CD40.FasL inhibits human T cells: evidence for an auto-inhibitory loop-back mechanism.

A chimeric CD40.FasL (CD40-CD95L) protein was designed with the combined capacities to bind to two surface receptors on activated T cells, CD40 ligand (CD40L; CD154) and Fas receptor (CD95). CD40.FasL, once tethered to the cell surface via one of its ends, can transmit a signal via its other end. In principle, simultaneous triggering from both ends is possible, and thus there is the intriguing potential for 'auto-inhibition' if such dual triggering occurs on the same cell itself. Several lines of evidence support this mechanism: (i) CD40.FasL is cytotoxic to Fas receptor-positive cell lines of different cell lineages, (ii) CD40.FasL's function is potentiated when there is enforced expression of CD40L on target cells, (iii) CD40.FasL inhibition does not require intercellular contact, as demonstrated by soft agar clone formation and cell dilution analysis and (iv) introduction of exogenous CD40 into the system interferes with CD40.FasL inhibition. Taken together, these data are consistent with a 'loop-back' inhibitory mechanism within individual activated (CD40L and Fas receptor expressing) T cells causing suicide of these T cells. Significantly, this type of fusion protein provides a unique way to confine immunoinhibition to activated T cells.

CTLA-4.FasL inhibits allogeneic responses in vivo.

CTLA-4.Fas ligand (CTLA-4.FasL), a paradigmatic 'trans signal converter protein (TSCP)', can attach to APC (via CTLA-4 binding to B7) and direct intercellular inhibitory signals to responding T cells (via FasL binding to Fas receptor), converting an activating APC-to-T cell signal into an inhibitory one. Our previous studies established that CTLA-4.FasL inhibits human primary mixed lymphocyte reactions (MLR) and induces alloantigen-specific hyporesponsiveness ex vivo. The present study extends this to an in vivo context. Using splenocytes from MHC-mismatched C57BL/6 and Balb/c mice, we demonstrated that his(6)CTLA-4.FasL, effectively inhibits murine MLR. Moving in vivo, we demonstrated that subcutaneously administered his(6)CTLA-4.FasL modulates the in vivo response of infused allogeneic splenocytes. his(6)CTLA-4.FasL reduces the number of cells in each cell division, and increases the percentage of dead cells in each division. These findings are consistent with an antigen-induced cell death of the alloreactive cells, and bolsters recombinant TCSP promise as a therapeutic for transplantation diseases.

A rheostatic mechanism for T-cell inhibition based on elevation of activation thresholds.

The activation of discrete T-cell responses depends on the triggering of individualized threshold numbers of T-cell receptors (TCRs). The results of this study indicate that the lipocalin placental protein 14 (PP14), a T-cell inhibitor produced by cells of the reproductive and hematopoietic systems, mediates its anti-inflammatory activity by elevating the T-cell activation threshold, thereby rendering T cells less sensitive to stimulation. Significantly, the data demonstrate hierarchical sensitivity of selected cytokine responses to PP14-mediated inhibition, with the hierarchy reflecting their respective activation thresholds. These findings suggest a novel paradigm for immunoinhibition wherein negative regulators can finely tune, rather than inactivate, T-cell responses, and thereby skew the cytokine output of immunologic responses.

alpha2-macroglobulin modulates the immunoregulatory function of the lipocalin placental protein 14.

Human placental protein 14 (PP14; also known as glycodelin and progesterone-associated endometrial protein) is an immunosuppressive protein of the lipocalin structural superfamily. Mechanisms regulating serum PP14's immunosuppressive activity remain to be elucidated. In the present study, an interaction between PP14 and a major serum protein carrier, alpha(2)-macroglobulin (alpha(2)M), was documented for the first time. Using native gel electrophoresis, we showed that PP14, as well as its alternative splice variant PP14.2, binds to both alpha(2)M and methylamine-activated (MA)-alpha(2)M. Cross-competition studies demonstrated that the variants compete for binding to alpha(2)M. PP14 bound to alpha(2)M and MA-alpha(2)M with K(d) values of 167+/-70 and 221+/-56 nM (means+/-S.D.) respectively, as determined by surface plasmon resonance. Significantly, the addition of alpha(2)M or MA-alpha(2)M to a T-cell proliferation assay strongly potentiated the inhibitory capacity of PP14. On the basis of these findings, alpha(2)M emerges as the first serum protein that can physically associate with, and thereby regulate, PP14. Moreover, this represents the first documented interaction between the protein carrier alpha(2)M and a lipocalin protein.

