The cytoskeletal protein LASP-1 differentially regulates migratory activities of choriocarcinoma cells.

PURPOSE: During healthy pregnancy, a distinct but limited invasion of trophoblast cells into the uterus occurs. In contrast, excessive trophoblast invasion is associated with placental choriocarcinoma (CC). Overexpression of the cytoskeletal protein LASP-1 was shown to contribute to cancer aggressiveness. Here, the yet unknown role of LASP-1 in CC cells is analysed. METHODS: Expression of LASP-1 in human primary carcinoma was assessed by immunohistochemistry and confirmed in CC-derived cell lines by immunocytochemistry, RT-PCR and Western blot. After down-regulation of LASP-1 expression with specific si-RNA in CC-derived cell lines, migratory and proliferative activities were analysed by matrigel migration assay and WST-8 test. RESULTS: LASP-1 expression was detected in human primary choriocarcinoma and in JEG-3, JAR and BeWo cells. Knock down of LASP-1 resulted in a decreased expression of LASP-1 protein in JEG-3 and JAR cells accompanied by a diminished migration and a decreased proliferative activity of these two cell lines. Knockdown of LASP-1 in BeWo cells failed. In consequence, migratory function and proliferation was unaffected. CONCLUSION: This is the first study describing LASP-1 expression in CC cells. Detecting an affection of migratory processes after LASP-1 silencing, we propose that LASP-1 could impact on metastasis of CC cells.

[Quality of life of young adults after a growth hormone therapy with childhood onset]. / Die Lebensqualität junger Erwachsener nach einer Wachstumshormonbehandlung im Kindesalter.

INTRODUCTION: Little is known about the health-related quality of life of young adults with childhood onset idiopathic growth hormone deficiency or neurosecretory dysfunction of growth hormone secretion, who have been treated with recombinant human growth hormone (GH). METHODS: Patients were diagnosed and treated with human growth hormone at the University Children´s Hospital in Erlangen (n=85). The data of both groups were merged for analysis, because no difference between idiopathic growth hormone deficiency and neurosecretory dysfunction of growth hormone secretion in auxological. Data were found. Health-related quality of life was cross- sectionally assessed after the end of growth hormone therapy with the Short Form-36 Health Survey and the Nottingham Health Profiles for which population based norm data are available. RESULTS: At the time of the survey, the patients (53 m, 32 f) were 23.5 ±â€…4.6 years old. At start of GH therapy, age was 10.5 ±â€…2.8 and at the end 16.3 ±â€…1,4 years. At start, height SDS was -3.20 ±â€…1.06. GH dose was 0,026 ±â€…0,012 mg/kg/d (daily s. c.-injections). The increase in height SDS after the end of GH therapy was 1.69 ±â€…1.22.  Compared to the reference population, patients reported significantly lower scores on the scales energy level, vitality, social functioning, indicating a greater social isolation, a stronger emotional reaction, an increased loss of mobility and a worse psychological state. CONCLUSION: Young adults report specific impairments after completion of GH therapy.

Thrombopoietin modulates the proliferation, migration and cytokine profile of decidual cell subsets during early gestation.

During early gestation, a considerable increase in different leukocyte subsets can be observed in the decidualized endometrium concomitantly to the invasion of cytotrophoblast cells (CTB). To date, it is still in question which factors induce this accumulation of immune cells and whether it is evoked by an in situ proliferation or by a migratory process. Studies on hepatoblastoma cells identified thrombopoietin (TPO) as a novel factor, which elicits dose-dependent chemotactic and chemokinetic effects. However, the impact and function of TPO on decidual cells has not been clarified yet. This study analyses the expression and function of TPO and its receptor c-Mpl in decidua during early gestation. Applying western blot analysis, we detected that TPO is expressed by decidual immune cells (uNK cells and CD14+ monocytes) as well as CTB and decidual stromal cells (DSCs). Expression of the different isoforms of c-Mpl was found in uNK cells, CD14+ monocytes and DSC. Studying the signalling pathway proteins in the uNK cells, an activation of STAT3/Tyr by TPO, was detected. The investigation of the proliferative effects of TPO on the decidual cell subsets revealed that TPO enhances the proliferation of uNK cells and CTB. No change of the proliferative activity after TPO incubation was found in DSC and even a decrease in CD14+ monocytes. In addition, TPO was observed to induce significantly the migratory activity of uNK cells, CD14+ monocytes and CTB. Investigating the effects of TPO on the cytokine profile of the isolated decidual cells, we observed a decrease in the secretion of IL-8, IL-10 and IL-1ß of isolated uNK cells, CD14+ monocytes and CTB, although these changes did not reach statistical significance. Thus, we here identified TPO as a novel factor modulating the proliferation, migration and possibly cytokine secretion of decidual cell subsets.

