Distribution of decidual natural killer cells and macrophages in the neighbourhood of the trophoblast invasion front: a quantitative evaluation.

STUDY QUESTION: Do decidual natural killer (dNK) cells and decidual macrophages (dMph) become enriched in the vicinity of the trophoblast invasion front? SUMMARY ANSWER: Morphometric image analysis and areal cell density calculations, which excluded observer bias, showed an enrichment of decidual leukocytes in the neighbourhood of the trophoblast invasion front. WHAT IS KNOWN ALREADY: In previous studies, the number of decidual leukocytes was visually counted in medium- or high power fields. These methods, however, cannot reveal the exact spatial relationship between leukocytes and invasive trophoblast cells, and are therefore prone to subjective errors. Thus, a more objective approach is required. STUDY DESIGN, SIZE, DURATION: Applying a new method of morphometric image analysis, leukocyte populations were studied in human tissue fragments derived from first trimester placentation sites (n = 7) as well as in co-cultures of first trimester decidual tissue with placental villi of the same pregnancy representing an appropriate in vitro model of trophoblast invasion (n = 15). PARTICIPANTS/MATERIALS, SETTINGS, METHODS: First trimester decidual tissue was obtained from women undergoing elective terminations of pregnancy at 7-10 weeks of gestational age. Tissue sections were double-stained immunohistochemically for markers of dNK cells or dMph on one hand, and for invasive extravillous trophoblast cells on the other. To analyse the distribution of leukocytes, distinct cell compartments as well as cell neighbourhood areas were defined. Finally, relative areal cell densities were calculated and these data were compared with those of an in vitro model of trophoblast invasion as well as with tissue fragments derived from decidua parietalis without trophoblast cells. MAIN RESULTS AND THE ROLE OF CHANCE: At first trimester placentation sites, a higher density of dNK cells as well as of dMph was found in close proximity to the invasive trophoblast (P ≤ 0.01), compared with the average areal cell density of decidual leukocytes in the tissue with exclusion of the trophoblast. The highest areal cell density of leukocytes was determined up to a distance of 20 µm from the trophoblast cells, whereas in more distant regions it was even lower than average, indicating a migration of these leukocytes towards the trophoblast invasion front. In the three-dimensional co-culture model, however, we found an enrichment of dMph (P ≤ 0.01) but not of dNK cells (P > 0,05) in the neighbourhood of the invasive trophoblast. LIMITATIONS, REASONS FOR CAUTION: The morphometric image analysis depends on intense immunohistochemical staining that is free of background and cross-reactivity. WIDER IMPLICATIONS OF THE FINDINGS: The presented method will be useful not only for the investigation of recurrent miscarriage but also in the fields of tumour immunology and inflammation. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the European Commission (Network of Excellence 'The Control of Embryo Implantation (EMBIC)', FP6-512040, lead researcher: P.S.), and by the Franz Lanyar Foundation of the Medical University of Graz, Austria (Grant #347). None of the authors declared a conflict of interests.

Human trophoblast cells express the immunomodulator progesterone-induced blocking factor.

Progesterone-induced blocking factor (PIBF) is an immunomoduatory factor with anti-abortive properties. In this study, we present evidence that PIBF is synthesized in the human placenta and determine its cellular source. Expression of PIBF was analysed with polyclonal rabbit anti-human PIBF antibodies against recombinant N-terminal 48kDa PIBF in first trimester and term placental tissues and in the choriocarcinoma cell line JAR by means of immunohistochemistry, confocal laser scanning microscopy of double immunofluorescence labelling, and Western blotting; RT-PCR was performed for analysis of PIBF mRNA in isolated trophoblast cells. PIBF protein is present in human first trimester and term placenta. Double immunofluorescence labelling localised PIBF to the extravillous cytotrophoblast. PIBF is also expressed heterogeneously by syncytiotrophoblast and part of the villous cytotrophoblast. Full-length PIBF mRNA encoded by exons 1-18 is present in isolated first trimester and term villous trophoblast and in the choriocarcinoma cell line JAR. The corresponding 90kDa protein is expressed by JAR cells, first trimester and term villous trophoblast cells. In addition, these cells express PIBF proteins of 50 and 34kDa. Trophoblast is a source of PIBF; its tissue distribution suggests a role both in systemic and local (decidual) immunoregulation.