Placental protein 14 functions as a direct T-cell inhibitor.

Human placental protein 14 (PP14, also referred to as glycodelin and progesterone-associated endometrial protein) inhibits phytohemagglutinin (PHA)-stimulated T-cell proliferation and monokine secretion within PBMC populations. However, the mechanisms underlying these and other PP14 immunoinhibitory activities remain unclear. In the present study, we asked whether PP14's T-cell inhibitory effect is a direct one or, alternatively, an indirect consequence of accessory cell (AC) perturbation. Using either immunopurified PP14 or first-trimester amniotic fluid (AF) as a rich source of PP14, we documented inhibition of the proliferation of highly purified peripheral blood T-cells when stimulated with anti-CD3 mAbs or PHA in the presence of paraformaldehyde-fixed AC. Significantly, PP14 inhibited T-cell proliferation and IL-2 secretion induced by immobilized anti-CD3 and anti-CD28 mAbs in the absence of AC. PP14 depletion (via immunoprecipitation) abrogated AF's T-cell inhibitory activity, indicating that the PP14 within the amniotic fluid is required for this immunoregulatory effect. These findings establish that PP14 can inhibit T-cell proliferation in the absence of AC and thus add PP14 to the relatively restricted set of immunoinhibitory proteins that are known to target T-cells directly. Additional data demonstrate that PP14's inhibitory effect can be overridden by stimuli which circumvent early events during T-cell receptor (TCR) activation, namely, protein kinase C activators in combination with Ca2+ ionophores. These latter results suggest that PP14 inhibits early events in the TCR signaling pathway.

Differential effects of chondroitin sulfates A and B on monocyte and B-cell activation: evidence for B-cell activation via a CD44-dependent pathway.

At inflammatory sites, proteoglycans are both secreted by activated mononuclear leukocytes and released as a consequence of extracellular matrix degradation. Chondroitin 4-sulfate proteoglycans constitute the predominant ones produced by activated human monocytes/macrophages. In this study, we show that two chondroitin 4-sulfate forms, CSA and CSB, can activate distinct peripheral blood mononuclear cell types. Whereas CSA activates monocytes (to secrete monokines), CSB activates B-cells (to proliferate). In contrast, the chondroitin 6-sulfate CSC and heparin do not exert these functional effects. We further show that CD44 monoclonal antibodies block CSB-induced B-cell proliferation. These findings point to glycosaminoglycans, and specifically chondroitin 4-sulfates, as a novel class of immunological mediators at inflammatory sites. Furthermore, the data link CD44 to B-cell activation, paralleling the established roles of CD44 in T-cell and monocyte activation.

Normal and atypical butyrylcholinesterases in placental development, function, and malfunction.

1. In utero exposure to poisons and drugs (e.g., anticholinesterases, cocaine) is frequently associated with spontaneous absorption and placental malfunction. The major protein interacting with these compounds is butyrylcholinesterase (BuChE), which attenuates the effects of such xenobiotics by their hydrolysis or sequestration. Therefore, we studied BuChE expression during placental development. 2. RT-PCR revealed both BuChEmRNA and acetylcholinesterase (AChE) mRNA throughout gestation. However, cytochemical staining detected primarily BuChE activity in first-trimester placenta but AChE activity in term placenta. 3. As the atypical variant of BuChE has a narrower specificity for substrates and inhibitors than the normal enzyme, we investigated its interactions with alpha-solanine and cocaine, and sought a correlation between the occurrence of this variant and placental malfunction. 4. Atypical BuChE of serum or recombinant origin presented > 10-fold weaker affinities than normal BuChE for cocaine and alpha-solanine. However, BuChE in the serum of the heterozygote and a homozygous normal were similar in their drug affinities. Therefore, heterozygous serum or placenta can protect the fetus from drug or poison exposure, unlike homozygous atypical serum or placenta. 5. Genotype analyses revealed that heterozygous carriers of atypical BuChE were threefold less frequent among 49 patients with placental malfunction than among 76 controls of the entire Israeli population. These observations exclude heterozygote carriers of atypical BuChE from being at high risk for placental malfunction under exposure to anticholinesterases.

Characterization of the imprinted IPW gene: allelic expression in normal and tumorigenic human tissues.