MIC-1 (a multifunctional modulator of dendritic cell phenotype and function) is produced by decidual stromal cells and trophoblasts.

BACKGROUND: Macrophage inhibitory cytokine-1 (MIC-1) is a multifunctional cytokine produced in high amounts by placental tissue. Inhibiting trophoblast invasion and suppressing inflammation through inhibition of macrophage activation, MIC-1 is thought to provide pleiotropic functions in the establishment and maintenance of pregnancy. So far, little is known about the decidual cell subsets producing MIC-1 and the effect of this cytokine on dendritic cells (DCs), which are known to play a distinct role in the development of pro-fetal tolerance in pregnancy. METHODS: To identify the decidual cell types expressing and secreting MIC-1, immunohistochemical staining, PCR experiments, western blot analysis and ELISAs were performed. Immature DCs (iDCs) were generated from peripheral blood-derived monocytes and differentiated in the presence of MIC-1 or dexamethasone (Dex) for control. Migratory and proliferative activity of DCs after MIC-1 exposure was investigated by migration and proliferation assay. Cytokine secretion after MIC-1 exposure was tested in isolated uNK cells, isolated CD14+ monocytes, monocyte-derived iDCs and mature DCs. Subsequently, the phenotype of DCs was studied using FACS analysis. To test the T-cell stimulatory capacity of pre-incubated DCs, mixed lymphocyte reaction was applied. Finally, the expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) after the exposure of MIC-1 to maturing DCs was analysed by western blot. RESULTS: Immunohistochemical staining, PCR and western blot experiments demonstrated that MIC-1 is mainly expressed by trophoblast cells and decidual stromal cells. Analysis of the MIC-1 secretion of decidual cell types by ELISA again characterized trophoblast and stromal cells as main producers. The migratory activity of iDCs was significantly induced by MIC-1. No changes in proliferative activity of DCs were observed after MIC-1 pre-incubation. The secretion of pro- or anti-inflammatory cytokines was not affected significantly by MIC-1. Studying the phenotype of DCs after MIC-1 exposure by FACS analysis, we observed that MIC-1 suppresses the expression of typical maturation molecules such as CD25 and CD83 as well as of CD86 during cytokine-induced DC maturation similar to Dex. In addition, T-cell stimulatory capacity of DCs was significantly reduced after MIC-1 exposure. MIC-1 was also able to increase slightly the expression of IDO (a key immunomodulatory enzyme promoting periphereal tolerance) in maturing DCs. CONCLUSIONS: We have identified MIC-1 as a novel factor (secreted by decidual cells in early pregnancy) that could promote the increase of a tolerogenic subtype of DC in decidua.

Human endogenous retrovirus K (HERV-K) is expressed in villous and extravillous cytotrophoblast cells of the human placenta.

Human endogenous retroviruses (HERVs) have been shown to be important in physiological and pathophysiological processes in humans. Several HERVs have been found to be expressed in the placenta-a tissue with special immunomodulatory functions that is responsible for nutrition of the embryo and the ability of the semiallogenic trophoblast to invade. The envelope proteins of HERV-W (also known as syncytin 1) and HERV-FRD (syncytin 2) were shown to be involved in cell fusion leading to the generation of the syncytiotrophoblast. Syncytin 2 was further shown to have immunosuppressive properties. Herein we analyse the expression of another HERV, HERV-K, which is characterised by open reading frames for all viral genes. Using immunohistochemistry and Western blot analysis, expression of the transmembrane envelope (TM) protein of HERV-K was studied in normal placental and decidual tissues obtained at different gestational ages. The TM protein was expressed exclusively in villous (VT) and extravillous cytotrophoblast (EVT) cells, but not in the syncytiotrophoblast or other cells. The expression of the TM protein of HERV-K in EVT cells was confirmed by Western blot analysis of isolated c-erbB2-expressing cytotrophoblast cells. Thus, this is the first report showing expression of the TM protein of HERV-K in normal human placental tissue with an exclusive expression in cytotrophoblast cells, suggesting a potential involvement of HERV-K in placentogenesis and pregnancy. Since retroviral TM proteins including the TM protein of HERV-K have immunosuppressive properties, expression of the TM protein of HERV-K may contribute to immune protection of the fetus.

