Zika Virus Infects Human Placental Mast Cells and the HMC-1 Cell Line, and Triggers Degranulation, Cytokine Release and Ultrastructural Changes.

Zika virus (ZIKV) is an emergent arthropod-borne virus whose outbreak in Brazil has brought major public health problems. Infected individuals have different symptoms, including rash and pruritus, which can be relieved by the administration of antiallergics. In the case of pregnant women, ZIKV can cross the placenta and infect the fetus leading to congenital defects. We have identified that mast cells in the placentae of patients who had Zika during pregnancy can be infected. This led to our investigation on the possible role of mast cells during a ZIKV infection, using the HMC-1 cell line. We analyzed their permissiveness to infection, release of mediators and ultrastructural changes. Flow cytometry detection of ZIKV-NS1 expression 24 h post infection in 45.3% of cells showed that HMC-1 cells are permissive to ZIKV infection. Following infection, ß-hexosaminidase was measured in the supernatant of the cells with a notable release at 30 min. In addition, an increase in TNF-α, IL-6, IL-10 and VEGF levels were measured at 6 h and 24 h post infection. Lastly, different intracellular changes were observed in an ultrastructural analysis of infected cells. Our findings suggest that mast cells may represent an important source of mediators that can activate other immune cell types during a ZIKV infection, which has the potential to be a major contributor in the spread of the virus in cases of vertical transmission.

Listeria monocytogenes induces mast cell extracellular traps.

Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when ß-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.

Mast cells in the colon of Trypanosoma cruzi-infected patients: are they involved in the recruitment, survival and/or activation of eosinophils?

Megacolon is frequently observed in patients who develop the digestive form of Chagas disease. It is characterized by dilation of the rectum-sigmoid portion and thickening of the colon wall. Microscopically, the affected organ presents denervation, which has been considered as consequence of an inflammatory process that begins at the acute phase and persists in the chronic phase of infection. Inflammatory infiltrates are composed of lymphocytes, macrophages, natural killer cells, mast cells, and eosinophils. In this study, we hypothesized that mast cells producing tryptase could influence the migration and the activation of eosinophils at the site, thereby contributing to the immunopathology of the chronic phase. We seek evidence of interactions between mast cells and eosinophils through (1) evaluation of eosinophils, regarding the expression of PAR2, a tryptase receptor; (2) correlation analysis between densities of mast cells and eosinophils; and (3) ultrastructural studies. The electron microscopy studies revealed signs of activation of mast cells and eosinophils, as well as physical interaction between these cells. Immunohistochemistry and correlation analyses point to the participation of tryptase immunoreactive mast cells in the migration and/or survival of eosinophils at the affected organ.

Mast cell activation by conidia of Sporothrix schenckii: role in the severity of infection.

Mast cells are abundant in the skin and other peripheral tissues, where they are one of the first immune cells to make contact with invading pathogens. As a result of pathogen recognition, mast cells can be activated and release different preformed and de novo-synthesized mediators. Sporothrix schenckii is the fungus that causes sporotrichosis, a worldwide-distributed subcutaneous mycosis considered as an important emerging health problem. It remains unknown whether or not mast cells are activated by S. schenckii. Here, we investigated the in vitro response of mast cells to conidia of S. schenckii and their in vivo involvement in sporotrichosis. Mast cells became activated after interaction with conidia, releasing early response cytokines as TNF-α and IL-6. Although histamine release was not significantly stimulated by S. schenckii, we determined that conidia potentiate histamine secretion induced by compound 48/80. Furthermore, functional depletion of peritoneal mast cells before S. schenckii infection significantly reduced the severity of cutaneous lesions of the sporotrichosis. These data demonstrate that mast cells are important contributors in the host response to S. schenckii infection, suggesting a role of these cells in the progress of clinical manifestations in sporotrichosis.

Activation of human leukemic mast cell line LAD2 is modulated by dehydroleucodine and xanthatin.

