The Alternative Faces of Macrophage Generate Osteoclasts.

The understanding of how osteoclasts are generated and whether they can be altered by inflammatory stimuli is a topic of particular interest for osteoclastogenesis. It is known that the monocyte/macrophage lineage gives rise to osteoclasts (OCs) by the action of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL), which induce cell differentiation through their receptors, c-fms and RANK, respectively. The multinucleated giant cells (MGCs) generated by the engagement of RANK/RANKL are typical OCs. Nevertheless, very few studies have addressed the question of which subset of macrophages generates OCs. Indeed, two main subsets of macrophages are postulated, the inflammatory or classically activated type (M1) and the anti-inflammatory or alternatively activated type (M2). It has been proposed that macrophages can be polarized in vitro towards a predominantly M1 or M2 phenotype with the addition of granulocyte macrophage- (GM-) CSF or M-CSF, respectively. Various inflammatory stimuli known to induce macrophage polarization, such as LPS or TNF-α, can alter the type of MGC obtained from RANKL-induced differentiation. This review aims to highlight the role of immune-related stimuli and factors in inducing macrophages towards the osteoclastogenesis choice.

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.

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.

Antigens of Euparipha pisana (snail). I. Identification of allergens by means of in vivo and in vitro analysis.

The data obtained in this study suggest that eating Euparipha pisana (snail), a common food in Mediterranean countries, could give serious allergic reaction such as asthma. We describe here the identification and partial characterization of allergenic molecules form this new source. An aqueous extract of snail was obtained by homogenization in distilled water, centrifugation, dialysis and defatting with ethyl ether. Skin prick test (SPT) performed with the snail extract on 70 subjects allergic to the more common allergens of the Mediterranean area gave a SPT positivity in 61% of the subjects tested, with a mean value of histamine-equivalent prick (HEP) equal to 0.81 +/- 0.25 (n = 43), while no SPT-snail-positive reactions were obtained by using the same extract on 30 not allergic subjects. To ascertain if such a sensitivity was IgE-mediated, sera from SPT-snail-positive subjects were analyzed by RAST, coupling the snail extract to polystyrene balls and to paper discs. 19% of the sera tested were RAST-positive, mean value of binding 4.8 +/- 2.8% (n = 13), while when using sera from SPT-snail-negative subjects, the RAST mean value was 0.49 +/- 0.18% (n = 27). Histamine release (HR) was also performed. Basophils prepared from SPT-snail-positive subjects were incubated with a snail extract. All of the SPT-snail-positive subjects gave a significant value of HR, mean value 21.8 +/- 7% using 1 micrograms of snail extract (n = 16), while 1.41 +/- 1.1% (n = 10) was the mean value obtained when SPT-snail-negative subjects were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)