Schwannoma of the epiglottis: case report focusing on clinico-pathological aspects.

Laryngeal schwannomas are uncommon lesions with only few cases reported. Herein we present a further case of a schwannoma of the epiglottis, occurring in a 62-year-old with a clinical history of a cutaneous malignant melanoma and laryngeal glottic keratosis. The schwannoma was incidentally discovered as a small polypoid lesion located on the laryngeal surface of the epiglottis and was removed endoscopically. The procedure was uneventful and the patient is well six months later. Authors focus on the diagnostic and therapeutic options for this unusual lesion and discuss the differential diagnosis of the spindle cell proliferation of the larynx.

Mast cell production of TNF-alpha induced by substance P evidence for a modulatory role of substance P-antagonists.

Unregulated increasing of Tumor necrosis factor-alpha (TNF-alpha) could be pathogenic in inflammatory diseases. The aim of this study was to investigate the anti-inflammatory role of the Substance P-antagonists (SPAs) through the inhibition of histamine release (HR) and TNF-alpha production from mast cell. Rat peritoneal mast cells (PMC) stimulated with Substance P (SP), in the presence of SPAs or not, were analyzed for HR and TNF-alpha protein production. Competitive Polymerase Chain Reaction, with an internal standard competing with target cDNA for the same primers, was used to determine the TNF-alpha mRNA expression. We show that the increase of either HR and TNF-alpha levels in peritoneal (PMC) after induction with SP was inhibited by pre-incubation with SPA or with the Peptide 101 (P101), while the [D-Pro2, D-Phe7, D-Trp9]-SP (dSP) had no effect. Neuraminidase treatment suggests that dSP, as well as SP, interacts with sialic acid residues on the cell surface. Moreover, SPA and P101 also inhibit the release of histamine and TNF-alpha induced by dSP suggesting that a receptor-independent mechanism is involved. These data could be useful to better understand the mechanisms involved in the mast cell activation and TNF-alpha production in the inflammatory diseases where SP is involved.

Histamine and tumor necrosis factor-alpha production from purified rat brain mast cells mediated by substance P.

The effect of cytokines and neuropeptides on neuroimmune functions has not been completely elucidated and recent evidence suggests an important role for these molecules linking the neuroimmune system and inflammatory events. The aim of this study was to analyse the effect of substance P (SP) on a pure population of hypothalamic brain mast cell (BMC). A pure population of BMC challenged with 10(-8) M SP gave 78% histamine release (HR) and secreted 600 pg/ml of tumor necrosis factor-alpha (TNF-alpha) as determined by ELISA. The production of TNF-alpha mRNA, measured by a competitive RT-PCR, was 14 times higher than that in unstimulated cells. The secretion of histamine and TNF-alpha from BMC after stimulation with SP supports the hypothesis that these mediators could induce an initial response in neuroinflammatory diseases.

Evidence that brain mast cells can modulate neuroinflammatory responses by tumour necrosis factor-alpha production.

Tumour necrosis factor-alpha (TNF-alpha) levels in mammalian brain increase during neuroinflammatory diseases. We used the competitive polymerase chain reaction (PCR) to quantify the amount of TNF-alpha in stimulated and unstimulated brain mast cells (BMC). A cDNA fragment shortened by a deletion of 56 bp was used as an internal TNF-alpha-specific standard. The immunological stimulus resulted in enhanced TNF-alpha mRNA expression and increased release of histamine and TNF-alpha. This is the first time that BMC showing functional FCepsilonRI-bound IgE receptors have been purified. Our results support the hypothesis that BMC mediators might induce an initial response in neuroinflammatory diseases.

Substance P selectively activates TNF-alpha mRNA in rat uterine immune cells: a neuroimmune link.