The aim of the present study was to determine whether dehydroleucodine, xanthatin and 3-benzyloxymethyl-5H-furan-2-one inhibit the activation of human leukemic LAD2 mast cells induced by compound 48/80 or the calcium ionophore A23187. LAD2 cells were preincubated in the presence of test drugs and then challenged with the secretagogues. This study provides the first evidence in favor of the view that dehydroleucodine and xanthatin inhibit the degranulation of LAD2 cells, thus acting as human mast cell stabilizers. These molecules could be effective in the treatment of human diseases associated with inappropriate mast cell activation.

Phospholipase D2: a pivotal player modulating RBL-2H3 mast cell structure.

The current study examined the role of PLD2 in the maintenance of mast cell structure. Phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine to produce choline and phosphatidic acid (PA). PLD has two isoforms, PLD1 and PLD2, which vary in expression and localization depending on the cell type. The mast cell line RBL-2H3 was transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2. The results of this study show that PLD2CI cells have a distinct star-shaped morphology, whereas PLD2CA and RBL-2H3 cells are spindle shaped. In PLD2CI cells, the Golgi complex was also disorganized with dilated cisternae, and more Golgi-associated vesicles were present as compared with the PLD2CA and RBL-2H3 cells. Treatment with exogenous PA led to the restoration of the wild-type Golgi complex phenotype in PLD2CI cells. Conversely, treatment of RBL-2H3 and PLD2CA cells with 1% 1-Butanol led to a disruption of the Golgi complex. The distribution of acidic compartments, including secretory granules and lysosomes, was also modified in PLD2CI cells, where they concentrated in the perinuclear region. These results suggest that the PA produced by PLD2 plays an important role in regulating cell morphology in mast cells.

Mast cell repopulation of the peritoneal cavity: contribution of mast cell progenitors versus bone marrow derived committed mast cell precursors.

BACKGROUND: Mast cells have recently gained new importance as immunoregulatory cells that are involved in numerous pathological processes. One result of these processes is an increase in mast cell numbers at peripheral sites. This study was undertaken to determine the mast cell response in the peritoneal cavity and bone marrow during repopulation of the peritoneal cavity in rats. RESULTS: Two mast cell specific antibodies, mAb AA4 and mAb BGD6, were used to distinguish the committed mast cell precursor from more mature mast cells. The peritoneal cavity was depleted of mast cells using distilled water. Twelve hours after distilled water injection, very immature mast cells could be isolated from the blood and by 48 hours were present in the peritoneal cavity. At this same time the percentage of mast cells in mitosis increased fourfold. Mast cell depletion of the peritoneal cavity also reduced the total number of mast cells in the bone marrow, but increased the number of mast cell committed precursors. CONCLUSIONS: In response to mast cell depletion of the peritoneal cavity, a mast cell progenitor is released into the circulation and participates in repopulation of the peritoneal cavity, while the committed mast cell precursor is retained in the bone marrow.

Novel anti-ulcer alpha,beta-unsaturated lactones inhibit compound 48/80-induced mast cell degranulation.

The present study was designed to examine the effects of a sesquiterpene lactone isolated from Artemisia douglasiana Besser (dehydroleucodine), a xanthanolide sesquiterpene isolated from Xanthium cavanillesii Schouw (xanthatin) and a semisynthetic butenolide (3-benzyloxymethyl-5H-furan-2-one) on mast cell degranulation induced by compound 48/80. Peritoneal mast cells from male adult Sprague-Dawley rats were purified in Percoll, preincubated in the presence of test lactones (dehydroleucodine, xanthatin or 3-benzyloxymethyl-5H-furan-2-one) and then challenged with the mast cell activator compound 48/80 (10 microg/ml). Concentration-response and kinetic studies of mast cell serotonin release evoked by compound 48/80, evaluation of mast cell viability and morphology by light and electron microscopy, and comparative studies using ketotifen and sodium chromoglycate were carried out. Serotonin release studies, carried out together with morphological studies, showed the effectiveness of the above lactones to stabilize mast cells. The comparative study with ketotifen and sodium chromoglycate, well known mast cell stabilizers, showed the following order of potency dehydroleucodine=xanthatin>3-benzyloxymethyl-5H-furan-2-one> or =ketotifen/sodium chromoglycate to inhibit mast cell serotonin release induced by compound 48/80. The present study provides the first strong evidence in favour of the hypothesis that dehydroleucodine, xanthatin and 3-benzyloxymethyl-5H-furan-2-one inhibit compound 48/80-induced serotonin release from peritoneal mast cells, acting thus as mast cell stabilizers. Our findings may provide an insight into the design of novel pharmacological agents which may be used to regulate the mast cell response.