Nuclear localisation of LASP-1 correlates with poor long-term survival in female breast cancer.

BACKGROUND: LIM and SH3 protein 1 (LASP-1) is a nucleo-cytoplasmatic signalling protein involved in cell proliferation and migration and is upregulated in breast cancer in vitro studies have shown that LASP-1 might be regulated by prostate-derived ETS factor (PDEF), p53 and/or LASP1 gene amplification. This current study analysed the prognostic significance of LASP-1 on overall survival (OS) in 177 breast cancer patients and addressed the suggested mechanisms of LASP-1-regulation. METHODS: Nucleo-cytoplasmatic LASP-1-positivity of breast carcinoma samples was correlated with long-term survival, clinicopathological parameters, Ki67-positivity and PDEF expression. Rate of LASP1 amplification was determined in micro-dissected primary breast cancer cells using quantitative RT-PCR. Cell-phase dependency of nuclear LASP-1-localisation was studied in synchronised cells. In addition, LASP-1, PDEF and p53 expression was compared in cell lines of different tumour entities to define principles for LASP-1-regulation. RESULTS: We showed that LASP-1 overexpression is not due to LASP1 gene amplification. Moreover, no correlation between p53-mutations or PDEF-expression and LASP-1-status was observed. However, nuclear LASP-1-localisation in breast carcinomas is increased during proliferation with peak in G2/M-phase and correlated significantly with Ki67-positivity and poor OS. CONCLUSION: Our results provide evidence that nuclear LASP-1-positivity may serve as a negative prognostic indicator for long-term survival of breast cancer patients.

Transketolase-like 1 expression correlates with subtypes of ovarian cancer and the presence of distant metastases.

Tumorbiology of ovarian cancer remains unclear. However, it is known that ovarian tumors, especially carcinomas, show elevated expression of glucose membrane transporters for facilitated glucose uptake. It can be assumed that increased glucose uptake leads to higher glucose metabolism. The energy resources of fully malignant transformed carcinomas are mainly supplied by aerobic glycolysis, for which several pathways are known. A key role in aerobic glycolysis is described for the transketolase enzymes. Recently, a novel transketolase-like enzyme called transketolase-like 1 (TKTL1) has been described that links aerobic glycolysis to the synthesis of fatty acids via production of acetyl-CoA. In order to investigate the role of TKTL1 for the progression of ovarian carcinomas, we examined paraffin sections of normal ovarian tissues, ovarian borderline tumors, and mucinous or serous papillary ovarian adenocarcinomas with respect to their expression of TKTL1. We identified a significantly elevated expression of TKTL1 in serous papillary ovarian adenocarcinomas, which correlates with poor prognostic parameters in the examined study group. Therefore, it can be assumed that TKTL1 plays a crucial role in ovarian cancer metabolism and that its expression predicts poor prognosis. Further investigations should be performed in order to evaluate whether this new enzyme is important for ovarian cancer tumorbiology and to analyze the potential role of TKTL1 as new target for specific antitumoral therapy.

Overexpression of LASP-1 mediates migration and proliferation of human ovarian cancer cells and influences zyxin localisation.

