Bone Marrow Cultured Mast Cells Research Articles

Characterization of WWOX expression and function in canine mast cell tumors and malignant mast cell lines

BackgroundThe WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of WWOX has been implicated as playing a key role in tumor cell survival, DNA damage repair, and genomic stability. The purpose of this study was to characterize WWOX expression in spontaneous canine mast cell tumors (MCTs) and malignant cell lines and investigate the potential contribution of WWOX loss on malignant mast cell behavior.Methods/resultsWWOX expression is decreased in primary canine MCTs and malignant mast cell lines compared to normal canine bone marrow-cultured mast cells. In transformed canine mastocytoma cell lines, overexpression of WWOX or WWOX knockdown had no effect on mast cell viability. Inhibition of WWOX enhanced clonogenic survival following treatment with ionizing radiation in the C2 mast cell line. Lastly, immunohistochemistry for WWOX was performed using a canine MCT tissue microarray, demonstrating that WWOX staining intensity and percent of cells staining for WWOX is decreased in high-grade MCTs compared to low-grade MCTs.ConclusionsThese data suggest that WWOX expression is attenuated or lost in primary canine MCTs and malignant mast cell lines. Given the observed increase in clonogenic survival in WWOX-deficient C2 mast cells treated with ionizing radiation, further investigation of WWOX and its role in mediating the DNA damage response in malignant mast cells is warranted.

Influenza virus induces mast cell death independent of mast cell activation

Abstract Influenza A virus (IAV) infects over a million individuals each year, resulting in significant morbidity, and during pandemic years, significant mortality. Severe IAV infection is characterized by a dysregulated cytokine response, termed a ‘cytokine storm’. Recent evidence indicates mast cells can play a central role in initiating this cytokine storm, though the long-term effects of IAV infection on mast cells is poorly understood. To this end, we infected bone marrow cultured mast cells with IAV at a low multiplicity of infection, allowing us to track IAV infection in mast cell cultures over time. Within hours mast cells express viral proteins, but fail to release appreciable levels of infectious viral particles throughout the course of infection. Despite the abortive nature of this infection, it is highly cytotoxic, with significant cell death occurring between 12–48 hours post infection. This cell death is the result of simultaneous activation of apoptosis and necroptosis pathways. Remarkably, while UV-inactivated virus activates mast cells, it does not induce cell death. Conversely, infection with a neuraminidase mutant is cytotoxic despite the fact this mutant fails to activate mast cells. These data suggest the induction of programmed cell death is dependent on viral replication, but independent of mast cell activation. In support of this hypothesis, the RIG-I and interferon signaling pathways are dispensable for the induction of cell death, as mast cells derived from MAVS−/−, AG129 (IFNAR1/IFNGR DKO), and STAT1−/− mice die at rates comparable to WT cells. Together, these data indicate mast cells detect IAV infection using multiple innate sensors, with each mode of detection resulting in the activation of a distinct response.

When signals get crossed: the consequences of dual allergen- and influenza-mediated signaling in mast cells

Abstract Despite the many advances in our understanding of viral infections, influenza A virus (IAV) continues to impose a significant heath burden and presents a serious threat for a worldwide pandemic. Severe IAV infection is characterized by a cytokine storm. Recent evidence indicates mast cell activation is a key initiator of this cytokine storm during severe IAV infection. During the 2009 H1N1 pandemic, asthma was the most common co-morbidity among hospitalized individuals suggesting a potential interplay between pre-existing mast cell disease and IAV infection. Experimental data examining the cross-talk that can occur during dual FcɛRI and IAV stimulation of mast cells is lacking. Here we show that signaling through FcɛRI concurrent with IAV infection (A/WSN/33) augments the mast cell response resulting in a highly synergist enhancement of cytokine synthesis and release by these cells in vitro. Surprisingly, while IAV induced cytokine production by A/WSN/33 is dependent on the MAVS pathway, using bone marrow cultured mast cells from MAVS and STING knockout mice, we determined this synergistic effect can occur independent of MAVS signaling and the STING adaptor. Furthermore, the synergy can be observed as early as 1 hour PI, prior to significant viral replication. These data suggest an early signaling event is responsible for the synergy. Our data supports a role for the lipid mediator sphingosine-1-phosphate, which is known to be important during allergen stimulation, and as our data demonstrates, is essential for proper IAV activation of mast cells. Taken together, our data suggest that IAV may incite asthmatic exacerbation through enhanced cytokine production from mast cells receiving two distinct stimuli simultaneously.