In vivo hydroquinone exposure impairs allergic lung inflammation in rats.

Hydroquinone (HQ) is naturally found in the diet, drugs, as an environmental contaminant and endogenously generated after benzene exposure. Considering that HQ alters the immune system and its several source of exposures in the environment, we hypothesized that prolonged exposure of HQ could affect the course of an immune-mediated inflammatory response. For this purpose, male Wistar rats were intraperitoneally exposed to vehicle or HQ once a day, for 22 days with a 2-day interval every 5 days. On day 10 after exposure with vehicle or HQ, animals were ovalbumin (OA)-sensitized and OA-aerosolized challenged on day 23. HQ exposure did not alter the number of circulating leukocytes but impaired allergic inflammation, evidenced by lower number of leukocytes in the bronchoalveolar lavage fluid 24h after OA-challenge. Reduced force contraction of ex vivo tracheal segments upon OA-challenge and impaired mesentery mast cell degranulation after in situ OA-challenge were also detected in tissues from HQ exposed animals. The OA-specificity on the decreased responses was corroborated by normal trachea contraction and mast cell degranulation in response to compound 48/80. In fact, lower levels of circulating OA-anaphylactic antibodies were found in HQ exposed rats, as assessed by passive cutaneous anaphylaxis assay. The reduced level of OA-anaphylactic antibody was not dependent on lower number or proliferation of lymphocytes. Nevertheless, lower expression of the co-stimulatory molecules CD6 and CD45R on OA-activated lymphocytes from HQ exposed rats indicate the interference of HQ exposure with signaling of the humoral response during allergic inflammation. Together, these data indicate specific effects of HQ exposure manifested during an immune host defense.

Mast cell heterogeneity between two different species of Hoplias sp. (Characiformes: Erythrinidae): response to fixatives, anatomical distribution, histochemical contents and ultrastructural features.

Mast cells from two erythrinid species: Hoplias malabaricus and Hoplias lacerdae, were studied in several tissues throughout the body using light and electron microscopy. Mast cells were found in all organs studied, but were especially abundant in the gastrointestinal tract, and were always in association with connective tissue. These cells showed different characteristics between the two species studied, like varied morphology, anatomical distribution, density, basophilic/eosinophilic staining and heparin content. In H. malabaricus, the tissues fixed with Helly's solution contained mast cells that were basophilic, metachromatic and had heparin in their cytoplasmic granules, while the tissues fixed with Karnovsky's solution contained eosinophilic and orthochromatic mast cells in which heparin was not detected. In H. lacerdae, the use of both fixatives resulted in mast cells that were eosinophilic, orthochromatic, with no identifiable heparin content. Exclusively in H. malabaricus oesophagus, the mast cells were additionally seen among the epithelial cells. The ultrastructural studies performed in hindgut fixed with Karnovsky's solution revealed that the cytoplasmic granules seen in H. lacerdae mast cells were better preserved than in H. malabaricus mast cells. The latter had electron-lucent granules that were often merged, forming channels. The present study demonstrated that mast cells from two species belonging to the same genus or even mast cells from the same species but under different fixatives can present heterogeneous characteristics, possibly due to their functional properties or to their sensitivity to fixatives.

The inflammatory response in drug-induced acute urticaria: ultrastructural study of the dermal microvascular unit.

