The synthesis and cellular localization of tumor necrosis factor-alpha (TNF-alpha) were studied in mouse GMG cells, which are activated NK cells in uterine decidual tissue during pregnancy. Synthesis of the protein was demonstrated in GMG cells on days 10 and 14 of pregnancy by in situ hybridization of TNF-alpha message. Immunostaining demonstrated that TNF-alpha protein was localized in the cytoplasmic granules of GMG cells at these times. The results suggest that the cytolytic activity of uterine NK cells may be due in part to TNF-alpha, and that this cytokine may be delivered to target cells intracellularly via transmembrane pores formed by perforin, which is also localized in uterine NK cell granules.

Macrophages and natural killer (NK)-like cells are the major hematopoietic cell populations in the cycling and pregnant mouse uterus and are also found in the embryo. In order to evaluate potential receptivity of these cells to interferon-gamma (IFN-gamma), tissues taken from cycling and pregnant mice were tested for IFN-gamma receptor (IFN-gamma R) mRNA and protein. Macrophages were identified immunohistochemically by using the specific monoclonal antibody F4/80. NK cells were identified by their large size, distinctive intracellular granules, and binding of a monoclonal antibody to the common leukocyte antigen. In cycling uteri, the abundance of IFN-gamma R mRNA relative to an invariant message (glyceraldehyde-3-phosphate dehydrogenase) increased during progression of the hormonally regulated estrous cycle. IPN-gamma R mRNA in situ hybridization signals were slightly higher in macrophage-like than in other types of endometrial stromal cells. In pregnant uteri, the highest proportions of IFN-gamma R mRNB were observed at gestation day (g.d.) 16. Specific message and protein were present in uterine macrophages by p.d. 7 and in NK cells by g.d. 9. IFN-gamma R expression in both lineages remained stable through the balance of pregnancy. In embryos, IFN-gamma R mRNA increased between g.d. 14 and 16. Specific transcripts were present in many cells at g.d. 14, but none were detected in embryonic liver macrophages until g.d. 16. The results of this study; suggest relationships between IFN-gamma R expression and ovarian hormones as well as cell maturation and support the postulate that IFN-gamma receptor-ligand interactions may improve the ability of uterine and embryonic hematopoietic cells to perform specific tasks during gestation.

Abnormal expression of polypeptide growth factors and their receptors is closely associated with tumorigenic transformation. In this study tumor necrosis factor-α (TNF-α) mRNA and protein were analyzed in polyps and proliferative lesions of endometrium as well as in low and high grade endometrial tumors by using in situ hybridization and immunocytochemistry. All samples contained products of the TNF-α gene. Histochemical scores (HS), which reflect the proportion of cells positive for TNF-α message or protein and the intensities of the signals, were higher for epithelial than for stromal cells. Benign lesions (endometrial polyps) contained little TNF-α mRNA or protein, whereas specific message was abundant in proliferative lesions (hyperplasia, adenofibroma). Although neoplastic cells in both low and high grade endometrial tumors contained TNF-α mRNA, two major differences were observed: HS for TNF-α mRNA were significantly less in low grade than in high grade neoplasms, and TNF-α message was restricted to the nucleus in low grade adenocarcinoma cells but was abundant in the cytoplasm of high grade tumor cells. In contrast to cells in benign and proliferative lesions, TNF-α protein scores in endometrial tumor cells were inversely rather than positively correlated with TNF-α mRNA scores. Collectively, the findings in this study are consistent with the postulate that TNF-α is useful to endometrial tumor cells and suggest that production may increase as cells diverge from normal.

Transforming growth factor-beta 1 (TGF-beta 1) has major effects on hematopoietic cell proliferation, migration and function. In an effort to learn whether or not TGF-beta 1 might be among the cytokines that influence uterine, placental and embryonic hematopoietic cells, in situ hybridization was used to map expression of the TGF-beta 1 gene through gestation in the rat. In cycling uteri, hybridization signals with a biotinylated TGF-beta 1 antisense RNA probe were comparatively weak and were restricted to myometrium. During pregnancy, TGF-beta 1 mRNA appeared in specific cell lineages in an ordered temporal sequence. In early post-implantation tissues (g.d. 5-8), TGF-beta 1 transcripts were prominent in uterine epithelial cells, muscle and decidual cells. At g.d. 15, hybridization signals were particularly strong in uterine epithelial cells, muscle and placental trophoblast cells. At this stage, TGF-beta 1 mRNA was also present in uterine cells resembling macrophages and in natural killer-like (GMG) cells. Steady state levels of specific message in uteri and placentas declined at late stages of gestation (g.d. 18, 21). In rat embryos, specific transcripts were identified at g.d. 9 and were prominently displayed in various types of embryonic cells from mid-gestation onwards. The results of this study are consistent with the postulate that throughout pregnancy in the rat, TGF-beta 1 may, in addition to its other functions, influence the proportions, patterns of distribution and activities of maternal and fetal hematopoietic cells.