Clonal differences in IgE antibodies affect cutaneous anaphylaxis-associated thermal sensitivity in mice

Cellular and molecular mediators of immune responses are increasingly implicated in acute and chronic pain pathophysiologies. Here we demonstrate that passive cutaneous IgE/Ag anaphylaxis provokes increased thermal sensitivity in the hind paw tissue of mice. The murine anti-DNP IgE antibodies SPE-7 and ɛ26 are known to induce differential cytokine production in bone marrow cultured mast cells in vitro without antigen challenge. We found a novel, antigen-dependent heterogeneity in the thermal pain responses elicited in the hind paws between SPE-7 and ɛ26 sensitized DNP-challenged mice. Mice experienced pronounced hind paw thermal sensitivity lasting 6h after DNP challenge when sensitized with SPE-7 but not ɛ26 IgE. The two IgE clones induced equivalent hind paw edema, neutrophil influx, cytokine production, and reduction in tissue histamine content in vivo, and bound to the same or overlapping epitopes on the DNP antigen in vitro. Therefore IgE antibodies against the same antigen can induce comparable inflammation, yet contribute to markedly different anaphylaxis-associated pain within an allergic response, suggesting that non-canonical IgE binding partners such as sensory neurons may play a role in allergy-related pain responses.

Role of mast cells in a chronic murine asthma model (HYP3P.348)

Abstract Mast cells are thought to be important in the cause of allergic diseases, but the role of mast cells in the airway remodeling of chronic long-term asthma remains controversial. In this study, using the mast cell “nock in” strategy to get the mast cell-deficient WBB6F1-W/Wv mice (W/Wv) selectively reconstituted bone marrow-cultured mast cells (BMCMCs) from normal congenic wild-type mice (+/+) (+/+ BMCMCs→W/Wv mice), mice were employed to study the roles of mast cell in a murine asthma model. The mice were sensitized with ovalbumin (OVA) and challenged with OVA for 14 weeks. The mice were assessed airway reactivity by PenH. The lungs and trachea were stained with H-E for pathologic alteration, toluidine blue for mast cell, congo red for eosinophil, PAS for goblet cell and Masson’s-trichrome for fibrosis. Mast cells were not observed in the lungs of control and OVA-sensitized and challenged W/WV mice, but OVA-sensitization and challenge increased mast cell number in +/+ mice and +/+BMCMCs→W/Wv mice. OVA-sensitization and challenge could enhance airway reactivity, pathologic alteration, eosinophil infiltration, goblet cell hyperplasia and fibrosis in +/+ mice. The pathologic changes of OVA treated W/Wv mice were less than that of +/+ mice, but +/+BMCMCs→W/Wv mice could restore the reaction to OVA-sensitization and challenge of +/+ mice. These results indicate that the mast cell play an important role in the OVA-induced chronic murine asthma model.

Infection and activation of mast cells by influenza A virus enhances viral-induced pathology (P6030)

Abstract Influenza A virus (IAV) is a seasonal respiratory pathogen that can result in severe lung pathology. The role of mast cells has been under explored during severe pulmonary viral infections. We found that both A/WSN/33 (WSN) and A/PR/8/34 (PR8) influenza viruses cause significant immunopathology in C57BL/6 mice, but only WSN induced pathology was mast cell dependent. Using in vitro-derived bone marrow cultured mast cells (BMCMC), we found that WSN, but not PR8, directly activated BMCMC to produce histamine, leukotrienes, inflammatory cytokines, and anti-viral chemokines. Moreover, human H1N1, H3N2, and influenza B virus isolates could activate both murine BMCMC and the human mast cell line HMC-1 in vitro. BMCMC activation required infection of mast cells by IAV, which was dependent on the viral hemagglutinin. Cytokine and chemokine production from BMCMC occurs in a RIG-I/MAVS-dependent fashion which required the de novo production of vRNA; conversely, degranulation occurs through a RIG-I/MAVS-independent mechanism. Reconstitution of mast cell deficient mice with RIG-I-/- BMCMC generated lung pathology similar to wild type BMCMC, suggesting that mast cell degranulation, rather than production of cytokines, causes WSN induced lung pathology. Thus, we have identified a unique inflammatory cascade which could be therapeutically targeted to limit morbidity following infection with influenza virus.