Substance P (SP) is a neuropeptide which influences the interaction between the nervous and immune systems. It is an important modulator of cytokines, including tumour necrosis factor-alpha (TNF-alpha) whose role during the reproductive processes has been established. We have investigated the effects of SP on TNF-alpha mRNA expression in macrophages and mast cells (MC) isolated from rat peritoneum and uterus. Cell supernatants were analysed for their histamine content as a measure of stimulation. SP alone increased TNF-alpha expression in peritoneal MC but not in peritoneal macrophages. The addition of SP resulted in a six-fold enhancement of TNF-alpha expression in uterine MC whereas no stimulation was observed in macrophages as determined by competitive polymerase chain reaction (PCR).

Inhibitory effect of neuraminidase on SP-induced histamine release and TNF-alpha mRNA in rat mast cells: evidence of a receptor-independent mechanism.

The neuropeptide substance P (SP) is a mediator of neuro-inflammation and can play a role by induction of histamine release (HR) and TNF-alpha. However, its effect on the heterogeneous response of mast cells (MC) has not been completely studied. We have established that the SR can induce 25% of HR in highly purified rat uterine MC at diestrous but not at proestrous phases of the reproductive cycle and 88% of HR in peritoneal mast cells (PMC). We also found 2.2 fold increase in TNF-alpha mRNA at diestrous, in SP stimulated uterine MC versus control and 2.7 fold increase in PMC; RT and competitive PCR were used to amplify the TNF-alpha mRNA. We have thereafter investigated the mechanism whereby the binding of SP to sialic acid on the MC membrane, could trigger secretion of histamine and induction of TNF-alpha mRNA. The neuraminidase pretreatment (0.1 U/ml) inhibited SP-stimulated HR from either uterine MC and PMC (98% and 50%, respectively) and totally inhibited SP-stimulated TNF-alpha mRNA levels. The neuraminidase effect was not toxic, since it was not observed in IgE mediated HR and TNF-alpha mRNA levels. In conclusion, the inhibitory effect of the neuraminidase on the SP-mediated increase of histamine and TNF-alpha mRNA, suggests that the SP-sialic acid interaction could have a role in the MC heterogeneous response.

A factor secreted by human embryo stimulates cytokine release by uterine mast cell.

The existence of a biochemical network of embryo-maternal communication implies that various secreted molecules constitute a signal-response mechanism, important for the process of embryo implantation in mammals. Here we report the purification of a protein with an apparent molecular weight of 136 kDa, responsible for a 2000-fold increase in embryo-derived histamine-releasing factor (EHRF) activity. This protein, purified from medium from the in-vitro culture of 2-8-cell human embryos, by means of affinity chromatography, was capable of binding immunoglobulin (Ig)E as demonstrated by immunoblotting and enzyme-linked immunosorbent assays. We found EHRF was capable of inducing release of histamine and cytokines in vitro from rat uterine tissue, collected on day 4 of pregnancy (preimplantation stage of embryo development). When EHRF was used as a secretagogue, granulocyte macrophage-colony stimulating factor (GM-CSF) release increased from 3 to 55 pg/g (P < 0.01) and tumour necrosis factor-alpha (TNF-alpha) release increased from 0 to 2.1 ng/g (P < 0.01), as detected by enzyme-linked immunosorbent assay. A simple method was used to purify uterine mast cells using an IgE-Sepharose affinity chromatography column and the purity (90%) was checked with Dynabeads coupled to specific rat IgE antibody. When purified mast cells were stimulated with EHRF in the same way as the uterine explants, a similar pattern of GM-CSF and TNF-alpha release was obtained. We also describe the reverse transcription-polymerase chain reaction (RT-PCR) of GM-CSF and TNF-alpha mRNA from purified uterine mast cells. On day 4 of pregnancy only the mRNA of TNF-alpha was found and this increased after stimulation with the EHRF. In conclusion, the data presented suggest that uterine mast cells isolated during the preimplantation stage release cytokines in vitro following interaction with an embryo factor.

Effect of substance P on uterine mast cell cytokine release during the reproductive cycle.