IPW (Imprinted gene in the Prader-Willi syndrome region) is a recently identified paternally expressed gene. Previous work has demonstrated IPW expression in the human fetus and adult, with monoallelic expression in adult lymphoblasts and fibroblasts, and in fetal tissues. To further examine the expression of IPW, a series of experiments were carried out using RT-PCR to measure IPW expression in placentae and various fetal and tumor tissues. Biallelic expression of IPW was found in testicular germ cell tumor and bladder cancer cells, suggesting loss of imprinting in the latter case. Both H19 and Insulin-like growth Factor 2 (IGF2), two additional imprinted genes, also showed biallelic expression in those same tumors that demonstrated IPW biallelic expression. Of note, the naturally occurring parthenogenetic-derived mature teratoma unexpectedly expressed large amounts of IPW. Lastly, the pluripotent embryonal cancer cell line Tera-2 expressed IPW at the same level before and after differentiation induced by retinoic acid, suggesting that this gene functions in a 'housekeeping' capacity throughout cell growth. This was in contradistinction to H19 and IGF2, both of which showed significant transcriptional upregulation after Tera-2 cell differentiation.

H19 expression and tumorigenicity of choriocarcinoma derived cell lines.

Certain embryonal tumors demonstrate a loss of heterozygosity at the parentally imprinted region of chromosome 11p15.5. It has been hypothesized that this implicates a tumor suppressor gene at this locus. The human H19 gene maps to 11p15.5, is expressed in fetal tissues including the placenta and is paternally imprinted. Here we show that the abundance of H19 transcripts in cells of two choriocarcinoma derived cell lines (JAr and JEG-3) differs greatly. While JAr cells express high levels of H19 RNA, the expression of H19 in JEG-3 cells is much lower than that of normal trophoblasts. Cells of these two cell lines were subcutaneously injected into nude mice with subsequent tumor formation. A fivefold increase in the H19 RNA level was measured in tumors derived from JEG-3 cell lines as compared to these cells before injection. However this increase in H19 RNA did not alter the clonogenicity in soft agar nor the growth rate of the cells derived from these tumors as compared to the original JEG-3 cells. Nevertheless, the cells retaining the elevated level of H19 transcripts were more tumorigenic than the original cells. We propose that there is a selection of cells expressing high levels of H19 from the total JEG-3 cell population during the microevolution of tumor formation. These observations, together with our previous publications on H19 expression in human cancers, do not support the notion of a tumor suppressor role for the H19 gene.

The interaction between cytotrophoblasts and their derived tumor cells.

Previous experiments demonstrated that human cytotrophoblasts and cells of the choriocarcinoma cell line JAr interact in vitro. As a result of this interaction there is an increased synthesis of CG and hPL, probably as a result of the increased CG and hPL synthesis by the cytotrophoblasts. In the present investigation we studied this interaction in greater detail and found that both cytotrophoblasts and JAr cells undergo changes in their biological properties as a result of this interaction. JAr cells and cytotrophoblasts cocultured for 72 hr were fractionated according to their size by centrifugal elutriation. The number of cells in the fraction which contain the largest cells was very significantly increased as a result of the coculture. This increase was due to an increase in the number of cells of both cell types. This fraction was the most active one in the synthesis of CG and hPL. The synthesis of DNA by the JAr nuclei in this fraction of the cocultured cells was almost completely inhibited but in the parallel fraction of the JAr cells cultivated alone the level of DNA synthesis was equal to that of all other JAr cell fractions. Heterokaryons are formed in the coculture. In these heterokaryons a factor which inhibits DNA synthesis in the cytotrophoblasts may inhibit DNA synthesis in JAr nuclei and at least be partly responsible for the inhibition of DNA synthesis observed.

ABL and BCR genes are not imprinted in androgenetic and gynogenetic human tissues.

In the translocation leading to the formation of the Philadelphia chromosome, the hallmark of chronic myeloid leukemia (CML), the translocated chromosome 9 (ABL), is of paternal descent whereas chromosome 22 (BCR) is of maternal origin (1). To study possible imprinting of the human ABL and BCR genes, we used human tissues exclusively endowed with their maternally (benign teratoma) or paternally (complete hydatidiform mole) inherited chromosomes. Using the sensitive PCR technique followed by northern blotting, we demonstrate here that ABL and BCR are expressed to a similar extent in androgenetic and gynogenetic human tissues, thus suggesting that ABL and BCR genes are not imprinted in these human tissues.

Ultrastructural characteristics of cytotrophoblast cells during stages of differentiation.

