An essential role for mast cells as modulators of neutrophils influx in collagen-induced arthritis in the mouse

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pulmonary neutrophil recruitment and bronchial reactivity in formaldehyde-exposed rats are modulated by mast cells and differentially by neuropeptides and nitric oxide

We have used a pharmacological approach to study the mechanisms underlying the rat lung injury and the airway reactivity changes induced by inhalation of formaldehyde (FA) (1% formalin solution, 90 min once a day, 4 days). The reactivity of isolated tracheae and intrapulmonary bronchi were assessed in dose-response curves to methacholine (MCh). Local and systemic inflammatory phenomena were evaluated in terms of leukocyte countings in bronchoalveolar lavage (BAL) fluid, blood, bone marrow lavage and spleen. Whereas the tracheal reactivity to MCh did not change, a significant bronchial hyporesponsiveness (BHR) was found after FA inhalation as compared with naive rats. Also, FA exposure significantly increased the total cell numbers in BAL, in peripheral blood and in the spleen, but did not modify the counts in bone marrow. Capsaicin hindered the increase of leukocyte number recovered in BAL fluid after FA exposure. Both compound 48/80 and indomethacin were able to prevent the lung neutrophil influx after FA, but indomethacin had no effect on that of mononuclear cells. Following FA inhalation, the treatment with sodium cromoglycate (SCG), but not with the nitric oxide (NO) synthase inhibitor L-NAME, significantly reduced the total cell number in BAL. Compound 48/80, L-NAME and SCG significantly prevented BHR to MCh after FA inhalation, whereas capsaicin was inactive in this regard. on the other hand, indomethacin exacerbated BHR. These data suggest that after FA inhalation, the resulting lung leukocyte influx and BHR may involve nitric oxide, airway sensory fibers and mast cell-derived mediators. The effect of NO seemed to be largely restricted to the bronchial tonus, whereas neuropeptides appeared to be linked to the inflammatory response, therefore indicating that the mechanisms responsible for the changes of airway responsiveness caused by FA may be separate from those underlying its inflammatory lung effects. (c) 2005 Elsevier B.V. All rights reserved.

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.

Gap junctions between mast cells and fibroblasts in the developing avian eye

Mast cells are present in the eye of chick embryos from the 14th day onward, displaying metachromatic granules, mainly in the iris anterior surface and pectinate ligament. Ultrastructurally these cells show electron-dense granules and a few thin and short cytoplasmic projections in close contact with fibroblasts. Sometimes these contacts are extensive, with long fibroblast projections partially involving the mast cells. Gap junctions between mast cells and fibroblasts are observed only in the eyes of 16- and 20-day-old embryos. These intercellular specializations are represented by a close apposition of cytoplasmic membranes with an extension up to 300 nm. Gap junctions between mast cells and fibroblasts were not observed previously in vivo or in vitro, although in vitro studies have shown that a number of functionally critical interactions may occur between these cells. Our morphological findings suggest that, in vivo, fibroblasts interact with mast cells and may influence their maturation.

Characteristics of the histamine release from hamster cheek pouch mast cells stimulated by lectins from Brazilian beans and concanavalin A

We have characterized the histamine releasing effects of lectins extracted from Brazilian beans, in comparison to concanavalin A, in hamster cheek pouch cell suspensions containing mast cells. The lectins from Dioclea virgata, Canavalia brasiliensis, and Dioclea rostrata induce histamine release in a similar manner to concanavalin A, but appear to differ in potency and efficacy. The effects depended on the temperature, pH, and metabolic energy, demonstrating the non-cytotoxic nature of the histamine release. It is suggested that the lectins studied act by the same mechanism as concanavalin A (interacting with sugars in the antibodies bound to the mast cells), since high concentrations of glucose inhibit the histamine release. The lectins at high concentrations quench the histamine release. This suppression is reversed by increasing calcium concentration, suggesting that the lectins bind to the calcium that is essential for the secretion, thereby confirming and extending our previous data using the lectin from Dioclea virgata in rat peritoneal mast cells.