Previous studies have shown that the TNF-alpha gene is transcribed and translated in fully differentiated human placental syncytiotrophoblast. In this study, TNF transcripts were identified by in situ hybridization in cytotrophoblastic cells, a progenitor subpopulation that proliferates rapidly in early gestation tissues. To establish molecular and biochemical characteristics of cytotrophoblastic TNF and to evaluate potential utilization, experiments were conducted on two cytotrophoblastic cell lines, Jar and JEG-3. Northern blot hybridization and immunocytochemical tests showed that Jar and JEG-3 cells contained TNF mRNA and specific protein. Enzyme immunoassays demonstrated production of TNF, and immunoprecipitation experiments showed that Jar cell TNF protein was the same molecular mass as macrophage TNF. DNA synthesis in both lines was promoted by rTNF, and experiments employing 17-mer TNF antisense and sense oligonucleotides showed specific inhibition of DNA synthesis by antisense sequences. Both p60 and p80 TNF-R mRNA were present in the choriocarcinoma cell lines, and DNA synthesis was inhibited by antibody to the p60 TNF-R. Although the two lines were similar in many respects, Jar cells produced more TNF and demonstrated a greater reliance on TNF for their growth. Collectively, the results indicate that: 1) the TNF gene is expressed in both normal and malignant cytotrophoblast; 2) certain molecular, immunologic, and biochemical characteristics of trophoblast-derived TNF are similar to macrophage TNF; and 3) the p60 TNF-R facilitates utilization of TNF as an autocrine growth factor by choriocarcinoma cells. Although TNF apparently serves important functions in cytotrophoblast during the course of placental development that might include promotion of proliferation and invasion, constitutive expression of this gene in neoplastic cells could account in part for the remarkable ability of trophoblastic tumors to overcome host defenses.

Enriched fractions of spermatogenic cells were isolated by unit gravity sedimentation and analyzed both for the presence of secreted tumor necrosis factor-alpha (TNF alpha) in vitro by bioassay and for the presence of TNF alpha mRNA by Northern blot analysis. Small quantities of bioactive TNF alpha were consistently detected in medium conditioned by round spermatid fractions. Both pachytene spermatocyte and round spermatid fractions contained RNA that hybridized with murine cDNA probes for TNF alpha, with pachytene spermatocytes containing a normal 1.9-kilobase (kb) transcript, while round spermatids contained principally an approximately 2.8-kb transcript. Both the normal size transcript and the larger haploid-specific transcript were enriched when total RNA from pachytene spermatocyte and round spermatid fractions was passed through an oligo(dT) column. The normal 1.9-kb transcript within pachytene spermatocytes could be induced by exposing the spermatogenic cells to lipopolysaccharides in vitro, yet the approximately 2.8-kb transcript within round spermatids appeared uninduced by LPS treatment. In situ hybridization for the TNF alpha message by using digoxigenin label antisense TNF alpha riboprobe labeled pachytene spermatocytes, round spermatids, and presumptive interstitial macrophages. Spermatogonia and elongating spermatids as well as other interstitial cells were unlabeled or very lightly labeled. Hybridization of 16-day-old prepuberal testis resulted in the labeling of spermatocytes and presumptive interstitial macrophages. RNA from Sertoli cells, but not pachytene spermatocytes or round spermatids, hybridized with human TNF alpha receptor p60 probe in Northern blot analysis. These results are consistent with the working hypothesis that spermatids release TNF alpha, which is detected by Sertoli cells and may serve as a paracrine factor, regulating an as yet unidentified process in spermatogenesis.