Isolated cytotrophoblast cells from term human placenta were separated into eleven fractions according to cell size, by centrifugal elutriation. Each fraction isolated was examined by electron microscopy to elucidate ultrastructural features consistent with differences in stages of cellular differentiation. As a rule, increasing cell size correlated with evidence of progressive intracellular differentiation. This was represented by the appearance of specialization structures in later fractions, and by changes in the density of organelles and other cellular constituents. Progressive development and maturation of the rough endoplasmic reticulum was also evident. These data are the first to demonstrate successful subfractionation of the heterogeneous cytotrophoblast cell population into distinct groups, each representing different levels of cellular differentiation. These morphologic features of differentiation correlate closely with established biochemical parameters associated with various stages of intermediate cytotrophoblast cell differentiation.

Parallel regulation of parentally imprinted H19 and insulin-like growth factor-II genes in cultured human fetal adrenal cells.

Adjacent, parentally imprinted, insulin-like growth factor-II (IGF-II) and H19 genes are highly expressed during embryogenesis and are important for fetal growth. Human fetal adrenals express abundantly both IGF-II and H19 genes. To clarify the significance and regulation of the H19 gene, we studied its expression in fetal adrenals. In situ hybridization experiments showed H19 RNA expression throughout the fetal adrenal cortex, with slightly higher expression in the outer definitive (adult) than in the inner fetal zone. In primary cultures of fetal adrenal cells, ACTH and other activators of the protein kinase-A signal transduction pathway increased both H19 and IGF-II RNA accumulation 1.7- to 10-fold. Staurosporine, a protein kinase-C inhibitor, increased H19 and IGF-II RNA to the same extent as did ACTH. The protein kinase-C activator 12-O-tetradecanoyl phorbol-13-acetate and cytokines, tumor necrosis factor-alpha and interferon-gamma, inhibited H19 and IGF-II RNA accumulation. Transforming growth factor-beta 1 caused a decrease in levels of H19 and IGF-II RNA, whereas the IGFs caused a slight increase. Our data show parallel multifactorial regulation of H19 and IGF-II RNAs in human fetal adrenal cells. This suggests common regulatory mechanisms for these adjacent genes.

Relaxation of imprinting in trophoblastic disease.

Genomic imprinting--the uniparental-dependent transmittance of a genetic trait--has been accepted in recent years as a major mechanism in mammalian genetics. We studied the expression of the H19 gene, a parentally imprinted (maternally expressed) gene, by in situ hybridization in human placenta and trophoblastic disease. Expression was found to be abundant, in a decreasing order, in the intermediate trophoblast (villous and interstitial), the cytotrophoblast, and the syncytiotrophoblast. The villous stroma was also prominently labeled. Partial hydatidiform mole showed a similar pattern of expression as normal placenta. As expected, complete hydatidiform mole, whose genome consists of two haploid sets of paternal origin, was not labeled in the villous stroma and surrounding trophoblastic layer. However, some of the large mononuclear cells in the proliferating groups sprouting from the villous surface were strongly labeled. Prominent expression of H19 was found in placental site trophoblastic tumor and gestational choriocarcinoma. The phenomenon of emergence of expression of alleles subject to repression according to their gamete of origin is termed relaxation of imprinting, and is considered to be relevant to tumorigenesis. We suggest that the expression of the maternally expressed H19 gene in the androgenetic tissue of complete hydatidiform mole represents relaxation of imprinting and may be associated with its malignant potential.

Induction of tumor necrosis factor and interleukin-6 mRNA in human cytotrophoblast cells exposed to lipopolysaccharide.

OBJECTIVE: The cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) have previously been identified in placental tissue and are known to be mediators of infection-associated induction of the host immune system. This study was undertaken to better characterize the in vitro regulation of these cytokines in cytotrophoblast cells when challenged with the bacterial product lipopolysaccharide (LPS). METHODS: Term placentas were freshly collected, digested with trypsin/DNase, and subjected to Percoll gradient centrifugation to isolate cytotrophoblasts. Either immediately or after overnight incubation, LPS (1 microg/ml) or media alone was added to the cell cultures for 0, 1, 2, 4, 8, 24, 48, and 72 h. Total cellular RNA was isolated by the guanidinium thiocyanate/cesium chloride methodology. RNA samples were run on 1% agarose-formaldehyde gels and subsequently transferred to nylon filters. Blots were hybridized with the appropriate P32-radiolabeled probe. RESULTS: In non-LPS-treated cells, minimal amounts of TNF mRNA could be detected at zero time, or throughout the incubation periods. Conversely, LPS exposure resulted in detectable signal starting at 1 h and peaking at 2 h after the addition of LPS. Overnight incubation gave stronger TNF signals in the LPS stimulated cells, although the kinetics of this response remained similar to zero time exposure. IL-6 was likewise minimally expressed at zero time, although non-stimulated cell cultures demonstrated progressive increases in mRNA expression which was maximal at 16 h after plating. LPS further augmented the transcription of IL-6 mRNA, with peak signals seen at 4 h after LPS stimulation. Again, overnight incubation of the cytotrophoblasts increased baseline and LPS-induced IL-6 mRNA responses. Long-term constant exposure of cells to LPS did not demonstrate any evidence of prolonged signaling. LPS did not alter mRNA expression of the placental gene H19 or the oncogene FOS. CONCLUSIONS: These data demonstrate the induction of TNF and IL-6 mRNA in cytotrophoblast cells with LPS. These transcriptional events are kinetically distinct and short term in nature. Overnight incubation accentuates the TNF and IL-6 mRNA signal and allows for an augmented response to LPS.

