Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines

Mast cells are immunoregulatory cells that participate in inflammatory processes. Cross-linking mast cell specific GD1b derived gangliosides by mAbAA4 results in partial activation of mast cells without the release of preformed mediators. The present study examines the release of newly formed and newly synthesized mediators following ganglioside cross-linking. Cross-linking the gangliosides with mAbAA4 released the newly formed lipid mediators, prostaglandins D2 and E2, without release of leukotrienes B4 and C4. The effect of cross-linking these gangliosides on the activation of enzymes in the arachidonate cascade was then investigated. Ganglioside cross-linking resulted in phosphorylation of cytosolic phospholipase A2 and increased expression of cyclooxygenase-2. Translocation of 5-lipoxygenase from the cytosol to the nucleus was not induced by ganglioside cross-linking. Cross-linking of GD1b derived gangliosides also resulted in the release of the newly synthesized mediators, interleukin-4, interleukin-6, and TNF-α. The effect of cross-linking the gangliosides on the MAP kinase pathway was then investigated. Cross-linking the gangliosides induced the phosphorylation of ERK1/2, JNK1/2, and p38 as well as activating both NFκB and NFAT in a Syk-dependent manner. Therefore, cross-linking the mast cell specific GD1b derived gangliosides results in the activation of signaling pathways that culminate with the release of newly formed and newly synthesized mediators

Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines

Mast cells are immunoregulatory cells that participate in inflammatory processes. Cross-linking mast cell specific GD1b derived gangliosides by mAbAA4 results in partial activation of mast cells without the release of preformed mediators. The present study examines the release of newly formed and newly synthesized mediators following ganglioside cross-linking. Cross-linking the gangliosides with mAbAA4 released the newly formed lipid mediators, prostaglandins D2 and E2, without release of leukotrienes B4 and C4. The effect of cross-linking these gangliosides on the activation of enzymes in the arachidonate cascade was then investigated. Ganglioside cross-linking resulted in phosphorylation of cytosolic phospholipase A2 and increased expression of cyclooxygenase-2. Translocation of 5-lipoxygenase from the cytosol to the nucleus was not induced by ganglioside cross-linking. Cross-linking of GD1b derived gangliosides also resulted in the release of the newly synthesized mediators, interleukin-4, interleukin-6, and TNF-α. The effect of cross-linking the gangliosides on the MAP kinase pathway was then investigated. Cross-linking the gangliosides induced the phosphorylation of ERK1/2, JNK1/2, and p38 as well as activating both NFκB and NFAT in a Syk-dependent manner. Therefore, cross-linking the mast cell specific GD1b derived gangliosides results in the activation of signaling pathways that culminate with the release of newly formed and newly synthesized mediators

The Role of Mast Cell Specific Chymases and Tryptases in Tumor Angiogenesis

An association between mast cells and tumor angiogenesis is known to exist, but the exact role that mast cells play in this process is still unclear. It is thought that the mediators released by mast cells are important in neovascularization. However, it is not known how individual mediators are involved in this process. The major constituents of mast cell secretory granules are the mast cell specific proteases chymase, tryptase, and carboxypeptidase A3. Several previous studies aimed to understand the way in which specific mast cell granule constituents act to induce tumor angiogenesis. A body of evidence indicates that mast cell proteases are the pivotal players in inducing tumor angiogenesis. In this review, the likely mechanisms by which tryptase and chymase can act directly or indirectly to induce tumor angiogenesis are discussed. Finally, information presented here in this review indicates that mast cell proteases significantly influence angiogenesis thus affecting tumor growth and progression. This also suggests that these proteases could serve as novel therapeutic targets for the treatment of various types of cancer

Adaptor protein-3: A key player in RBL-2H3 mast cell mediator release.