Characterization of a monoclonal antibody recognizing mast cells

Immunohistochemical screening for monoclonal antibodies prepared by immunization of mice with a rat osteoblastic cell population led to identification of one antibody that reacted against a small population of cells present in the soft connective tissue compartment of 21 days fetal rat calvaria. The morphology of the cells and the immunohistochemical staining characteristics (a distinct intracellular granular pattern) suggested that the antibody might be reacting specifically against mast cells. We used combined histochemistry and immunohistochemistry to further characterize this antibody, designated RCJ102. Cryosections containing calvaria bone, soft connective tissues and skin were prepared from the top of the head of 21 days fetal rats, and from adult rats cryosections of lung, muscle, adipose tissue and small intestine were prepared. Some sections were labelled by indirect immunofluorescence with RCJ102; corresponding sections were labelled histochemically with toluidine blue. There was a direct correspondence between mast cells identified histochemically and cells labelling with RCJ102 in all tissues except intestine, in which the mast cell detectable by histochemistry were not labelled by RCJ102. These results suggest that the RCJ102 antibody will be a valuable new reagent for further elucidation of the heterogeneity described between connective tissue and intestinal mucosal mast cells.

Neutrophil migration induced by IL-1β depends upon LTB4 released by macrophages and upon TNF-α and IL-1β released by mast cells

In the present study, we investigate whether mast cells and macrophages are involved in the control of IL-1β-induced neutrophil migration, as well as the participation of chemotactic mediators. IL-1β induced a dose-dependent neutrophil migration to the peritoneal cavity of rats which depends on LTB 4, PAF and cytokines, since the animal treatment with inhibitors of these mediators (MK 886, PCA 4248 and dexamethasone respectively) inhibited IL-1β-induced neutrophil migration. The neutrophil migration induced by IL-1β is dependent on mast cells and macrophages, since depletion of mast cells reduced the process whereas the increase of macrophage population enhanced the migration. Moreover, mast cells or macrophages stimulated with IL-1β released a neutrophil chemotactic factor, which mimicked the neutrophil migration induced by IL-1β. The chemotactic activity of the supernatant of IL-1β-stimulated macrophages is due to the presence of LTB4, since MK 886 inhibited its release. Moreover, the chemotactic activity of IL-1β-stimulated mast cells supernatant is due to the presence of IL-1β and TNF-α, since antibodies against these cytokines inhibited its activity. Furthermore, significant amounts of these cytokines were detected in the supernatant. In conclusion, our results suggest that neutrophil migration induced by IL-1β depends upon LTB4 released by macrophages and upon IL-1β and TNFα released by mast cells. © 2007 Springer Science+Business Media, LLC.

Mast cells modulate the inflammatory process in endotoxin-induced uveitis

Purpose: To investigate the role of mast cells and annexin-A1 (Anxa1) in endotoxin-induced uveitis (EIU). Methods: EIU was induced by injection of lipopolysaccharide (LPS) into the paws of rats, which were then sacrificed after 24 and 48 h. To assess EIU in the absence of mast cells, groups of animals were pretreated with compound 48/80 (c48/80) and sacrificed after 24 h after no treatment or EIU induction. The eyes were used for histological studies and the aqueous humor (AqH) pool was used for the analysis of transmigrated cells and Anxa1 levels. In inflammatory cells, Anxa1 expression was monitored by immunohistochemistry. Results: After 24 h, rats with EIU exhibited degranulated mast cells, associated with elevated numbers of infiltrating leukocytes and the high expression of Anxa1 in the AqH and the neutrophils. After 48 h of EIU, the mast cells were intact, indicating granule re-synthesis, and there was a reduction of neutrophil transmigration and an increase in the number of mononuclear phagocytic cells in ocular tissues. Anxa1 expression was decreased in neutrophils but increased in mononuclear phagocytic cells. In the animals pretreated with c48/80 and subjected to EIU, mast cells responded to this secretagogue by degranulating and few transmigrated neutrophils were observed. Conclustions: We report that mast cells are a potential source of pharmacological mediators that are strongly linked to the pathophysiology of EIU, and the endogenous protein Anxa1 is a mediator in the homeostasis of the inflammatory process with anti-migratory effects on leukocytes, which supports further studies of this protein as an innovative therapy for uveitis. © 2011 Molecular Vision.

