The mouse macrophage activation-associated marker protein, p71/73, is an inducible prostaglandin endoperoxide synthase (cyclooxygenase).

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‐γ (IFN‐γ). 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 antiserum 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.

Characterization and use of monoclonal and polyclonal antibodies against the mouse interferon-γ receptor

To facilitate investigation of its physical and functional properties, 11 monoclonal antibodies (mAbs) and a goat polyclonal IgG specific for the mouse interferon‐ (IFN‐γ) receptor were characterized and their potential uses studied. Eight of the mAbs interacted with epitopes on the extracellular domain of the receptor, two interacted with epitopes on the intracellular domain, and one interacted with an epitope that could not be localized definitively to either region. Of the 11 mAbs, the majority (8) were IgGs, 2 were IgMs, and 1 was an IgA. Relative avidities of the seven that could be determined ranged from 333 to 0.002 μM‐1. Both the polyclonal goat IgG and mAb GR‐20 (the latter specific for an epitope in the binding site for IFN‐γ) blocked binding of the ligand and, as expected, prevented induction by IFN‐γ of priming of macrophages for tumor cell killing. None of the other mAbs had an effect despite the fact that GR‐22 partially (> 50%) blocked binding of IFN‐γ. Neither the polyclonal IgG nor any of the mAbs had an agonist effect. The relative usefulness of the antibodies for immunoprecipitation, immunoblotting, immunoassay, and cell staining with and without prior fixation is described. The results of immunocytochemical staining directly confirmed that the majority of immunologically reactive receptor protein expressed by cells is intracellular. To facilitate use by other investigators, the hybridomas that produce these mAbs will be offered to the American Type Culture Collection.

Suppressive effect of interferon-beta on development of tumoricidal activity in mouse macrophages.

The suppressive effect of IFN‐alpha and IFN‐beta on the induction of tumoricidal activity in mouse bone marrow‐derived macrophages was investigated. Macrophages incubated for 24 hr with IFN‐beta developed lower levels of cytolytic activity when stimulated with IFN‐gamma and LPS, in comparison with macrophages pretreated with medium. The suppressive effect was dependent on the pretreatment dose of IFN‐beta over a concentration range of 1 to 1,000 U/ml. Analysis of IFN‐gamma dose response curves of IFN‐beta treated macrophages showed that these cells were less sensitive to IFN‐gamma. The suppressive effects were fully neutralized by an antiserum to IFN‐alpha/beta. Prostaglandins were apparently not involved in this process since the addition of indomethacin to IFN‐beta treated macrophages did not prevent the loss of responsiveness to activating stimuli. In contrast to the results obtained with IFN‐alpha and IFN‐beta, macrophages pretreated with IFN‐gamma did not develop lower levels of cytolytic activity when again stimulated with IFN‐gamma and LPS. These observations provide evidence for a potentially important negative regulatory role for IFN‐alpha and IFN‐beta in macrophage activation for tumor cell killing.

Synergistic interactions between IFN-gamma and IFN-beta in priming murine macrophages for tumor cell killing.

The ability of MulFN‐β and MulFN‐γ to potentiate the development of tumoricidal activity in proteose peptone‐elicited murine peritoneal macrophages was investigated. Macrophages were stimulated with increasing concentrations of either MulFN‐β or MulFN‐γ, alone and in combination, in the presence of 1 ng/ml lipopolysaccharide (LPS). The priming activities attributable to the interferons (IFNs) were quantified using the dose‐response curves obtained for these samples. The priming activity observed for mixtures of MulFN‐β and MulFN‐γ was greater than that expected if MulFN‐β and MulFN‐γ had acted in an additive manner. Isobologram analysis of data obtained when macrophages were stimulated with combinations of IFNs demonstrated that MulFN‐β and MulFN‐γ acted synergistically to prime macrophages for tumor cell killing. The greatest degree of synergy was observed when macrophages were stimulated with suboptimal and nearly equivalent concentrations of each class of IFN. Further studies demonstrated that macrophages stimulated with combinations of MulFN‐β and MulFN‐γ were more sensitive to the trigger signal provided by LPS than were cells primed with either IFN alone. Thus, the synergistic effects observed were quantitative in nature in that macrophages perceived combinations of MulFN‐β and MulFN‐γ as having higher priming activities than expected.

Immunoregulatory molecules in human placentas: potential for diverse roles in pregnancy.

Molecules with immunological functions abound in hemochorial mammalian placentas where maternal blood and tissues are in direct contact with fetal placental cells. For the most part, investigators have focused on the possibility that these molecules are primarily in place for the purpose of preventing maternal immune mechanisms from attacking the genetically different fetal cells. Yet information collected in recent years indicates that these immunological mediators may serve other, non-immunological functions in placentas. In this article we discuss two families of these molecules investigated in our and other laboratories, namely the tumor necrosis factor superfamily (TNFSF) and the human leukocyte antigen (HLA) family, and present accumulating evidence for dichotomy of function during gestation.

Prolonged exposure of mouse macrophages to IFN-β suppresses transcription of the inducible nitric oxide synthase gene: Altered availability of transcription factor Stat1α

Previous studies from our laboratory have shown that prolonged exposure of mouse macrophages to IFN‐β interferes with their subsequent ability to become activated for tumor cell killing. Data reported here show that such inhibition is due to reduced production of NO, resulting from decreased transcription of the gene that encodes inducible NO synthase (iNOS; EC 1.14.13.39). The molecular basis for such suppression was shown to be, at least in part, decreased nuclear accumulation of tyrosine‐phosphorylated Stat1α (pStat1α), and a consequent change in the nuclear ratio of pStat1α to non‐transactivating pStat1β. Reduced phosphorylation was observed despite the fact that time‐course studies revealed greater than normal quantities of both Stat1α and Stat1β proteins in macrophages that had been pre‐exposed to IFN‐β. The decrease in nuclear pStat1α was demonstrated to involve an increase in the rate of turnover of phosphorylated protein. The homodimeric form of pStat1α is essential for the expression of both the iNOS and IFN‐regulatory factor‐1 genes (the product of the latter is necessary for full expression of the iNOS gene). These results have broad implications, because they suggest that limiting the availability of homodimeric pStat1α is a means by which down‐regulation of genes containing promoter‐linked IFN‐γ‐activated sites might be achieved.