There is increasing evidence that neuropeptides, steroid hormones and inflammatory cytokines influence the immune response during the reproductive cycle. In the present study, we focus on the effects of neuropeptide Substance P (SP) during the pre-implantation stage of embryo development (day 4 of pregnancy), at pro-estrus and di-estrus (two phases with different hormonal states). We found heterogeneous responses to SP and anti-IgE by the rat uterine mast cells (MCs), as detected by ELISA. In fact, MCs purified from uteri on day 4 of pregnancy released histamine, granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) in response to anti-IgE, but not to SP. When pre-incubated with SP, the release to anti-IgE was significantly enhanced compared to anti-IgE alone. Exposure of SP to antibodies to SP, prior to pre-incubation with MCs, negated the SP effect on IgE-mediated release. At the pro-estrus phase SP showed similar behavior as on day 4 of pregnancy, whereas at the di-estrus phase SP alone was capable of inducing release of histamine and cytokines from purified uterine MCs. Moreover, non-quantitative RT-PCR analysis of the TNF-alpha mRNA level suggested an SP stimulation at the di-estrus phase, but neither on day 4 of pregnancy nor at the pro-estrus phase. Taken together, these data strongly suggest that SP can modulate IgE-mediated uterine MC release of histamine and inflammatory cytokines in different ways, depending on the phase of the reproductive cycle.

Oestradiol enhances in vitro the histamine release induced by embryonic histamine-releasing factor (EHRF) from uterine mast cells.

The relationship between maternal hormones and factors secreted by the implanting embryo is still controversial. We have analysed the in-vitro effect of oestradiol and human embryo-derived histamine-releasing factor (EHRF) on histamine release from rat uterine mast cells. Rat uterine mast cells which were preincubated with oestradiol and then challenged with human EHRF gave histamine release values two- to threefold higher than those without preincubation. The enhancement observed was time- and temperature-dependent. A similar enhancement was obtained with human sensitized basophils but not with rat peritoneal mast cells. Oestradiol, used as a direct challenge, did not induce any histamine release from either rat uterine or peritoneal mast cells, or from human sensitized basophils. Oestradiol preincubation also enhanced the histamine release induced by anti-IgE but did not enhance the histamine release induced by substance P or compound 48/80, two secretagogues that are not mediated by IgE. Moreover, uterine fragments derived from rats at various oestrus phases, with different amounts of endogenous oestrogen, were challenged in vitro with EHRF. The release of histamine by mast cells was higher at the proestrus and preimplantation phases than at dioestrus. All these findings suggest that the interaction of oestradiol with rat uterine mast cells was capable of enhancing in vitro the histamine releasing effect of EHRF.

Modulation of rat peritoneal mast cell and human basophil histamine release by estrogens.

This study was undertaken to investigate the effect of estrogens on the histamine release mediated by IgE in rat peritoneal mast cells (PMC) and in sensitized human basophils. The estrogens were found to enhance the histamine release of either rat PMC and sensitized human basophils upon stimulation with anti-IgE. The enhancement was estrogens dose-dependent reaching the maximum value of 23% for rat PMC and 41% for sensitized human basophils stimulated with anti-IgE upon preincubation with 10(-8) M estrogens. Moreover, when purified PMC were used, the enhancing effect was still detected, suggesting a direct interaction between estrogens and mast cells. The enhancing effect took place quite rapidly reaching plateau levels in about 60 min. Basophils preincubated at 4 instead of 37 degrees C did not give any appreciable enhancement, suggesting that it was temperature-dependent and that the effect observed was not due to cytotoxicity. Incubation of PMC or human basophils with estrogens alone, without challenge with anti-IgE, did not give any detectable histamine release. The enhancement of histamine release by estrogens is probably mediated by IgE molecules present on the cell membrane, since this effect was not observed on challenge with substance P or compound 48/80, two segretagogues known to induce histamine release not via IgE.

Dispersal of rat uterine mast cells and their functional response to an embryo-derived histamine releasing factor: a possible model for embryo implantation.