LIM and SH3 protein 1 (LASP-1), initially identified from human breast cancer, is a specific focal adhesion protein involved in cell proliferation and migration. In the present work, we analysed the effect of LASP-1 on biology and function of human ovarian cancer cell line SKOV-3 using small interfering RNA technique (siRNA). Transfection with LASP-1-specific siRNA resulted in a reduced protein level of LASP-1 in SKOV-3 cells. The siRNA-treated cells were arrested in G(2)/M phase of the cell cycle and proliferation of the tumour cells was suppressed by 60-90% corresponding to around 70% of the cells being transfected successfully as seen by immunofluorescence. Moreover, transfected tumour cells showed a 40% reduced migration. LASP-1 silencing is accompanied by a reduced binding of the LASP-1-binding partner zyxin to focal contacts without changes in actin stress fibre and microtubule organisation or focal adhesion morphology as observed by immunofluorescence. In contrast, silencing of zyxin is not influencing cell migration and had neither influence on LASP-1 expression nor actin cytoskeleton and focal contact morphology suggesting that LASP-1 is necessary and sufficient for recruiting zyxin to focal contacts. The data provide evidence for an essential role of LASP-1 in tumour cell growth and migration, possibly through influencing zyxin localization.

Increased expression of galectin-1 during the progression of cervical neoplasia.

Galectin-1, a member of the beta-galactoside-binding family, is widely expressed in epithelial and immune cells. It is involved in several normal and pathologic processes, such as cancer progression, metastasis, and immunobiology. Galectin-1 was found to be overexpressed in various cancer cells and the corresponding benign tissue. Therefore, it has been described as a marker for tumor progression in some malignancies. In the current study, the expression of galectin-1 was examined in 80 formalin-fixed, paraffin-embedded cervical tissues: 20 benign cervical specimen, 20 low-grade squamous intraepithelial lesions (LGSIL), 20 high-grade squamous intraepithelial lesions (HGSIL), and 20 invasive squamous cell carcinomas (ISCC). Immunohistochemical analyses showed that the intensity of the galectin-1 expression on stromal cells next to the transformed cells increased according to the pathologic grade: benign cervical tissue < LGSIL < HGSIL < ISCC (P < 0.001). The epithelial cells were always negative for galectin-1. These results suggest that galectin-1 expression on stromal cells increases with the histopathologic grade of cervical tissues, and it can be concluded that this increase is associated with the progression of cervical neoplasia.

Increased tartrate-resistant acid phosphatase (TRAP) expression in malignant breast, ovarian and melanoma tissue: an investigational study.

BACKGROUND: Tartrate-resistant acid phosphatase (TRAP) is a metalloprotein enzyme that belongs to the acid phosphatases and is known to be expressed by osteoclasts. It has already been investigated as a marker of bone metastases in cancer patients. In this study, which examined the value of serum TRAP concentrations as a marker of bone disease in breast cancer patients, we observed high concentrations of TRAP even in patients without bone metastases. To elucidate this phenomenon, we examined the expression of TRAP in breast cancer cells and the cells of several other malignancies. METHODS: TRAP concentrations in the serum of tumor patients were determined by ELISA. The expression of TRAP in breast, ovarian, and cervical cancer and malignant melanoma was analyzed by immunohistochemistry. RT-PCR and immunocytology were used to evaluate TRAP expression in cultured tumor cells. RESULTS: A marked increase in serum TRAP concentrations was observed in patients with breast and ovarian cancer, regardless of the presence or absence of bone disease. TRAP expression was found in breast and ovarian cancers and malignant melanoma, while cervical cancer showed only minimal expression of TRAP. Expression of TRAP was absent in benign tissue or was much less marked than in the corresponding malignant tissue. TRAP expression was also demonstrated in cultured primary cancer cells and in commercially available cell lines. CONCLUSION: Overexpression of TRAP was detected in the cells of various different tumors. TRAP might be useful as a marker of progression of malignant disease. It could also be a potential target for future cancer therapies.

Natural killer cells and dendritic cells at the human feto-maternal interface: an effective cooperation?

Human endometrium and in particular decidua, harbours a considerable population of immunocompetent cells. The most prominent of these are uterine natural killer (uNK) cells, which differ considerably from their peripheral blood counterparts in terms of both gene expression and function. Recently, the existence of DC-SIGN positive immature dendritic cells (DCs) in human decidua has been demonstrated. Evidence exists that immature DCs are required for the initiation and maintenance of peripheral tolerance, whereas mature DCs, which are only found in minimal amounts in human decidua, are associated with a Th1 polarization of T cells. Although the study of uNK-DC cross-talk is only beginning, it may in the future provide important insights into how acceptance of the fetus by the maternal immune system is mediated.