Mast cell (MC) secretory granules are Lysosome-Related Organelles (LROs) whose biogenesis is associated with the post-Golgi secretory and endocytic pathways in which the sorting of proteins destined for a specific organelle relies on the recognition of sorting signals by adaptor proteins that direct their incorporation into transport vesicles. The adaptor protein 3 (AP-3) complex mediates protein trafficking between the trans-Golgi network (TGN) and late endosomes, lysosomes, and LROs. AP-3 has a recognized role in LROs biogenesis and regulated secretion in several cell types, including many immune cells such as neutrophils, natural killer cells, and cytotoxic T lymphocytes. However, the relevance of AP-3 for these processes in MCs has not been previously investigated. AP-3 was found to be expressed and distributed in a punctate fashion in rat peritoneal mast cells ex vivo. The rat MC line RBL-2H3 was used as a model system to investigate the role of AP-3 in mast cell secretory granule biogenesis and mediator release. By immunofluorescence and immunoelectron microscopy, AP-3 was localized both to the TGN and early endosomes indicating that AP-3 dependent sorting of proteins to MC secretory granules originates in these organelles. ShRNA mediated depletion of the AP-3 δ subunit was shown to destabilize the AP-3 complex in RBL-2H3 MCs. AP-3 knockdown significantly affected MC regulated secretion of β-hexosaminidase without affecting total cellular enzyme levels. Morphometric evaluation of MC secretory granules by electron microscopy revealed that the area of MC secretory granules in AP-3 knockdown MCs was significantly increased, indicating that AP-3 is involved in MC secretory granule biogenesis. Furthermore, AP-3 knockdown had a selective impact on the secretion of newly formed and newly synthesized mediators. These results show for the first time that AP-3 plays a critical role in secretory granule biogenesis and mediator release in MCs

rPbPga1 from Paracoccidioides brasiliensis Activates Mast Cells and Macrophages via NFkB.

BACKGROUND:The fungus Paracoccidioides brasiliensis is the leading etiological agent of paracoccidioidomycosis (PCM), a systemic granulomatous disease that typically affects the lungs. Cell wall components of P. brasiliensis interact with host cells and influence the pathogenesis of PCM. In yeast, many glycosylphosphatidylinositol (GPI)-anchored proteins are important in the initial contact with the host, mediating host-yeast interactions that culminate with the disease. PbPga1 is a GPI anchored protein located on the surface of the yeast P. brasiliensis that is recognized by sera from PCM patients. METHODOLOGY/PRINCIPAL FINDINGS:Endogenous PbPga1 was localized to the surface of P. brasiliensis yeast cells in the lungs of infected mice using a polyclonal anti-rPbPga1 antibody. Furthermore, macrophages stained with anti-CD38 were associated with P. brasiliensis containing granulomas. Additionally, rPbPga1 activated the transcription factor NFkB in the macrophage cell line Raw 264.7 Luc cells, containing the luciferase gene downstream of the NFkB promoter. After 24 h of incubation with rPbPga1, alveolar macrophages from BALB/c mice were stimulated to release TNF-α, IL-4 and NO. Mast cells, identified by toluidine blue staining, were also associated with P. brasiliensis containing granulomas. Co-culture of P. Brasiliensis yeast cells with RBL-2H3 mast cells induced morphological changes on the surface of the mast cells. Furthermore, RBL-2H3 mast cells were degranulated by P. brasiliensis yeast cells, but not by rPbPga1, as determined by the release of beta-hexosaminidase. However, RBL-2H3 cells activated by rPbPga1 released the inflammatory interleukin IL-6 and also activated the transcription factor NFkB in GFP-reporter mast cells. The transcription factor NFAT was not activated when the mast cells were incubated with rPbPga1. CONCLUSIONS/SIGNIFICANCE:The results indicate that PbPga1 may act as a modulator protein in PCM pathogenesis and serve as a useful target for additional studies on the pathogenesis of P. brasiliensis

Rat embryonic mast cells originate in the AGM.

Mast cells originate from pluripotent hematopoietic stem cells. Two mast cell specific antibodies, mAbsAA4 and BGD6, have previously been used to identify and study committed mast cell precursors (MCcps) in the bone marrow of adult mice and rats. However, the embryonic origin of MCcps is still not known. In the present study, we identified MCcps in rat embryos using these previously characterized mast cell specific antibodies. The MCcps were found in the AGM (aorta-gonad-mesonephros) region of rat embryos at E11.5. These cells were BGD6+, CD34(+), c-kit(+), CD13(+), FcεRI(-), AA4(-) CD40(-), and Thy-1(-). By PCR the cells contained message for the α and β subunits of FcεRI and mast cell specific proteases. In vitro, the MCcps differentiated into metachromatic mast cells. With age of gestation the percent of MCcps diminished while the percent of mast cell progenitors increased. An increased knowledge of the biology and embryonic origin of mast cells may contribute to a greater understanding of allergy, asthma, and other mast cell related diseases