Inhibition of lung carcinoma cell growth by high density lipoprotein-associated alpha-tocopheryl-succinate.

Alpha-tocopheryl-succinate (alphaTS) is a synthetic, anti-neoplastic derivative of alpha-tocopherol. Here we studied the effects of free and high-density lipoprotein subclass 3 (HDL3)-associated alphaTS on the growth of human (A549) and mouse Lewis (LL2) lung carcinoma cells. Both free and HDL3-associated alphaTS inhibited A549 growth in a time- and concentration-dependent manner. Treatment of A549 cells with alphaTS-enriched HDL3 led to DNA fragmentation and a time-dependent decrease in immunoreactivity of poly(ADP-ribose)polymerase. Uptake experiments revealed a high capacity for selective alphaTS uptake in excess of holoparticle endocytosis. Overexpression of scavenger receptor class B, type I (SR-BI), the prime receptor mediating selective lipid uptake, in A549 cells resulted in significantly increased selective alphaTS uptake, a finding associated with complete cellular growth arrest. The present in vitro findings were verified in an in vivo model: tumor inoculation in C57BL6 was performed with either wild-type, beta-galactosidase- or SR-BI-overexpressing LL2 cells. After tumor inoculation, the animals received six consecutive intravenous injections of alphaTS. This experimental setup resulted in significantly reduced tumor burden in animals that were inoculated with SR-BI-overexpressing LL2 cells but not in animals inoculated with wild-type or beta-galactocidase-transfected cells. Based on our in vitro and in vivo findings, we propose that SR-BI could provide a novel route for HDL3-mediated drug delivery of anti-neoplastic drugs.

Sustained hypoglycemia affects glucose transporter expression of human blood leukocytes.

The scarce data available on leukocyte glucose transporter expression are contradictory and nothing is known about its regulation by glycemic state. Therefore, cytospin preparations of blood leukocytes were searched immunocytochemically for the high-affinity glucose transporters GLUT1, 3, and 4. Hypoglycemia-associated quantitative changes in transporter expression were assessed by flow cytometry. Granulocytes and monocytes stained for GLUT1, 3, and 4. Granulocyte GLUT4 levels were increased by 73% (P < 0.05) under hypoglycemic conditions, which was paralleled by a reduction in GLUT1 and a rise in GLUT3. In monocytes, GLUT3 was elevated by 134% (P < 0.05), whereas GLUT1 and GLUT4 remained unaffected upon hypoglycemia. Apart from a minor subpopulation, lymphocytes were negative for these carriers. In conclusion, GLUT1, 3, and 4 are abundantly expressed in granulocytes and monocytes. The differential response of individual isoforms to hypoglycemia may represent a mechanism to protect the cells from the stress of glucose deprivation.

Localization of indoleamine 2,3-dioxygenase in human female reproductive organs and the placenta.

Indoleamine 2,3-dioxygenase (IDO) has been implicated in regulation of feto-maternal tolerance and protection against intracellular and extracellular pathogens. We have studied the expression of IDO in the human female reproductive tract and the placenta by immunohistochemistry. Endometrial glandular and surface epithelial cells showed increasing IDO expression during the course of the menstrual cycle. In term placenta, IDO was irregularly localized to the mesenchymal core and found in isolated areas of the syncytiotrophoblast. In first trimester pregnancy, IDO was not present in placental villi, but was present in glandular epithelium of the decidua, and there were distinctly positive cells scattered in the connective tissue, sometimes in conjunction with lymphoid aggregates. The endothelium of spiral arteries and of capillaries showed some, albeit no generalized, reactivity. IDO was also present in the epithelium of cervical glands and of Fallopian tubes. Specificity of antibody binding was confirmed by Western blot analysis. IDO mRNA was detected in first trimester decidua as determined by RT-PCR. IDO is secreted, as determined by analysis of cervical mucus by high pressure liquid chromatography for the presence of the tryptophan metabolite L-kynurenine, indicating IDO activity. Our results support the concept of IDO providing a mechanism of innate immunity protecting against ascending infections in the female reproductive tract.

Report from the IFPA meeting in conjunction with the 8th EPG meeting, Schladming, Austria, September 26-29, 1999.