BACKGROUND: Drug exposure is one of the main aetiologies of urticaria and represents the second most common cause in acute urticarias. Studies involving the ultrastructural aspects of urticaria are relatively rare in the literature. Most of the articles published report on skin biopsies of experimentally induced urticaria, and acute urticaria has been studied even less from a morphological point of view. OBJECTIVES: The aims of this study were to observe ultrastructural cell characteristics in five patients with drug-induced acute urticaria and possible aspects of the inflammatory skin response. METHODS: Clinical manifestations, light microscopy and transmission electron microscopy were evaluated. RESULTS: With light microscopy, a mild perivascular lymphocyte-monocyte infiltrate was observed with few neutrophils and dermal oedema in skin biopsies of five patients. With electron microscopy, a mild vascular dilatation was observed, with platelets in the lumen and several lymphocytes and dendritic cells close to the superficial dermal vessels. Some mast cells appeared normal, whereas others were granule-depleted. In some areas, mast cells, lymphocytes and satellite dendritic cells were closely associated, as well as some macrophages. A significant number of plasma cells, eosinophils and polymorphonuclear neutrophils were not observed; however, the presence of lymphocytes and macrophages was significant. The epidermis and the dermal-epidermal junction were preserved, except for a discrete oedema in keratinocytes. CONCLUSIONS: The ultrastructural aspect of drug-induced acute urticaria is similar to that observed in urticaria caused by Urtica dioica, intradermal histamine and cold urticaria. The presence of the cellular triad with mast cells, dendritic (or satellite) cells and lymphocytes suggests a functional interaction of these cells. These findings support the possible existence of mechanisms in the dermis that may participate in protective and/or injurious vasocentric immune reactions.

Pentacyclic triterpenoids, alpha,beta-amyrins, suppress the scratching behavior in a mouse model of pruritus.

In the search for natural compounds useful against pruritus, alpha,beta-amyrins, the pentacyclic triterpenes isolated from the resin of popular medicinal plant Protium heptaphyllum were examined on scratching behavior induced by dextran T40 and compound 48/80 in mice. The animals were pretreated orally with alpha,beta-amyrins (50, 100 and 200 mg/kg) or cyproheptadine (10 mg/kg), an antagonist of histamine and serotonin receptors and 2 h later, they were given subcutaneous injections of dextran T40 (75 mg/kg) or compound 48/80 (3 mg/kg) into the rostral back, and scratching was quantified for 20 min. The scratching behavior induced by dextran T40 and compound 48/80 was significantly inhibited in mice pretreated with alpha,beta-amyrins (100 and 200 mg/kg) or cyproheptadine (10 mg/kg), In addition, the compound 48/80-elicited degranulation of rat peritoneal mast cells (ex vivo) was also markedly reduced in animals pretreated with alpha,beta-amyrins (100 mg/kg) or ketotifen (1 mg/kg), a known mast cell stabilizer. In the open-field test, alpha,beta-amyrins (100 and 200 mg/kg)-pretreated mice showed no impairment of spontaneous locomotion, suggesting that these triterpenoids possess no sedative activity that could account for suppression of scratching behavior. These results clearly indicate the antipruritic effect of alpha,beta-amyrins and suggest that this effect may be related to a stabilizing action on mast cell membrane.

Toxoplasma gondii and mast cell interactions in vivo and in vitro: experimental infection approaches in Calomys callosus (Rodentia, Cricetidae).

In the present work, we studied some qualitative and quantitative characteristics of mast cells located in the peritoneal cavity, submandibular and dorsal lymph nodes and ileum of Calomys callosus experimentally infected by Toxoplasma gondii. In uninfected animals, the majority of mast cells had similar ultra-structural characteristics, including several cytoplasmic granules with homogeneous and electron dense contents. However, after 1 h of infection, a significant influx of mast cells into peritoneal cavity was observed. The number of mast cells in this compartment decreased progressively in infected animals, and was significantly lower than the number of mast cells in control animals after 48 h of infection. Mast cells from infected animals or from purified suspensions that were infected in vitro presented significant morphological modifications, suggesting a degranulation process: cytoplasmic granules with electron dense content, fusion of the cytoplasmic granules, intracytoplasmic channels, cytoplasmic granules with flocculent material, plasma membrane rupture and granule contents in the extracellular environment. A remarkable increase in the influx of neutrophils toward the peritoneal cavity of the infected animals was observed after 12 h of infection. Moreover, this event occurred after the mast cell degranulation process took place. The relative increase in the number of mast cells and neutrophils was also followed by an increase in the number of macrophages, but there was a significant decrease in lymphocyte influx. After 48 h of infection, the parasite had spread from the peritoneal cavity to all organs examined. Also, mast cells from these organs showed evident morphological alterations, indicating the presence of the degranulation process. These results suggest that mast cells are deeply involved with the acute phase of the inflammatory response in this experimental model.