Brain metastases in breast cancer--an in vitro study to evaluate new systemic chemotherapeutic options.

BACKGROUND: Fifteen-30% of breast cancer patients develop central nervous system (CNS) metastases. The most potent drugs for the treatment of breast cancer like taxanes, anthracyclines and trastuzumab have limited efficacy for brain metastases. No standardized therapy has yet been established for this condition. Drugs with proven efficacy in the CNS and which are commonly used for primary brain tumors were applied. We evaluated the capacity of these drugs to inhibit breast tumor cell growth in vitro. MATERIALS AND METHODS: Twelve primary cell cultures of pulmonary/pleural metastases of breast cancer and 3 commercially available cell lines were used for non-radioactive cytotoxicity assays to evaluate the efficacy of 3 different concentrations of Topotecan, Cisplatin, Nimustine, Vincristine, Irinothecan, Caelyx (pegylated liposomal Doxorubicin) and Etoposide. RESULTS: Topotecan, Cisplatin, Caelyx and Vincristine showed significantly higher cytostatic activity in vitro than Irinotecan, Etoposide and Nimustine. With regard to the median cytotoxicity, the order of drugs in our assays was Topotecan, Cisplatin, Vincristine, Caelyx, Irinotecan, Etoposide and Nimustine. Nimustine showed almost no efficacy against breast cancer cells. CONCLUSION: Topotecan, Cisplatin, Vincristine and Caelyx seem to be suitable candidates for further clinical evaluation. The data and the "liposomal packaging" suggest that Caelyx might be effective in the CNS. Since pulmonary metastases are often associated with brain metastases, evaluatingprimary cell cultures from malignant pleural effusions could be a valuable approach for the testing of new cytostatic drugs for brain metastases.

S100 as an immunohistochemically-detected marker with prognostic significance in endometrial carcinoma.

BACKGROUND: Several studies have indicated that dendritic cells (DC) participate in anti-tumor immunity, possibly influencing the course of malignant disease. We tested whether tumor infiltration by S100+ DC could be a prognostic marker for endometrial cancer. MATERIALS AND METHODS: A retrospective study analyzing 115 tissue samples from patients with endometrial carcinoma and known histological grading as well as hormone receptor, Ki-67, Her-2/neu and p53 expression. Sections of paraffin-embedded endometrial tissue were immunohistochemically-stained with anti S100 antibody. Tumor infiltrating S100+ DC were counted via microscopic examination and calculated as S100+ DC per mm2 of tissue. RESULTS: Samples were divided into group 1: less than 10 S100+DC/mm2 (n = 44) and group 2: 10 or more S100+ DC/mm2 (n = 71). Correlation with clinico-pathological markers was calculated by Chi-square test. Compared to group 1, the DC-rich group 2 showed a higher level of differentiation (p=0.045), a lower overexpression of p53 (p=0.021) and less proliferation (p=0.028). DC infiltration was not correlated with Her-2/neu, hormone receptor status and FIGO-stage. Although no significant correlation could be seen, the DC-poor group samples seemed to correlate with a higher FIGO-stage compared to the DC-rich group. In uni- and multivariate analysis, DC infiltration proved to be a significant prognostic marker for adjusted survival but not for overall survival. CONCLUSION: Our results indicate that the immunohistochemical determination of S100+ DC could contribute to the identification of a high-risk subgroup and, therefore, would be a favorable prognostic factor for endometrial carcinoma. Our observation that pronounced DC infiltration is associated with good prognosis points to an important role of the host's immune system response for the clinical course of endometrial cancer.

Antigen-presenting cells in human endometrium during the menstrual cycle compared to early pregnancy.