Mast cell-dependent inflammatory response during influenza A virus infection (168.31)

Abstract The lung is protected from pathogens by alveolar epithelial cells, tissue resident alveolar macrophages, dendritic cells, and mast cells. The role of the mast cell has been under explored during severe pulmonary viral infections. In mice that lack functional mast cells, pathological immune responses are altered depending on the influenza strain. We found that both A/WSN/33 (WSN) and A/PR/8/34 (PR8) influenza viruses generate significant immunopathology in C57BL/6 mice, but only WSN induced pathology was mast cell dependent. To assess the differences in mast cell dependent inflammation response due to influenza strain, we examined the ability of each strain to activate in vitro-derived bone marrow cultured mast cells (BMCMC). We found that WSN, but not PR8, influenza virus activated BMCMC to produce inflammatory cytokines/chemokines, anti-viral chemokines, and degranulate. Importantly, a recombinant PR8 virus that expresses the WSN hemagglutinin (HA) glycoprotein instead of its own could induce cytokine production in BMCMC, demonstrating an important role for the HA glycoprotein for mast cell activation. Mast cell infection and viral RNA synthesis and export were necessary for mast cell activation and occurred in a MAVS-dependent mechanism. Thus, we have identified a unique inflammatory cascade which could be targeted to limit morbidity following infection with influenza virus.

Mast Cell-Fibroblast Interactions Induce Matrix Metalloproteinase-9 Release from Fibroblasts: Role for IgE-Mediated Mast Cell Activation

Mast cells adhere to fibroblasts, but the biological effects of adhesion are not well understood. We hypothesized that these adhesive interactions are important for tissue remodeling through the release of matrix metalloproteinases (MMP). Murine bone marrow cultured mast cells (BMCMC) were cocultured with NIH-3T3 fibroblasts or murine lung fibroblasts (CCL-206) and supernatants analyzed for MMP-9 release by gelatin zymography. Coculture of BMCMC for 24 h with NIH-3T3 or CCL-206 fibroblasts increased the release of MMP-9 from fibroblasts by 1.7+/-0.2 and 2.0+/-0.7-fold, respectively. Coculture of BMCMC and fibroblasts in the presence of IgE increased further MMP-9 release, which was released by fibroblasts. MMP-9 release was dependent on TNF released from IgE activated BMCMC and on adhesive interactions between BMCMC and fibroblasts. Increased MMP-9 release was also p44/42-dependent, as was MMP-9 up-regulation during coculture of fibroblasts with resting BMCMC. Finally, IgE injection into the mouse ear increased MMP-9 content of the ear tissue in the absence of Ag, indicating that IgE-mediated remodeling may play a pathogenic role in allergic conditions even in the absence of exposure to allergens. In conclusion, mast cell-fibroblast interactions induce the release of proteases important for tissue remodeling, such as MMP-9. MMP-9 release was further increased in the presence of IgE during coculture, suggesting a role for mast cell-fibroblast interactions in atopic conditions.

Mast Cell-Derived Plasminogen Activator Inhibitor-1 in the Airways of a Murine Asthma Model and Its Regulation by Cysteinyl Leukotrienes

RATIONALE: We previously demonstrated that plasminogen activator inhibitor (PAI)-1 is produced in mast cells (MCs) and promotes the airway remodeling in a murine asthma model. In the present study, we investigated the regulation of MC-derived PAI-1 by cysteinyl leukotrienes (cysLTs). METHODS: MC-deficient W/Wv mice were reconstituted with bone marrow cultured MCs (BMCMCs) from PAI-1-deficient (PAI-1-/-) or PAI-1+/+ mice (PAI-1-/-→ W/Wv or PAI-1+/+→ W/Wv mice, respectively). The effect of commonly used asthma drugs, including albuterol, corticosteroids, and a cysLT receptor 1 antagonist (montelukast) were tested on PAI-1 production in MCs. The regulation of PAI-1 production by cysLTs was investigated in MCs and a murine asthma model using real time RT-PCR and ELISA. RESULTS: The PAI-1 levels in PAI-1+/+→ W/Wv mice were restored similar to those in PAI-1+/+ mice, whereas the PAI-1 levels in PAI-1-/-→ W/Wv mice were less than 50% of those in PAI-1+/+→ W/Wv mice. Only montelukast reduced PAI-1 secretion by 30%. Leukotriene D4, a potent cysLT, augmented PAI-1 production in primary cultured human MCs (PCHMCs) primed with interleukin-4 or stimulated by FcεRI cross-linking, but not in unstimulated PCHMCs. Montelukast reduced PAI-1 production in FcεRI-stimulated PCHMCs and in the airways of a murine asthma model. CONCLUSIONS: These data suggest that MCs are a main source of PAI-1 in the airways of a murine asthma model and cysLTR1 is an important regulator of MC-derived PAI-1 production.