Mast cell degranulation induced by lectins: effect on neutrophil recruitment.

The mammalian lectin macrophage-derived neutrophil chemotactic factor (MNCF) and the plant lectin KM+ were characterized for their ability to activate and degranulate mast cells. The association between mast cell activation and the induction of neutrophil migration was also investigated. Incubation of rat peritoneal mast cells with these lectins resulted in degranulation and mediator release. By confocal microscopy, both lectins were evenly distributed on the cell surface. MNCF activated RBL-2H3 mast cells only if the cells had been sensitized with IgE. KM+ was able to activate either unsensitized or IgE sensitized RBL-2H3 cells. In microplate assays MNCF, but not KM+, bound to rat IgE. In rats that were depleted of mast cells, neutrophil recruitment by MNCF and KM+ were significantly reduced indicating that mast cell activation provides an amplification loop for the neutrophil recruitment induced by these lectins. The present study supports the concept that mammalian lectins play a fundamental role in innate immunity.

Effect of dehydroleucodine on histamine and serotonin release from mast cells in the isolated mouse jejunum.

OBJECTIVE AND DESIGN: DhL, a lactone isolated from Artemisia douglasiana, prevents gastrointestinal damage elicited by necrosis-inducing agents and exhibits antiinflammatory action. This work examines the effect of DhL on compound 48/80-induced histamine and serotonin release in the isolated mouse jejunum, to determine whether DhL inhibits mediator release from mast cells at the enteric level. MATERIAL: Thirty jejuna from male Balb-c mice were used for the studies. TREATMENT: Samples were incubated sequentially in 9 test tubes containing RBS or 10 microg/ml compound 48/80 or 1.6 mmol/l + 10 microg/ml compound 48/80 at 37 degrees C for 90 minutes (10 min per tube). METHODS: Histamine and serotonin release studies, quantification of granulated mast cells, and evaluation of mast cell ultrastructure were carried out. Differences between groups were determined using analysis of variance followed by Tukey-Kramer multiple comparisons test. RESULTS: Compound 48/80 increased histamine and serotonin release by the tissue (141.95 +/- 62.58 pg/mg tissue vs basal 5.45 +/- 1.04, P<0.01 and 20.04 +/- 2.81 vs basal 9.24 +/- 1.56 ng/ mg tissue, P<0.01, respectively), decreased the number of granulated submucosal mast cells (0.077 +/- 0.0035 vs basal 0.14 +/- 0.015, P<0.05), and elicited evident granule ultrastructural changes. These effects were reduced by dehydroleucodine (19.51 +/- 7.88, P<0.01; 12.69 +/- 1, P<0.05 and 0.143 +/- 0.014, P<0.05, respectively). CONCLUSION: The lactone inhibits compound 48/80-induced histamine and serotonin release from mast cells in the isolated mouse jejunum.

Mast cell activation by Mycobacterium tuberculosis: mediator release and role of CD48.