Some 350 participants from 35 countries gathered in the small town of Schladming in the Austrian Alps, where the meeting of the International Federation of Placental Associations took place in conjunction with the 8th meeting of the European Placenta Group. The congress was organized by Gernot Desoye and Gottfried Dohr (both from Graz, Austria). The topics covered all aspects of placenta research. One symposium, two workshops and a number of posters were specifically devoted to placental immunology.

Reaction patterns of monoclonal antibodies to HLA-G in human tissues and on cell lines: a comparative study.

We compared the immunohistochemical reaction patterns of HLA-G-specific antibodies 87G, 4H84, G233, 16G1, and BFL.1 on human placentas under three different preparative conditions and on cryosections of other human tissues. Human and murine cell lines, either naturally expressing or transfected with HLA-G, were analyzed for their reaction patterns by immunocytochemistry and flow cytometry. Antibodies HCA2, TP25.99, W6/32 to classical HLA class I, anti-beta(2)-m and various non-HLA-G expressing cell lines were used as controls. The binding ability of the antibodies depends on the histotechnical procedure used. 4H84 and HCA2 bind to HLA-G despite aldehyde fixation and also paraffin embedding. 87G does not bind HLA-G in studies involving fixation with aldehydes. G233 labels HLA-G in aldehyde fixed but not paraffin embedded tissues. By immunocytochemistry HLA-G2 is merely detected with antibodies 4H84 and HCA2. MAb 16G1 binds to HLA-Gsol transfected cell lines only. The HLA-G specificity of mAb BFL.1 was considered as doubtful because it failed to react with most of the HLA-G transfected cell lines. Binding of 87G to the surface of monocytes or U-937 cells stimulated with IFN-gamma and GM-CSF is an Fc-receptor mediated phenomenon.

HLA-G in reproduction: studies on the maternal-fetal interface.

For more than a decade, investigators have known that membrane-bound and soluble isoforms of the HLA class Ib molecule, HLA-G, are present at the maternal-fetal interface. Although it is clear that extravillous cytotrophoblast cells are major producers, other cells may also contribute. Recent studies in our laboratory raised the question of whether soluble isoforms might reach the maternal and/or fetal blood circulation. A capture enzyme-linked immunoabsorbent assay (ELISA) identified soluble HLA-G (sHLA-G) in maternal blood throughout pregnancy but failed to detect sHLA-G in cord sera. Further studies suggested that the circulating proteins may be either free heavy chain (sHLA-G1 and/or sHLA-G2) or exclusively sHLA-G2. To study the potential function(s) of the soluble isoforms to modulate local or systemic immunity in mothers, we generated recombinant sHLA-G1 and -G2 in both prokaryotic and eukaryotic systems. Preliminary experiments conducted using DNA microarray analysis suggest that sHLA-G is capable of modulating gene expression in blood mononuclear leukocytes. Potential local targets were also identified; decidual and placental macrophages but not trophoblast cells contained mRNA encoding two of the known receptors for HLA-G, ILT2 and ILT4. Collectively, the studies are consistent with the hypothesis that sHLA-G produced at the maternal-fetal interface targets to the cells of the monocyte/macrophage lineage and modulates their functions for the benefit of pregnancy.

Translocation (3;5)(p26;q13) in a patient with chronic T-cell lymphoproliferative disorder.

A 67-year-old patient with large granular lymphocyte (LGL) leukemia is described. At fluorescence-activated cell sorting (FACS) analysis of the peripheral blood, the lymphocytes were positive for CD3, CD4, CD5, CD29, CD45RA, CD57, and TCR alpha/beta and negative for CD7, CD8, CD16, CD56, CD19, CD22, and TCR gamma/delta. Bone marrow histology and immunohistochemistry did not reveal any lymphocyte infiltration. Cytogenetic examination of peripheral blood cultures showed a clone with the karyotype 46,XY,t(3;5)(p26;q13). Molecular analysis revealed rearrangement of the gamma-T-cell-receptor chain. The region 3p25-3p26 which harbors the von Hippel-Lindau tumor suppressor gene and the RAF1 oncogene has been rearranged in a few cases of T-cell leukemia. The translocation in this case has not yet been described and may reflect an alternative mechanism in the pathogenesis of these disorders.