Rat uterine tissue was dissociated by enzymatic digestion with collagenase and viable mast cells were obtained. Their viability was assessed by the ability to exclude trypan blue dye and to respond functionally to different stimuli. Challenge with anti-IgE gave a calcium-dependent histamine release of 49%, whilst the undigested uterine fragments gave 23%. Moreover, they were capable of releasing histamine on challenge with the compound 48/80, suggesting a similarity with connective tissue mast cells. This similarity was further supported by their insensitivity to aldehyde blocking of dye binding. The final dispersed cell preparation contained 3 X 10(5) mast cells/g of uterine tissue, representing about 2% of total nucleated cells. The total histamine content of the undigested uterus was 2.5 micrograms/g of tissue, whilst after digestion the histamine determined was 1.2 pg per mast cell with a yield of 14%. The total histamine content of the uterus changed throughout the reproductive cycle, increasing before ovulation, reaching a maximum during ovulation and then decreasing after embryo implantation. This suggests that the implanting embryo, interacting with the uterus, may be capable of inducing the release of histamine. The embryo-derived histamine releasing factor (EHRF) that we have described previously is capable of inducing 22% histamine-release on uterine mast cells, thus supporting this hypothesis.

Identification of a histamine-releasing factor secreted by human pre-implantation embryos grown in vitro.

Human pre-implantation stage embryos cultured in vitro spontaneously secreted a factor capable of inducing histamine-release from human blood basophils. The embryo-derived histamine-releasing factor (EHRF) has been isolated from the culture medium by means of heparin-Sepharose affinity chromatography. The factor bound to the column and was then eluted by increasing the buffer molarity to 1.5 M NaCl. EHRF was detected using an enzymatic-isotopic microassay and sensitized basophils known to undergo release with anti-IgE. The EHRF-induced histamine-release was calcium and temperature dependent and the relatively slow kinetics (10 min) were similar to those obtained with anti-IgE. EHRF caused the release of a substantial amount of histamine (48%, n = 18) in a dose-dependent manner. The equivalent fraction isolated from medium containing unfertilized oocytes gave less than 10% of histamine-release using the same source of basophils, suggesting that EHRF was secreted after fertilization. EHRF was very stable since it was resistant to boiling, lyophilization, and to several freeze and thaw treatments. The histamine-releasing activity induced by EHRF was measured in vitro also by means of purified leukocytes containing sensitized basophils. EHRF could represent a message sent by the embryo to the mother to induce histamine release at the time of implantation.

Isolation of a histamine releasing factor from human embryo culture medium after in-vitro fertilization.

In-vitro fertilization (IVF) of human oocytes in our laboratories gave a percentage pregnancy rate per transfer close to 20% during 1985. Embryos were grown until the two-four cell stage and then transferred to the maternal uterus. The media from these embryo cultures were collected and subjected to chromatography on heparin-Sepharose affinity columns. The bound protein fraction contained a factor capable of inducing histamine release from sensitized basophils. The effect of this embryo-derived histamine-releasing factor (EHRF) was to induce a maximum 56 +/- 7% release of the total histamine available. This value varied between 20 and 60%, resulting from 10-30 micrograms/ml of EHRF. Since the histamine release assay performed with basophils from non-atopic donors gave no positive results, we conclude that the release was not due to a cytotoxic mechanism. This was also supported by the absence of histamine release when the assay was performed at 0 degree C, or in the presence of 2 mM EDTA, suggesting that release was dependent on an immunological interaction between EHRF and some receptor on the basophils. The immunosuppressive role of histamine is well known, and a model involving EHRF and histamine is suggested here to explain the mechanism mounted by the embryo to escape maternal immune rejection.

Isolation of a histamine-releasing factor from two-cell human embryo.

An embryo-derived histamine-releasing factor (EHRF) was identified and partially purified from media in which two-cell human embryos were cultured. The EHRF at 5 micrograms/ml was capable of inducing 22 +/- 7% release of histamine from sensitized human leukocytes, reaching a maximum of 56 +/- 4% over an EHRF concentration range of 1-30 micrograms/ml. The EHRF was not detected in media where unfertilized oocytes were cultured or in medium alone. The effect of EHRF was not due to cytotoxicity since unsensitized leukocytes were unreactive. Histamine release did not occur when the assay was performed at 4 degrees C or in presence of EDTA.