Mast cell-fibroblast interactions: Role of monomeric IgE in MMP-9 upregulation

Rationale Mast cells (MC) adhere to fibroblasts, but the stimuli inducing adhesion and the biological effects of adhesion are not well understood. We hypothesized that these adhesive interactions are important for tissue remodeling through the release of matrix metalloproteinases (MMP). Methods Murine bone marrow cultured mast cells (BMCMC) generated from Balb/c mice were co-cultured with NIH-3T3 fibroblasts or murine lung fibroblasts (CCL-208) for up to 24h. Adhesion was evaluated with a fluorescent assay. Release of TNF and IL-6 was evaluated with ELISA. MMP-2 and MMP-9 release was assessed with gelatin zymography. Results Co-culture of BMCMC with fibroblasts for 24h increased the release of MMP-9 from NIH-3T3 and CCL-208 fibroblasts by 1.89 ± 0.43 fold and 2.31 ± 0.77 fold respectively. Co-culture had no effect on MMP-2 release from fibroblasts. BMCMC adhesion to fibroblasts was approximately 20%. Monomeric IgE (1-10 mg/ml), although increased MC adhesion to fibronectin, had no effect on MC adhesion to fibroblasts. However, co-culture of BMCMC and fibroblasts in the presence of monomeric IgE (10 mg/ml) increased MMP-9 release from fibroblasts by 8.15 ± 1.55 fold. IgE did not induce MMP-9 release from BMCMC cultured alone. Monomeric IgE induced the release of TNF from BMCMC. Anti-TNF antibodies inhibited partially the effect of IgE activated BMCMC on fibroblast MMP-9 release. Anti-c-kit antibodies also partially inhibited MMP-9 release indicating a role for adhesion. Conclusions MC-fibroblast interactions activate fibroblasts to release proteases important for tissue remodeling. These effects are upregulated by monomeric IgE pointing towards a role for MC-fibroblast interactions in atopic conditions.

Mast cell-derived plasminogen activator inhibitor-1 in the airways of a murine asthma model and its regulation by cysteinyl leukotrienes

Rationale Plasminogen activator inhibitor (PAI)-1 is secreted from activated mast cells (MCs). In the present study, we further investigated the role of MC-derived PAI-1 in the airways of a murine model of asthma. Methods MC-deficient WBB6F 1- Kit W/Kit W-v (W/W v) mice were reconstituted with bone marrow cultured MCs (BMCMCs) from PAI-1 −/− or PAI-1 +/+ mice (PAI-1 −/−→W/W v or PAI-1 +/+→W/W v mice, respectively) and PAI-1 level was measured by ELISA. Human MC line (HMC-1) cells were stimulated by PMA/A3187 and primary cultured hMCs (PCHMCs) by FcϵRI crosslinking with or without asthma drugs and cysteinyl leukotrienes (cysLTs). Results The secretion of PAI-1 in the airways was greatly reduced in W/W v mice as compared with wild type (+/+) mice in a murine asthma model. PAI-1 levels in PAI-1 +/+→W/W v mice were restored almost similar to the levels of +/+ mice, whereas PAI-1 levels in PAI-1 −/−→W/W v mice were less than 50% of those in +/+ mice. To determine the effect of commonly used asthma drugs on PAI-1 regulation in MCs, albuterol, corticosteroids, and Montelukast were tested. Only Montelukast reduced PAI-1 secretion in stimulated HMC-1 cells by 30.7%. Upon stimulation by FcϵRI crosslinking, LTD 4 augmented PAI-1 production in PCHMCs and MK571 reduced the augmentative effect of LTD 4. Furthermore, we found that Montelukast reduced the secretion of PAI-1 in the airways of a murine model of asthma. Conclusions These findings suggest that MCs are a main source of PAI-1 in the airways of a murine asthma model and cysLT receptor 1 is an important regulator of MC-derived PAI-1 production.