OBJECTIVE: Human endometrium and early pregnancy decidua harbor a considerable and diverse population of antigen-presenting cells (APC). Changes in the number and distribution of macrophages and dendritic cells (DC) could point to a possible role of these immunocompetent cells in implantation and success of early pregnancy. METHODS: Uterine tissue was obtained from 22 women undergoing hysterectomy for bleeding disorders or dysmenorrhea and from 11 women undergoing legal abortion. Tissue was investigated with antibodies against CD14, CD68, CD83, DC-SIGN, Ki-67, and human leukocyte antigen (HLA)-DR using single and double immunohistochemical staining techniques. RESULTS: The number of CD14(+) cells was stable during all phases of the menstrual cycle and early pregnancy. In comparison to nonpregnant endometrium, DC-SIGN(+) cells showed a higher proliferation rate and were found associated in clusters with CD56(+) natural killer (NK) cells in early pregnancy. In the late secretory phase of the menstrual cycle, numbers of CD83(+) (P <.01) cells were significantly higher than in other endometrial phases and early pregnancy. HLA-DR(+) expression was significantly increased in early pregnancy but remained unchanged throughout the menstrual cycle. CONCLUSION: The presence of DC-SIGN(+) cells during the menstrual cycle and their proliferation in early pregnancy suggests an important role of these cells with regard to the balance between defense against pathogens and tolerance of the fetal allograft. Whether the increase of CD83(+) mature DC and CD68(+) macrophages in the late secretory phase is caused by hormonal stimuli and/or is due to changes of the cytokine/chemokine micromilieu remains to be investigated.

Immunocompetent cells in the endometrium of fetuses and children.

BACKGROUND: Although the immunocompetent cells of the adult human endometrium are well characterized, there is little information about these cells in the developing uterus. This study was undertaken to investigate the distribution of leukocyte subpopulations in the endometrium of fetuses and children. METHODS: Uterine tissue obtained at autopsy from fetuses (n = 11) and neonates/children (n = 9) between 17 weeks gestation and 5(1/2) years of age was investigated with antibodies against various leukocyte subsets by immunohistochemical staining techniques. RESULTS: The densities of CD45+ and CD68+ cells were significantly higher in the endometrium of neonates/children than in that of fetuses. CD14+ monocytes represented the largest leukocyte subpopulation in both groups. CD56+ natural killer cells and HLA-DR+ antigen-presenting cells were absent from fetal endometrium. There were no differences in density of CD3+ T cells between the two groups, but CD4+ T helper cells were found only in fetal endometrium. CONCLUSIONS: The endometrial leukocyte population of fetuses and small children is different from that seen in adult women. The appearance of CD56+ and HLA-DR+ cells in endometrium seems to be a post-natal event, which may be induced by the changes in hormone levels and/or the adaptation of the local immune system to the changing microenvironment.

[Expression of the molecules HLA-G and HLA-E modulates cytokine production of monocyte generated dendritic cells]. / Die Expression der Moleküle HLA-G und HLA-E moduliert die Zytokinproduktion monozytengenerierter dendritischer Zellen.

OBJECTIVE: Preferential secretion of Th1-like cytokine is mainly a property of monocyte derived dendritic cells (DC). Since normal early pregnancy is characterized by a shift towards a Th2-like cytokine pattern, it may be assumed that cytokine secretion by DC during early pregnancy could be modulated by the non-classical HLA molecules G and E present on invasive trophoblast. MATERIAL AND METHODS: DC were cultivated from monocytes isolated from peripheral blood mononuclear cells. DC were cocultured with K-562 leukemia cells lacking the class I and II HLA antigens transfected with either HLA-G or HLA-E or ultratransfected cells (controls) and the concentrations of IL-8, IL-10, IL-12p70, IL-18 and TNF-alpha were measured in the supernatants by ELISA. RESULTS: Coculture with ultratransfected cells resulted in a significant increase of the production of IL-8 and TNF-alpha by mature and immature DC and of IL-10 by immature DC (p < 0.01). When cocultured with HLA-G and HLA-E transfected K-562 cells, the secretion of IL-8 by immature and mature DC and that of IL-10 and TNF-alpha by immature DC was significantly (p < 0.01) decreased. The contact with HLA-G and HLA-E transfected cells had no effect on the production of IL-12p70 and IL-18 by DC. CONCLUSIONS: These results show that DC react with an increased cytokine release upon contact with cells lacking HLA class I and II antigens. The suppressive effect of HLA-G and HLA-E on the secretion of TNF-alpha (Th1 cytokine), IL-10 (Th2 cytokine) and IL-8 (chemokine) by immature DC could be interpreted as further evidence for the central immunotolerance role of HLA-G and HLA-E during early pregnancy.