Phenotypic comparison of natural killer cells from peripheral blood and from early pregnancy decidua.

In this descriptive flow cytometric study we analyzed the phenotype of human large granular lymphocytes from the decidua (DLGL) of first-trimester pregnancy. Expression of CD56 at high density on DLGL suggests a relationship to the small CD56bright+ subpopulation of peripheral blood natural killer (PBNK) cells. In comparison, these cell types differ in respect to the expression of a variety of adhesion molecules and receptors implicated in homing, migration and activation. In contrast to CD56bright+ PBNK cells, DLGL were still brighter for CD56 and show higher expression for CD29 and CD45RO. Less expression was found for CD15s, CD43, CD44, CD45RA, CD62L and HLA-DR. CD11a to c and CD18 were distributed in bimodal form on DLGL, part of the cells being negative. In summary, we found considerable differences between the cell surface marker profiles of DLGL and PBNK cells (subpopulations of the latter being separately analyzed).

Hyporeactivity of neonatal platelets is not caused by preactivation during birth.

UNLABELLED: Little information exists concerning platelet function in neonates due to the small blood volume. Most studies using conventional aggregation methods have shown a diminished response to various agonists. This is in contrast to the lack of a bleeding tendency and to a short bleeding time in healthy neonates. In previous work we have shown that in healthy term neonates even after an uncomplicated delivery signs of thrombin generation can be demonstrated. This activation of the clotting system may also lead to platelet activation in the neonate. We investigated by means of flow cytometry the expression of activation markers GMP 140 and GP 53 on the surface of neonatal platelets and GP 53 intracellularly by means of a newly developed assay. The expression of GMP 140 and of GP 53 after thrombin stimulation was significantly higher on adult rather than neonatal platelets, while there was no difference between neonatal and adult platelets using GP 53 as intracellular marker. In none of the healthy term neonates after an uncomplicated delivery were activated platelets demonstrated. CONCLUSION: Our data show that the decreased reactivity of neonatal platelets is not caused by preactivation during birth but rather represents a developmental phenomenon. Possibly the observed hyporeactivity of neonatal platelets to thrombin helps to prevent harmful effects of birth stress on the clotting system of neonates.

Differential phenotypic properties of human peripheral blood CD56dim+ and CD56bright+ natural killer cell subpopulations.

We investigated the presence of integrins and various other cell surface markers on the surface of freshly isolated CD56dim+ and CD56bright+ NK cells, the latter comprising a mean of 6.5% of total peripheral blood (PB) CD3-CD56+ NK cells. This small subpopulation stained more intensively for CD2, CD11c, CD15s, CD44, CD49e, CD55, CD62L, HLA-DR, and GZS-1, whereas there was no expression of CD49f in contrast to CD56dim+ cells. The myeloid antigen CDw65 was present on 11% of CD56bright+ cells, other myeloid markers were not found. 28% of CD56bright+ cells were positive for CD45R0. These results support the notion that CD56bright+ NK cells, based on their different marker profile, may possess different functional and biodistributional properties and--due to the signal-transducing capabilities of several adhesion molecules--differential patterns of stimulatability compared to the majority of PB-NK cells.

The monoclonal antibody GZS-1 detects a maturation-associated antigen of human spermatozoa that is also present on the surface of human mononuclear blood cells.

A monoclonal antibody (GZS-1) has been generated by fusion of mouse myeloma cells with spleen cells from BALB/c mice immunised with human sperm cells. The antibody was determined to be an IgG1. The corresponding antigen is present on the whole surface of ejaculated human spermatozoa. It is not detectable on spermatozoa of other mammalian species (rabbit, cat, dog, sheep, boar, bull, horse). In human male genital organs, immunostaining with GZS-1 is observed on sperm cells in the epididymis and the ductus deferens together with the lining epithelium of those organs. No reactivity of sperm cells or germ cell precursors in the testis has been detected. Functional tests using the antibody show a strong inhibitory effect of human sperm in the hamster egg penetration assay. Furthermore, the GZS-1 antigen is detectable on the surface of human lymphocytes and monocytes by immunogold electron microscopy and FACS analysis. By Western blotting of human sperm and seminal plasma performed under reducing conditions immunostaining was detected at 21-25, 31, 51-54, and 62 kDa. The reaction with human lymphocytes shows one major band at 62 kDa and additional bands at 31 and 54 kDa. The results suggest that the monoclonal antibody GZS-1 may recognise an antigen which is secreted from the epithelial cells of the epididymis and binds to ejaculated spermatozoa as a sperm coating antigen. This component may be involved in the capacitation of the sperm and the acrosome reaction. Molecules that are expressed both on sperm and on immunocompetent cells may be relevant for the regulation of immunological processes or for the development of the related immunological tolerance of sperm in the female reproductive tract.