Thrombin induces mast cell adhesion to fibronectin: evidence for involvement of protease-activated receptor-1.

Thrombin activates mast cells to release inflammatory mediators through a mechanism involving protease-activated receptor-1 (PAR-1). We hypothesized that PAR-1 activation would induce mast cell adhesion to fibronectin (FN). Fluorescent adhesion assay was performed in 96-well plates coated with FN (20 microg/ml). Murine bone marrow cultured mast cells (BMCMC) were used after 3-5 wk of culture (>98% mast cells by flow cytometry for c-Kit expression). Thrombin induced beta-hexosaminidase, IL-6, and matrix metalloproteinase-9 release from BMCMC. Thrombin and the PAR-1-activating peptide AparafluoroFRCyclohexylACitY-NH(2) (cit) induced BMCMC adhesion to FN in a dose-dependent fashion, while the PAR-1-inactive peptide FSLLRY-NH(2) had no effect. Thrombin and cit induced also BMCMC adhesion to laminin. Thrombin-mediated adhesion to FN was inhibited by anti-alpha(5) integrin Ab (51.1 +/- 6.7%; n = 5). The combination of anti-alpha(5) and anti-alpha(4) Abs induced higher inhibition (65.7 +/- 7.1%; n = 5). Unlike what is known for FcepsilonRI-mediated adhesion, PAR-1-mediated adhesion to FN did not increase mediator release. We then explored the signaling pathways involved in PAR-1-mediated mast cell adhesion. Thrombin and cit induced p44/42 and p38 phosphorylation. Pertussis toxin inhibited PAR-1-mediated BMCMC adhesion by 57.3 +/- 7.3% (n = 4), indicating that G(i) proteins are involved. Wortmannin and calphostin almost completely inhibited PAR-1-mediated mast cell adhesion, indicating that PI-3 kinase and protein kinase C are involved. Adhesion was partially inhibited by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 (24.5 +/- 3.3%; n = 3) and the p38 inhibitor SB203580 (25.1 +/- 10.4%; n = 3). The two inhibitors had additive effects. Therefore, thrombin mediates mast cell adhesion through the activation of G(i) proteins, phosphoinositol 3-kinase, protein kinase C, and mitogen-activated protein kinase pathways.

Stem cell factor influences neuro-immune interactions: The response of mast cells to pituitary adenylate cyclase activating polypeptide is altered by stem cell factor

Mast cells degranulation can be elicited by a number of biologically important neuropeptides, but the mechanisms involved in mast cell–neuropeptide interactions have not been fully elucidated. Stem cell factor (SCF), also known as c- kit or kit ligand, induces multiple effects on mast cells, including proliferation, differentiation, maturation, and prevents apoptosis. We investigated the ability of SCF to affect mast cell responsiveness to the neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). PACAP 1-27, PACAP1-38, or VIP failed to induced preformed mediator release from mouse bone-marrow-cultured mast cells (BMCMC) derived in concanavalin A-stimulated spleen conditioned medium (CM). By contrast, BMCMC grown in SCF-containing medium or freshly isolated peritoneal mast cells exhibited significant 3H-hydroxytrypamine (5-HT) release in response to PACAP peptides or VIP. Deoxyglucose and the mitochondrial inhibitor antimycin significantly inhibited PACAP-induced 5-HT release indicating that the central event induced by PACAP peptides was exocytosis. The G αi inhibitor, pertussis toxin, significantly diminished PACAP-induced 5-HT release from BMCMCs in SCF suggesting the involvement of heterotrimeric G-proteins. Western blot analysis using antibodies directed against the human VIP type I/PACAP type II receptor demonstrated a 70–72 kD immunoreactive protein expressed in greater amounts in BMCMC grown in SCF compared with BMCMC in CM. We conclude that SCF induces a mast cell population that is responsive to PACAPs and VIP involving a heterotrimeric G-protein-dependent mechanism.