Mast cells (MC) are abundant in the lung and other peripheral tissue, where they participate in inflammatory processes against bacterial infections. Like other effector cells of the innate immune system, MC interact directly with a wide variety of infectious agents. This interaction results in MC activation and inflammatory mediator release. We demonstrated that MC interact with Mycobacterium tuberculosis, triggering the release of several prestored reagents, such as histamine and beta-hexosaminidase, and de novo synthesized cytokines, such as TNF-alpha and IL-6. A number of M. tuberculosis Ags, ESAT-6, MTSA-10, and MPT-63, have been implicated in MC activation and mediator release. A MC plasmalemmal protein, CD48, was implicated in interactions with mycobacteria because CD48 appeared to aggregate in the MC membrane at sites of bacterial binding and because Abs to CD48 inhibited the MC histamine response to mycobacteria. Cumulatively, these findings suggest that MC, even in the absence of opsonins, can directly recognize M. tuberculosis and its Ags and have the potential to play an active role in mediating the host's innate response to M. tuberculosis infection.

Mast cells in the eyes of Calomys callosus(Rodentia: Cricetidae) infected by Toxoplasma gondii.

The mast cell is a powerful effector cell for the innate immune system, acting through the secretion of several distinct mediators. Few studies have demonstrated the relationship between mast cells and toxoplasmosis. In this study, mast cells were investigated in two experimental Toxoplasma infections using Calomys callosus (Rodentia: Cricetidae) as the host. Animals were inoculated either intraperitoneally or via the conjunctiva with tachyzoites of Toxoplasma gondii (RH strain) and sacrificed after 5 days or 24 h, respectively. Enucleated eyes were processed for histological and ultrastructural analysis. Neither experimental infection altered the localization of mast cells compared to control eyes, but they did lead to an accumulation in some tissues as well as to their activation. There was a significant increase in the number of mast cells within 5 days and 24 h after infection. The ocular lesions were characterized by the presence of tachyzoites, inflammatory cells and vasodilatation in the iris and retina. In conclusion, mast cells were mobilized in these experimental infections, suggesting that they play an important role in the host inflammatory response after infection with T. gondii.

[Suggestive evidences for a microanatomical relationship between mast cells and nerve in the tongue of the toad Bufo marinus demonstrated by means of high resolution light microscopy]. / Evidencias que sugieren una asociación microanatómica mastocito-nervio en la lengua del sapo Bufo marinus demostradas mediante microscopia de luz de alta resolucion.

Mast cells are connective tissue cells, present in all vertebrates and characterized by the metachromatic stain of their granules. Nowadays mast cells have been recognized as a potent cellular source of multiple cytokines, suggesting an important role in immunoregulation and host defense. These cells have been described as preferentially located around blood vessels but more recently close spatial relationship between mast cells and nerves has been reported mostly in mammalian species. The microanatomy of nerve tissue and associated mast cells in the toad Bufo marinus tongue have been studied here by means of high resolution light microscopy. Mast cell population was identified by the metachromatic staining of their cytoplasm granules in Epon embedded semithin sections counterstained with Toluidine Blue and Azure A. Numerous mast cells were observed scattered throughout the submucosal region, adjacent and/or within of nerve bundles and nerve ganglia, near skeletal muscle fibers and adjacent of blood vessels. Additionally, mast cells adjacent to single but conspicuous myelinated nerve fibers were seen under endothelia of lymphatic vessels wall and this is apparently a unreported event. Results suggest nerve-mast cell associations are functionally important in the toad tongue.

Morphological alteration of peritoneal mast cells and macrophages in the mouse peritoneal cavity during the early phases of an allergic inflammatory reaction.

We investigated the presence of mast cell granules in macrophages following an in vivo model of an allergic reaction. Injection of ovalbumin (100 microg) into the peritoneal cavity of sensitised mice produced a rapid (within 2 h) influx of neutrophils followed by a slower (after >4 h) eosinophil migration. Ovalbumin treatment induced a high incidence (approximately 50%) of mast cell degranulation compared to control phosphated-buffered saline-treated mice. The majority (approximately 90%) of peritoneal macrophages contained mast cell granules as early as 2 h post-ovalbumin, with lower values at later time-points, as determined by staining with Toluidine blue and Berberine sulphate. This was confirmed by electron microscopy which enabled us to identify the complex mast cell granule sub-structural components in macrophage phagosomes. In conclusion, we used histochemical and ultrastructural analyses to show that mast cell granules become internalised with macrophages during the early stages of an experimental allergic reaction.