Flow cytometric detection of intracellular platelet antigens.

Exocytosis following platelet activation leads to translocation of CD62P (P-selectin), CD63, and thrombospondin from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. We report the detectability of these molecules preformed--prior to platelet activation--inside the cytoplasm of resting platelets. Two different methods are described, using either methanol or the Fix&Perm kit for cell membrane permeabilization. In addition, interleukin(IL-)1 alpha is shown to be present in platelet cytoplasm after methanol treatment but not after permeabilization using Fix&Perm. Whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining only. Our data demonstrate the feasibility of the methods described for the detection of intracellular platelet molecules.

Platelets contain interleukin-1 alpha and beta which are detectable on the cell surface after activation.

Activated platelets have been shown previously to exhibit membrane-bound IL-1 bioactivity, which leads to the question of localization of the cytokine in platelets. Using immunocytological and flow cytometric techniques, we found IL-1 alpha and IL-1 beta in the cytoplasma of both resting and thrombin-activated platelets. Immunogold-silver staining of the cell surface of activated platelets as well as preembedding antibody treatment of platelets revealed the presence of IL-1 (alpha and beta) in low density on the surface of intact cells in contrast to distinct enrichment in the cytoplasma of damaged platelets. Fibrin fibres present between cells indicated adsorbance of IL-1. There was also weak binding of anti-IL-1 alpha to the surface of thrombin-activated platelets as shown by flow cytometry. Following activation there appears to be some transfer of IL-1 onto the cell surface of activated cells, the bulk of the cytokine, however, is probably not released prior to platelet disintegration. In summary, we present evidence for the presence of both IL-1 alpha and IL-1 beta in resting and activated platelets without being able to demonstrate localization of the cytokines to specific subcellular structures.

Response of human NK cells to IL-6 alterations of the cell surface phenotype, adhesion to fibronectin and laminin, and tumor necrosis factor-alpha/beta secretion.

In vitro effects of human recombinant IL-6 (1-1000 U/ml) on highly enriched human NK CD3-CD56+ cells (94% +/- 2; mean +/- SEM; n = 8), obtained from PBL were studied. IL-6 induced low levels of NK cell proliferation (7- to 30-fold during 6-day incubation), which was IL-2-independent, because IL-6 did not induce detectable IL-2 production by NK cells. Two-color flow cytometry analysis demonstrated that incubation of NK cells with IL-6 at the optimal concentration of 250 U/ml for 6 days significantly increased the proportion of NK cells expressing the following activation Ag: CD25 (26% +/- 17, mean +/- SEM vs 4% +/- 1 in control, n = 5), CD54 (44% +/- 17 vs 9% +/- 3), HLA-DR (29% +/- 13 vs 12% +/- 4), CD69 (45% +/- 7 vs 12% +/- 3), and CD71 (34% +/- 17 vs 6% +/- 2). The mean fluorescence intensity of these activation Ag was increased as well. IL-6 induced expression of CD49b (alpha-chain of VLA-2, 20% +/- 11 vs 2% +/- 1) and CD49c (alpha-chain of VLA-3, 43% +/- 17 vs 5% +/- 3), which are not expressed on resting NK cells. IL-6 also enhanced the fluorescence intensity of beta 1 integrins, CD49d, CD49e, and CD49f, expressed on NK cells. IL-6-stimulated NK cells showed significantly increased integrin-mediated adhesion to fibronectin- or laminin-coated plates (26 +/- 3 mean % cells adhering +/- SEM vs 15 +/- 4 in control for FN and 19 +/- 1 vs 11 +/- 1 for LM, p < 0.05 for both) as determined in a 3 h binding assay. As assessed by inhibition of adhesion using mAb to the VLA-2, -3, -4, -5, and -6, NK cell adhesion to fibronectin was mediated by VLA-4 and 5, and their adhesion to laminin by VLA-3 and -6. NK cells incubated in the presence of IL-6 were found to produce a factor cytostatic to WEHI-164 clone 13 target cells. This effect was partly, although significantly, blocked by neutralizing antibodies to TNF-alpha or TNF-beta. Our data demonstrate that IL-6 can directly activate human NK cells, but is a less potent NK cell activator, for all activation and functional parameters studied, than IL-2.