Chemotaxis of Rat Mast Cells Toward Adenine Nucleotides

Rat mucosal mast cells express P2 purinoceptors, occupation of which mobilizes cytosolic Ca2+ and activates a potassium conductance. The primary function of this P2 system in mast cell biology remains unknown. Here, we show that extracellular ADP causes morphological changes in rat bone marrow-cultured mast cells (BMMC) typical of those occurring in cells stimulated by chemotaxins, and that the nucleotides ADP, ATP, and UTP are effective chemoattractants for rat BMMC. ADP was also a chemotaxin for murine J774 monocytes. The nucleotide selectivity and pertussis toxin sensitivity of the rat BMMC migratory response suggest the involvement of P2U receptors. Poorly hydrolyzable derivatives of ADP and ATP were effective chemotaxins, obviating a role for adenosine receptors. Buffering of external Ca2+ at 100 nM or reduction of the electrical gradient driving Ca2+ entry (by elevating external K+) blocked ADP-driven chemotaxis, suggesting a role for Ca2+ influx in this process. Anaphylatoxin C5a was a potent chemotaxin (EC50 approximately 0.5 nM) for J774 monocytes, but it was inactive on rat BMMC in the presence or absence of laminin. Ca2+ removal or elevated [K+] had modest effects on C5a-driven chemotaxis of J774 cells, implicating markedly different requirements for Ca2+ signaling in C5a- vs ADP-mediated chemotaxis. This is supported by the observation that depletion of Ca2+ stores with thapsigargin completely blocked migration induced by ADP but not C5a. These findings suggest that adenine nucleotides liberated from parasite-infested tissue could participate in the recruitment of mast cells by intestinal mucosa.

Mast cells express connexins on their cytoplasmic membrane

Background: Because of the close association between mast cells and fibroblasts in the microenvironment and the importance of connexins (Cxs) in fibroblast communication with other cells, we hypothesized that mast cells also express Cxs, allowing them to similarly communicate with other cells through gap junctions. Objectives: We sought to identify the expression of Cxs (particularly Cx43, Cx32, and Cx26) by murine mast cells. Methods: The expression of Cxs was studied by RT-PCR, Northern blot analysis, Western blot analysis, flow cytometry, and confocal laser scanning microscopy. Results: In this report we demonstrate that murine bone marrow cultured mast cells and the growth factor–independent murine mast cell line C57, express Cx43 and Cx32 as assessed by RT-PCR, Northern blot analysis, Western blot analysis, and flow cytometry, but do not express Cx26. We also show, by confocal laser scanning microscopy, that Cx43 localizes to the cytoplasmic membrane of mast cells in a pattern similar to that seen in fibroblasts. Conclusions: Mast cells express Cx43 and Cx32, and Cx43 is associated with the cytoplasmic membrane, suggesting that mast cells have the potential to communicate with other cells in their microenvironment in part through gap junctions. (J Allergy Clin Immunol 1999;103:656-62.)

Tyrosine kinase-deficient Wv c-kit induces mast cell adhesion and chemotaxis.

W/Wv mice are deficient in tissue mast cells, and mast cells cultured from these mice do not proliferate in response to the c-kit ligand, stem cell factor (SCF). In this paper, we report that mouse bone marrow cultured mast cells derived from W/Wv mice do adhere to fibronectin in the presence of SCF and exhibit chemotaxis to SCF, and we explore this model for the understanding of c-kit-mediated signaling pathways. Both in vitro and in vivo (in intact cells) phosphorylation experiments demonstrated a low residual level of W/Wv c-kit protein phosphorylation. SCF-induced responses in W/Wv mast cells were abolished by the tyrosine kinase inhibitor herbimycin A and by the phospatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin but were not affected by protein kinase C inhibitors. These observations are consistent with the conclusions that Wv c-kit initiates a signaling process that is PI 3-kinase dependent and that mutated Wv c-kit retains the ability to initiate mast cell adhesion and migration.