Th1- and Th2-like cytokine production by first trimester decidual large granular lymphocytes is influenced by HLA-G and HLA-E.

During normal early pregnancy, a particular immune environment in the decidua and the expression of non-classical HLA-G and HLA-E molecules on the invading trophoblast are assumed to be essential for the tolerance of the fetus. To assess whether HLA-G and HLA-E influence the cytokine production of their putative target cells [large granular lymphocytes (LGL)], we analysed the concentrations of tumour necrosis factor (TNF-alpha), interferon (IFN)-gamma, interleukin (IL)-10, IL-13 and granulocyte-macrophage colony stimulating factor (GM-CSF) in supernatants of isolated first trimester LGL co-cultured with HLA-G or HLA-E transfected K-562 leukaemia cells lacking the classical HLA class I and II molecules. In comparison with that observed with untransfected K-562 cells, co-culture of LGL with HLA-G-expressing cells significantly reduced the concentration of all cytokines investigated (TNF-alpha, IL-10 and GM-CSF, P < 0.01; IFN-gamma and IL-13, P < 0.05). In contrast, co-culture of LGL with HLA-E-expressing cells significantly (P < 0.01) decreased only IL-10 production, although a strong tendency towards reduced IL-13 levels was also observed. In the co-culture system presented, membrane-bound HLA-G and, to a lesser extent, HLA-E expression affected cytokine release by decidual LGL in a manner not consistent with the Th1/Th2 paradigm. In conclusion, our data are indicative of a general immune-suppressive effect of HLA-G on LGL activity.

Interaction of Neisseria meningitidis with human dendritic cells.

Infection with Neisseria meningitidis serogroup B is responsible for fatal septicemia and meningococcal meningitis. The severity of disease directly correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-8. However, the source of these cytokines has not been clearly defined yet. Since bacterial infection involves the activation of dendritic cells (DCs), we analyzed the interaction of N. meningitidis with monocyte-derived DCs. Using N. meningitidis serogroup B wild-type and unencapsulated bacteria, we found that capsule expression significantly impaired neisserial adherence to DCs. In addition, phagocytic killing of the bacteria in the phagosome is reduced by at least 10- to 100-fold. However, all strains induced strong secretion of proinflammatory cytokines TNF-alpha, IL-6, and IL-8 by DCs (at least 1,000-fold at 20 h postinfection [p.i.]), with significantly increased cytokine levels being measurable by as early as 6 h p.i. Levels of IL-1beta, in contrast, were increased only 200- to 400-fold at 20 h p.i. with barely measurable induction at 6 h p.i. Moreover, comparable amounts of cytokines were induced by bacterium-free supernatants of Neisseria cultures containing neisserial lipooligosaccharide as the main factor. Our data suggest that activated DCs may be a significant source of high levels of proinflammatory cytokines in neisserial infection and thereby may contribute to the pathology of meningococcal disease.

Choriocarcinoma cells modulate the cytokine production of decidual large granular lymphocytes in coculture.

PROBLEM: How do major histocompatibility complex (MHC) I positive/negative choriocarcinoma (CC) cells affect the production of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-10 (IL-10), interleukin-4 (IL-4), and granulocyte-macrophage colony-stimulating factor (GM-CSF) by decidual CD56++ cells (large granular lymphocytes [LGL]), LGL-depleted decidual cells (WASH) and unseparated decidual cells (DEC) in cocultures? METHOD OF STUDY: Decidual tissue was obtained by legal abortions. CD 56++ LGL were isolated in a magnetic cell separator. Cytokines were measured by ELISA. Differences were analyzed for significance by Wilcoxons test. RESULTS: We found a significant increase of IL-10 in LGL/JEG-3 and of TNF-alpha in LGL/JAR cocultures compared to noncocultured decidual cells. There was a significant increase of IL-10 and TNF-alpha and a significant decrease of GM-CSF in WASH and DEC cocultures with both JEG-3 and JAR. IFN-gamma was only found in 3/11 cases of LGL/JAR cocultures. No IL-4 was found in any experiment. CONCLUSION: MHC class I positive/negative CC modulate the cytokine production of decidual LGL in different ways.