In order to evaluate the potential of placental cells to bind the multifunctional cytokine, interferon-γ (IFN-γ), tissues collected from pregnant Swiss mice were analysed for IFN-γ receptor (IFN-γR) mRNA and protein. Northern blot hybridization studies indicated that the relative abundance of IFN-γR mRNA increased as gestation progressed to term. Analysis by in situ hybridization revealed that trophoblast cells first contained high steady state levels of IFN-γRmRNA at g.d. 12. At g.d. 12 and 14, transcription was restricted to cells in the spongiotrophoblast region and nests of similar cells in the labyrinthine region. These cells also contained immunoreactive IFN-γR protein. By g.d. 18, IFN-γR mRNA was clearly detectable in large spongiotrophoblast cells and labyrinthine trophoblast. IFN-γR mRNA was low to absent in giant trophoblast cells at all stages of gestation. Specific mRNA was present in parietal and visceral yolk sac cells by g.d 14. Thus, expression of the IFN-γR gene in mouse placental cells is influenced by stage of gestation, cell lineage and state of differentiation. Whether or not these cells respond vigorously to IFN-γ with induction, of antiviral proteins, increased MHC class I antigens and growth modulation may therefore be determined by their expression of specific receptors for this pluripotent cytokine.

Although TNF-α is traditionally associated with macrophage activation during neoplasia and acute inflammation, recent Northern blot hybridization studies indicate that gene expression occurs in the absence of pathology. In order to identify the cellular sources of endogenous message and protein, normal mouse tissues were tested for TNF-α mRNA using in situ hybridization and for the corresponding protein by immunocytochemistry. Unexpectedly, specific TNF-α message was readily detected in hepatocytes, kidney tubule epithelial cells, various populations of spleen cells and neurons. TNF-α protein was present in the same liver and kidney cells as those that contained TNF-α mRNA, was low in spleen cells, and was absent in neurons. These results suggest that cells other than macrophages are the major sources of TNF-α gene products in normal tissues, indicate that regulation is accomplished by more than one mechanism, and are consistent with the postulate that products of this gene contribute to normal physiological processes.

The pregnant uterus contains TNF-alpha, a potent cytokine with pleotrophic effects. The uterus also contains numerous macrophages, which are well described sources of TNF-alpha. In order to determine if uterine TNF-alpha originated with these macrophages, patterns of macrophage tissue distribution and population densities were first established in rat uterine tissues from early, mid, and late stages of gestation by immunohistology. The potential of these and other uterine and placental cells to synthesize TNF-alpha was then tested by in situ hybridization with the use of biotinylated antisense and sense RNA probes. Although TNF-alpha mRNA was present during all stages of pregnancy, hybridization signals were highest in gestation day 15 tissues. The predominant TNF-alpha mRNA-containing cells were uterine epithelium, decidual cells, and placental trophoblast cells; these cells also contained immunoreactive TNF. Transcription of the TNF gene in the uterus and placenta was also documented by Northern blotting experiments, which showed that the transcript sizes for uterine, placental, and macrophage TNF mRNA were similar. Although stromal cells that were located in macrophage-rich uterine compartments (myometrium, metrial gland) contained TNF-alpha mRNA, the cells did not contain high levels of immunoreactive TNF-alpha. Thus, cells other than macrophages are likely to be the major contributors of TNF-alpha during uncomplicated pregnancy in the rat.

Tumor necrosis factor alpha (TNF-alpha), a polypeptide that regulates cellular growth and modulates the synthesis of various cell surface and secreted molecules, has been identified in the pregnant uterus. To determine which specific cells transcribed and translated this gene, extraembryonic fetal tissues (placenta and membranes) and uterine tissue from early and late stages of gestation were analyzed for TNF-alpha mRNA by in situ hybridization using biotinylated antisense and sense TNF-alpha probes, and for immunoreactive TNF-alpha using two monoclonal antibodies. Tumor necrosis factor-alpha transcripts and protein were identified in both extraembryonic and maternal cells. In first-trimester placental villi, TNF-alpha mRNA was present in syncytiotrophoblast but was low to absent in cytotrophoblast and villous stromal cells. Decidual and epithelial cells in maternal tissues contained TNF-alpha transcripts. In term placentas, both syncytiotrophoblast and villous stromal cells contained TNF-alpha mRNA, and transcripts were present in maternal cells in the decidua adjacent to the extraplacental membranes. In both first-trimester and term tissues, coincident expression of TNF-alpha mRNA and immunoreactive TNF-alpha was demonstrated. The results of this study show that TNF-alpha is synthesized by cells in both extraembryonic membranes and maternal tissues during human gestation and that transcription in specific types of cells is influenced by gestational age. These observations are consistent with a major role for TNF-alpha in the dynamic developmental events of human pregnancy.