Use of a novel system for defining a gene imprinting region.

Gene imprinting involves the expression of a single allele, depending on its parental origin. In this report, we describe the use of a novel system, implementing human tissues exclusively endowed with either maternally or paternally inherited chromosomes, to better define a known gene imprinting region. Specific RNA transcripts for Placental Ribonuclease Inhibitor (PRI) and Cathepsin D were analysed by northern blotting for expression in complete hydatidiform mole, mature teratoma, and normal placenta. These genes are in close proximity to the reciprocally imprinted H19 and IGF-2 genes found on chromosome 11p15.5. Since all the tissues studied expressed Cathepsin D and PRI, these are not, by definition, imprinted, but as yet we cannot define the borders of the imprinting area on chromosome 11p15.5.

Genetic imprinting in human evolution: the decisive role of maternal lineage.

The modern study of human evolution must take into account physical anthropology, which examines phenotypic expression, and molecular evolution, which examines genotypic change. Recent independent investigations have shown that the process of genetic imprinting, defined as parental-dependent transmission of genetic traits, plays a pivotal role in human evolution. We draw on data from various scientific disciplines to support the hypothesis that maternal lineage via preferential genetic contribution, plays a decisive role in this regard. This concept is of more than theoretical interest, in that, current human disease states can be better understood and studied in the context of loss of genetically-defined evolutionary advantage.

Intermediate cells during cytotrophoblast differentiation in vitro.

Differentiation of the human placental trophoblast cell involves a multistep process, with the generation of several distinct types of intermediate cytotrophoblast cells. Using a short term in vitro cell culture system and centrifugal elutriation, we studied the isolation and morphological and biochemical differentiation of these separated intermediate cell populations. Freshly isolated cell fractions, incubated for 24 h, are heterogeneous in their differentiation stages as determined by the secretion of the proteins chorionic gonadotropin alpha and beta, human placental lactogen, and pregnancy specific beta 1-glycoprotein. Maintenance in cell culture allows for the further differentiation of these intermediate cells and for syncytium formation. With the use of sequential trypsinizations, our data also suggest the parallel differentiation of cytotrophoblast cells into two distinct subsets: one which, through differentiation, gets committed to syncytium formation, and the other, which remains mononuclear despite high degrees of biochemical differentiation. These latter cells retain the capacity for syncytium formation when reintroduced into appropriate culture conditions. These findings refine the use of the term "intermediate cell" by previous investigators. We suggest that our in vitro system defines normal intermediate stages of trophoblast differentiation, and also serves as a model to simulate adverse conditions of syncytial degeneration or injury.

The expression of the H-19 and IGF-2 genes during human embryogenesis and placental development.

The H-19 gene in mice is maternally imprinted and its ectopic expression causes prenatal lethality. We have recently identified H-19 transcript in differentiating human placental cells and showed that its expression increases concomitantly with differentiation of cytotrophoblasts in vitro. Placental and embryonal specimens were collected from conception products derived from normal first and second trimester pregnancy terminations. We investigated the abundance of H-19 mRNA throughout placental development in vivo and compared it to the expression of other genes linked to placental differentiation. Furthermore, the expression of H-19 transcript in different organs of human fetuses, aborted during the second trimester, was examined by RNA isolation from separated fetal organs. Since IGF-2 is known to play an important role in embryogenesis, identical blots were hybridized with IGF-2 probe. H-19 expression in human placenta from the different trimesters of pregnancy remains practically constant. A high amount of H-19 gene product was found in the fetoplacental unit with the highest level measured in the adrenal gland. These findings argue that H-19 gene may play a role in human embryogenesis.