Generation of adherent lymphokine activated killer (A-LAK) cells from patients with acute myelogenous leukaemia.

Successful generation of adherent lymphokine-activated killer (A-LAK) cells, highly-enriched in CD3-CD56+ antitumour effector cells, from the peripheral blood of ten patients with acute myelogenous leukaemia (AML) is described. The AML patients were either untreated or in remission. In vitro proliferation of A-LAK cells in patients with AML was generally poor, unless the cells were cocultured with irradiated concanavalin A (ConA)--prestimulated allogeneic PBL or selected lymphoblastoid cell lines (LCL) as feeder cells. Using this method, the median fold proliferation was 290 for A-LAK cells cultured with ConA-activated feeders and 291 for those grown with LCL, both significantly higher (both P less than 0.001) than the median of 2-fold expansion observed in cultures without feeders. A-LAK cultures generated in the presence of feeders consistently showed good enrichment (up to 90%) in CD3-CD56+ NK cells. Although NK activity was not significantly increased on a per cell basis in A-LAK cells grown with feeder cells, total lytic activities against both NK-sensitive target, K562, and NK-resistant target, Daudi, were significantly greater (P less than 0.02 for ConA-PBL feeders and P less than 0.005 for LCL feeders) as compared to those in paired cultures without feeders. In the presence of irradiated allogeneic feeder cells, 7/10 AML patients generated A-LAK cultures characterised by good proliferation and increased purity as well as cytotoxic activity.

Depressed ability of patients with melanoma or renal cell carcinoma to generate adherent lymphokine-activated killer cells.

Adherent lymphokine-activated killer (A-LAK) cells, selected from peripheral blood lymphocytes (PBL) of normal human donors by adherence to plastic, and cultured in the presence of interleukin 2 (IL-2), are highly enriched in CD3-CD56+ natural killer (NK) cells. These IL-2-activated NK cells proliferate extensively upon further culture in conditioned medium containing IL-2. In contrast, we previously found that with PBL of some patients with advanced cancer, the same procedure often failed to yield high enrichment of NK cells or substantial expansion in the numbers of these effector cells. To obtain sufficient numbers of A-LAK cells for adoptive immunotherapy in cancer patients, an improved method for generation of human A-LAK cells with irradiated mitogen-stimulated allogeneic PBL- or Epstein-Barr virus-transformed lymphoblastoid cell lines was introduced. In paired experiments, A-LAK cultures with feeder cells showed significantly enhanced IL-2-driven proliferation of A-LAK cells obtained from normal donors or patients with metastatic melanoma, renal cell carcinoma, and other types of solid cancers. The growth-promoting effect of feeders for A-LAK cells resulted in significantly improved expansion of CD3-CD56+ (NK) effector cells in A-LAK cultures established from normal donors. Cells in these cultures also had significantly higher levels of antitumor cytotoxicity against K562 and Daudi targets than did A-LAK cells grown in the absence of feeder cells. Enrichment in CD3-CD56+ cells and antitumor activity also occurred in patient A-LAK cultures supplemented with mitogen-stimulated feeder cells, but was not statistically significant. Overall, despite improved proliferation and CD3-CD56+ cell content of A-LAK cultures established in the presence of mitogen-activated feeder cells, only 39% (21/54) of patients tested generated A-LAK cells that would be judged acceptable for large-scale therapeutic use by criteria based on fold expansion and purity of A-LAK cells. These results suggest that in comparison to normal individuals, NK cells of many patients with advanced solid tumors are defective in their ability to respond by proliferation to IL-2 even in the presence of exogenously supplied growth factors.