Phospholipase D2 Modulates the Secretory Pathway in RBL-2H3 Mast Cells

<div><p>Phospholipase D (PLD) hydrolyses phosphatidylcholine to produce phosphatidic acid (PA) and choline. It has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type. In mast cells it plays an important role in signal transduction. The aim of the present study was to clarify the role of PLD2 in the secretory pathway. RBL-2H3 cells, a mast cell line, transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2 were used. Previous observations showed that the Golgi complex was well organized in CA cells, but was disorganized and dispersed in CI cells. Furthermore, in CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized. These previous observations demonstrated that PLD2 is important for maintaining the morphology and organization of the Golgi complex. To further understand the role of PLD2 in secretory and vesicular trafficking, the role of PLD2 in the secretory process was investigated. Incorporation of sialic acid was used to follow the synthesis and transport of glycoconjugates in the cell lines. The modified sialic acid was subsequently detected by labeling with a fluorophore or biotin to visualize the localization of the molecule after a pulse-chase for various times. Glycoconjugate trafficking was slower in the CI cells and labeled glycans took longer to reach the plasma membrane. Furthermore, in CI cells sialic acid glycans remained at the plasma membrane for longer periods of time compared to RBL-2H3 cells. These results suggest that PLD2 activity plays an important role in regulating glycoconjugate trafficking in mast cells.</p></div

Knockdown of PLD2 in RBL-2H3 cells results in a phenotype similar to PLD2CI cells.

<p>(A) Cells were pulsed for 1 h with ManNAz and chased for 1 and 3 h (Bars: 10μm). (B) RBL-2H3 cells transfected with PLD2 siRNA had a 65% reduction in PLD2 expression when compared to control siRNA cells. P = 0.0279. (C) The fluorescence intensity of ManNAz in PLD2 siRNA cells was significantly lower than in control siRNA cells after 3 h of chase. P = 0.014 for 3 hours. For confocal microscopy, ManNAz was coupled to Alexa 488.</p

Trafficking of ManNAz towards the plasma membrane is regulated by PLD2.

<p>(A) Cells were pulse-labeled for 1 h with ManNAz, washed and chased for the indicated times. After 1 h of chase only PLD2CA cells had ManNAz labeling on the plasma membrane (arrow). With 3 h of chase all cell lines had ManNAz labeling on the plasma membrane (arrows) (Bars: 10μm). (B) Quantification of ManNAz at the cell surface. In PLD2CA cells, the amount of ManNAz on the plasma membrane was higher when compared to the other cells. P<0.0001 for 1 hour and 3 hours. (C) Cells were pulse-labeled for 1 h with ManNAz, washed and chased for the indicated times. ManNAz labeled the plasma membrane (arrows) (Bars: 10μm). (D) In PLD2CI cells there was more ManNAz on the cell surface throughout the investigated periods when compared with RBL-2H3 and PLD2CA. Moreover, in PLD2CA cells, there was a decreased amount of ManNAz on the cell surface in all the chase periods. P = 0.0122 for 12 hours. P = 0.0094 for 24 hours. P = 0.0337 for 36 hours. P = 0.0278 for 48 hours. P = 0.0020 for 60 hours. For confocal microscopy, ManNAz was coupled to Alexa 488.</p

ManNAz is incorporated in the biosynthetic secretory pathway in RBL-2H3 mast cells.

<p>(A) After 3 days of ManNAz incubation, cells were lysed and blotted with streptavidin-HRP. In the control lane RBL-2H3 cell lysate was cultured in the absence of ManNAz. (B) ManNAz was incorporated in all cell lines, with a higher degree in PLD2CA and PLD2CI cells. P = 0.0148. (C) The cell lysates were applied to polyacrylamide gels and stained with Coomassie blue to verify equal protein loading. (D) RBL-2H3 cells were incubated with ManNAz for 1 or 3 days. ManNAz-Alexa 488 was localized at the plasma membrane (arrows). The right column shows one plane of the confocal image, to better visualize ManNAz-Alexa 488 on the membrane (Bars: 10μm). (E) RBL-2H3 cells were incubated with ManNAz for 1 or 3 days. To visualize ManNAz subcellular localization cells were permeabilized. ManNAz-Alexa 488 can be seen in a juxtanuclear region and inside cytoplasmic vesicles (arrows). The right column shows one plane of the confocal image, to better visualize ManNAz-Alexa 488 inside the cell (Bars: 10μm). (F) ManNAz-Alexa 488 colocalizes with GD1b derived gangliosides labeled with anti-mouse IgG conjugated to Alexa 594 on the plasma membrane (Bars: 10μm).</p