Early embryonic histamine-releasing factor: a new model for human implantation.

In this paper are presented preliminary results on the identification of a histamine-releasing factor. This factor was partially purified by means of affinity heparin-Sepharose chromatography from media in which 2-cell human embryos were cultured. The culture was performed in preparation for an in-vitro fertilization (IVF) programme. The mean value of histamine release evoked by the embryo-derived histamine-releasing factor (EHRF) was 56.7%. The release was not due to cytotoxicity since no histamine release was obtained with unsensitized cells and when the assay was performed at 4 degrees C. As a control, no histamine release was obtained using medium from unfertilized oocytes or medium alone. The EHRF could be one of the first signals from the embryo to the uterus. The immunosuppressive activity of histamine is well known, and we suggest that the local secretion of histamine in vivo by uterine mast cells, alone or in cooperation with other factors and/or mechanisms could play a role in preventing maternal immuno-rejection at the implantation stage.

Immunosuppressive effect of early pregnancy factor on early expression of cell surface membrane IgG.

We have adopted a new assay to investigate the influence of early pregnancy factor (EPF) on the modulation of lymphocyte activity. Lymphocytes were attached to the plastic surfaces of microplates in serum-free medium in the presence of Sepharose-Con A. After 2-3 days incubation with EPF, and ELISA assay was used to detect the expression of surface membrane IgG (smIgG); this was done in the same microplates used for the culture, thus avoiding cell manipulation. Using only a few picograms of EPF a significant inhibition (in the range 26-40%) was obtained. The variation in the inhibition observed was mainly due to the different sources of lymphocytes used. Unrelated proteins and hormones, tested at the same concentration as EPF, did not show any inhibitory activity. Using the F(ab)2 fragment of anti-human IgG instead of the whole molecule the same levels of inhibition were obtained, suggesting that the observed inhibition by EPF was not due to a non-specific interaction between the anti-human IgG and the Fc receptors on the cell. Such inhibitory activity detected in vitro by this method provides additional support for a suppressive role for EPF during pregnancy.

A new method to detect directly in culture cell surface membrane immunoglobulins.

An ELISA assay is described for the measurement of the smIgG. The method is based on the detection of cell-smIgG directly on the same microplate used for the culture. The cells, preincubated at 37 degrees C for one hour, were cultured in the presence of S-ConA and serum-free medium for two days. Using this strategy, the background noise due to non specific adsorption of IgG to plastic wells and cytophilic antibodies was eliminated. The cells in the presence of S-ConA and serum-free medium adhered to the plastic wells, and the cell-smIgG were detected using an anti-human IgG covalently linked to alkaline phosphatase or its F(ab')2 fragment. The possibility of measuring the modulation of the expression of the cell-smIgG without any additional manipulation is stressed.

Binding of human EPF to receptors on PMBC as a first signal of pregnancy immunomodulation.

The interaction between human early pregnancy factor (EPF) and a specific receptor present on human peripheral blood mononuclear cells (PMBC) has been assessed. EPF was radioiodiated by the lactoperoxidase method to a high specific activity, and the [125I]EPF obtained bound in a specific and saturable manner to the receptors on PBMC. Saturation of the binding sites occurred at 10(-9) M. There were 6600 specific binding sites per cell in cells partially purified from pregnant women and fewer in those from non-pregnant women. Unlabelled EPF was capable of competing for binding sites with [125I]EPF, while the binding of [125I]EPF was not inhibited by human chorionic gonadotrophin. A new assay was used that permits the culture of PBMC and the detection of the surface IgG expression in the same microplate. With this method the influence in vitro of human EPF on lymphocyte expression was tested. The results demonstrated a specific inhibition of surface IgG expression of PBMC using a very low concentration of EPF (10 pg/ml).