Characterization of a monoclonal antibody recognizing mast cells

Immunohistochemical screening for monoclonal antibodies prepared by immunization of mice with a rat osteoblastic cell population led to identification of one antibody that reacted against a small population of cells present in the soft connective tissue compartment of 21 days fetal rat calvaria. The morphology of the cells and the immunohistochemical staining characteristics (a distinct intracellular granular pattern) suggested that the antibody might be reacting specifically against mast cells. We used combined histochemistry and immunohistochemistry to further characterize this antibody, designated RCJ102. Cryosections containing calvaria bone, soft connective tissues and skin were prepared from the top of the head of 21 days fetal rats, and from adult rats cryosections of lung, muscle, adipose tissue and small intestine were prepared. Some sections were labelled by indirect immunofluorescence with RCJ102; corresponding sections were labelled histochemically with toluidine blue. There was a direct correspondence between mast cells identified histochemically and cells labelling with RCJ102 in all tissues except intestine, in which the mast cell detectable by histochemistry were not labelled by RCJ102. These results suggest that the RCJ102 antibody will be a valuable new reagent for further elucidation of the heterogeneity described between connective tissue and intestinal mucosal mast cells.

Stem cell factor: a hemopoietic cytokine with important targets in asthma

We review evidence that Stem Cell Factor (SCF) plays an important role in the pathophysiology of asthma. SCF is produced by a wide variety of cells present in asthmatic lung, including mast cells and eosinophils. Its receptor, c-kit, is broadly expressed on mature mast cells and eosinophils. SCF promotes recruitment of mast cell progenitors into tissues, as well as their local maturation and activation. It also promotes eosinophil survival, maturation and functional activation. SCF enhances IgE-dependent release of mediators from mast cells, including histamine, leukotrienes, cytokines (TNF-alpha, IL-5, GM-CSF) and chemokines (RANTES/CCL5, MCP-1/CCL2, TARC/CCL17 e MDC/CCL22); it is required for IL-4 production in mast cells. SCF, acting in concert with IgE, also upregulates the expression and function of CC chemokine receptors in mast cells. Structural and resident airway cells express increased levels of SCF in the bronchus of asthmatic patients. In a murine model of asthma, allergen exposure increased production of SCF by epithelial cells and alveolar macrophages, which was transient and paralleled by histamine release. SCF induced long-lived airway hyperreactivity, which was prevented by local neutralization of SCF, as well as by inhibitors of the production or activity of cysteinyl-leukotrienes. Together, these observations suggest that SCF has an important role in asthma.

Effect of umbelliferone (7-hydroxycoumarin, 7-HOC) on the enzymatic, edematogenic and necrotic activities of secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus collilineatus venom

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Preliminary cryocrystallography analysis of an eumenine mastoparan toxin isolated from the venom of the wasp Anterhynchium flavomarginatum micado

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 Angstrom resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 Angstrom. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation. (C) 3001 Elsevier B.V. B.V. All rights reserved.

Neuropatia paraneoplásica associada ao mastocitoma canino

As síndromes paraneoplásicas compreendem um grupo diverso de alterações clínicas associadas a neoplasias e ocorrem em sítios distantes do tumor primário ou de suas metástases. As neuropatias paraneoplásicas são distúrbios raros em cães, mas representam morbidade significativa e servem como importantes indicadores diagnósticos e prognósticos. O presente trabalho relata a ocorrência de dois casos de neuropatia paraneoplásica em cães com mastocitoma, considerando a apresentação clínica, o diagnóstico e as formas de tratamento utilizadas.

Neoplasias de língua em cinco cães

Este trabalho tem como objetivo relatar a ocorrência de cinco casos de neoplasias de língua em cães. Os sinais clínicos mais observados foram ptialismo, halitose e hiporexia. Após biópsia excisional para exame histopatológico, os resultados revelaram dois casos de melanoma, um caso de histiocitoma, um caso de fibrossarcoma e um caso de mastocitoma grau II, sendo que neste o proprietário não autorizou qualquer forma de tratamento. O tratamento para os demais foi a glossectomia parcial e, no caso de fibrossarcoma, associou-se a quimioterapia. Dois animais apresentaram deiscência de sutura no pós-operatório, não havendo necessidade da realização de nova intervenção. em relação ao prognóstico, os pacientes com histiocitoma, fibrossarcoma e um com melanoma não apresentaram recidiva, nem metástase da doença 12 meses após a cirurgia. No outro caso de melanoma, o paciente apresentou metástase na pele e nos pulmões 30 dias após a ressecção cirúrgica.

Ultra-estrutura dos mastócitos de diferentes tipos histológicos de mastocitoma em cães

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)