Surface-expressed enolase contributes to the adhesion of Paracoccidioides brasiliensis to host cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Time-dependent expression of annexin 1 in a model of chronic granulomatous inflammation

Objective and design: To determine the expression pattern and distribution of the glucocorticoid-inducible protein annexin 1 (ANXA1) in a murine model of chronic granulomatous inflammation.Materials or subjects: TO Mouse.Treatment: Chronic granulomatous inflammation was induced by injecting into dorsal sub-cutaneous air-pouches in mice, a mixture of croton oil and Freund's complete adjuvant (CO/FCA).Methods: Western and northern analysis, corticosterone assay, and immunohistochemistry. Statistical analysis was performed using ANOVA followed by Tukey's pair-wise comparisons or Dunnett's multiple comparisons.Results: ANXA1 protein levels changed significantly throughout the 4-week time course, with an initial peak at day 7 and a later elevation at 28 days. ANXA1 mRNA levels peaked at days 1 and 3, with a significant decline at day 7 followed by an upward trend to day 28. Plasma corticosterone measurements taken throughout the time course revealed an increase from 14 days onward, suggesting that corticosterone does not influence ANXA1 expression during the initial stages of the model. Immunogold staining revealed that ANXA1 expression in the inflamed tissue was mainly in extravasated neutrophils, with intact protein (37 kDa) being predominantly observed on the cell membrane.Conclusions: the pattern of ANXA1 expression indicates that infiltrated neutrophils are responsible for the majority of ANXA1 present both at early and later stages of this model of granulomatous inflammation.

Interaction of human neutrophils with endothelial cells regulates the expression of endogenous proteins annexin 1, galectin-1 and galectin-3

Annexin 1 (ANXA1), galectin-1 (Gal-1) and galectin-3 (Gal-3) proteins have been identified as important mediators that promote or inhibit leukocyte migration. The expression of these proteins was studied in human neutrophils and endothelial cells (ECs) during a transmigration process induced by IL-8. Upon neutrophil adhesion to EC, a significant increase in the cleaved ANXA1 (LCS3, raised against all ANXA1 isoforms) expression was detected in the plasma membrane of adhered neutrophils and ECs compared to intact ANXA1 isoform (LCPS1, against N-terminus of protein). Adherent neutrophils had elevated Gal-3 levels in the nucleus and cytoplasm, and ECs in their plasma membranes. In contrast, a decrease in the total amounts of Gal-1 was detected in migrated compared to non-migrated neutrophils. Therefore, ANXA1 and Gal-3 changed in their content and localization when neutrophils adhere to endothelia, suggesting a process of sensitive-balance between two endogenous anti- and pro-inflammatory mediators. (c) 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.

Immunocytochemical localization of heparin in secretory granules of rat peritoneal mast cells using a monoclonal anti-heparin antibody (ST-1)

We performed immunogold labeling with an ST-1 monoclonal antibody (IgM), specific for intact heparin, to define the subcellular localization of heparin in mast cells. Rat peritoneal mast cells were fixed by a modified Karnovsky method and embedded in Araldite. Ultrathin sections were first treated with sodium periodate and then sequentially incubated with MAb ST-1, rabbit anti-mouse IgM, and protein A-gold. By transmission electron microscopy, gold particles were localized inside cytoplasmic granules of peritoneal mast cells. In contrast, with the same procedure, no labeling was observed in mast cells from rat intestinal mucosa. Control sections of rat peritoneal or intestinal mucosa mast Mast cells cells treated with an irrelevant MAb (IgM) did not show any labeling. Treatment with nitrous Heparin acid abolished the reactivity of MAb ST-1 with peritoneal mast cells. These results Granules show that different mast cells can be identified regarding their heparin content by immunochemical procedures using MAb ST-1.

Inflammation-induced modulation of cellular galectin-1 and-3 expression in a model of rat peritonitis

Objective and design: To investigate the effect of galectin-1 (Gal-1) and -3 (Gal-3) on leukocyte migration and analyze the expression of both galectins in inflammatory cells using a model of rat peritonitis.Material or Subjects: Sprague-Dawley rats (n = 4 per group).Treatment: Peritonitis was induced in animals through intraperitoneal injection of carrageenin (1.5 mg/kg) and rat mesenteries were analyzed at different time points (0, 4, 24 and 48h). For pharmacological treatment, rats received intravenous injection of Gal-1 or -3 (3 mu g/kg) followed by carrageenin.Methods: Western blotting and immunoelectron microscopy analysis. Statistical analysis was performed using ANOVA followed by Bonferroni test.Results: Pharmacological treatment with Gal-1, but not Gal-3, inhibited (similar to 50%) leukocyte recruitment into the peritoneal cavity at 4h time-point. In this early phase, immunogold staining of mesenteries showed a diminished Gal-3 expression in degranulated mast cells and Gal-1 in transmigrated neutrophils (similar to 20% reduction compared to intravascular cells). In the later phases (24 and 48 h), leukocyte turnover was associated with augmented Gal-1 expression in neutrophils and macrophages and Gal-3 in mast cells and macrophages.Conclusions: These results point to a balanced expression of cell-associated-Gal-1/Gal-3 and might impact on the development of new therapeutic strategies for inflammatory diseases.

The intricate role of mast cell proteases and the annexin A1-FPR1 system in abdominal wall endometriosis

Endometriosis is a continuous and progressive disease with a poorly understood aetiology, pathophysiology and natural history. This study evaluated the histological differences between eutopic and ectopic endometria (abdominal wall endometriosis) and the expression of mast cell proteases (tryptase and chymase), annexin A1 (ANXA1) and formyl peptide receptor 1 (FPR1). Ectopic endometrium from 18 women with abdominal wall endometriosis and eutopic endometrium from 10 women without endometriosis were obtained. The endometrial samples were analysed by histopathology, immunohistochemistry and ultrastructural immunogold labeling to determine mast cell heterogeneity (tryptase and chymase positive cells) and the expression levels of ANXA1 and FPR1. Histopathological analysis of the endometriotic lesions showed a glandular pattern of mixed differentiation and an undifferentiated morphology with a significant influx of inflammatory cells and a change in mast cell heterogeneity, as evidenced by a significant increase in the number of chymase-positive cells and endogenous chymase expression. The undifferentiated glandular pattern of endometriotic lesions was positively associated with a marked increase and co-localization of ANXA1 and FPR1 in the epithelial cells. In conclusion, the co-upregulated expression of mast cell chymase and ANXA1–FPR1 system in ectopic endometrium suggests their involvement in the development of endometriotic lesions.

Immunomodulatory Effects of Galectin-1 on an IgE-Mediated Allergic Conjunctivitis Model

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