Stem Cell Factor Influences Mast Cell Mediator Release in Response to Eosinophil-Derived Granule Major Basic Protein

Stem cell factor (SCF) is an important mast cell growth, differentiation, and survival factor. We investigated whether SCF influenced the response of mouse mast cells to an IgE-independent stimulus, eosinophil-derived granule major basic protein (MBP). Mouse bone marrow cultured mast cells (BMCMC) were derived in either concanavalin-stimulated mouse spleen conditioned medium (CM) or SCF. The cloned growth, factor-independent mast cell line Cl.MC/C57.1 was also studied. BMCMC in SCF exhibited cytochemical staining properties, protease and histamine content, and increased serotonin uptake consistent with more mature differentiated mast cells as compared with BMCMC in CM or Cl.MC/ C57.1 cells. BMCMC in SCF released serotonin,14C-labeled arachidonic acid metabolites and tumor necrosis factor-α (TNF-α) on stimulation with MBP, while no response was seen from either BMCMC in CM or Cl.MC/C57.1 cells. All three mast cell populations released mediators on stimulation with the cationic MBP analog, poly-L-arginine, indicating that the cationic charge did not explain the selective response of BMCMC in SCF to eosinophil-derived granule MBP. These findings show that SCF significantly influences mast cell differentiation and the responsiveness of mast cells to eosinophil-derived granule MBP.© 1998 by The American Society of Hematology.

Stem Cell Factor Influences Mast Cell Mediator Release in Response to Eosinophil-Derived Granule Major Basic Protein

AbstractStem cell factor (SCF) is an important mast cell growth, differentiation, and survival factor. We investigated whether SCF influenced the response of mouse mast cells to an IgE-independent stimulus, eosinophil-derived granule major basic protein (MBP). Mouse bone marrow cultured mast cells (BMCMC) were derived in either concanavalin-stimulated mouse spleen conditioned medium (CM) or SCF. The cloned growth, factor-independent mast cell line Cl.MC/C57.1 was also studied. BMCMC in SCF exhibited cytochemical staining properties, protease and histamine content, and increased serotonin uptake consistent with more mature differentiated mast cells as compared with BMCMC in CM or Cl.MC/ C57.1 cells. BMCMC in SCF released serotonin,14C-labeled arachidonic acid metabolites and tumor necrosis factor-α (TNF-α) on stimulation with MBP, while no response was seen from either BMCMC in CM or Cl.MC/C57.1 cells. All three mast cell populations released mediators on stimulation with the cationic MBP analog, poly-L-arginine, indicating that the cationic charge did not explain the selective response of BMCMC in SCF to eosinophil-derived granule MBP. These findings show that SCF significantly influences mast cell differentiation and the responsiveness of mast cells to eosinophil-derived granule MBP.© 1998 by The American Society of Hematology.

Fas ligand (FasL, CD95L, APO-1L) expression in murine mast cells.

Fas ligand (FasL, CD95L, Apo-1L), a type II membrane protein belonging to the tumour necrosis factor family, induces apoptosis in Fas-bearing cells. As murine mast cells have been shown to express Fas antigen, we hypothesized that mast cells might also express FasL. To explore this possibility, we first demonstrated FasL mRNA in mast cells by reverse transcription-polymerase chain reaction and FasL protein by immunoblot analysis. FasL protein was shown to be exclusively located within the cell by flow cytometry. In agreement with this observation, bone marrow cultured mast cells were unable to kill Jurkat T cells. Our results demonstrate that FasL is expressed in murine mast cells and suggest that this murine mast cell FasL is not lytic, owing to the intracellular localization.

SUBSTANCE P-INDUCED MAST CELL MEDIATOR RELEASE IS ASSOCIATED WITH STEM CELL FACTOR-DEPENDENT UPREGULATION OF Gαi-2 AND Gαi-3 PROTEINS AND DOWNSTREAM SIGNALING THROUGH A PROTEIN KINASE C-DEPENDENT PATHWAY

51 Aims: Substance P (SP)-induced mast cell activation has been implicated in the pathogenesis of gastrointestinal inflammation, but the precise nature of mast cell/SP interactions has not been completely defined. We previously reported that stem cell factor (SCF) conferred reactivity to mast cells stimulated by substance P through a signaling pathway involving G-proteins (Gastroenterology 1996; 110: A911). We therefore assessed whether SCF influenced the expression of G-proteins in mast cells and examined the role of protein kinase C (PKC) in SP-induced mast cell activation. Methods: Bone marrow cultured mast cells (BMCMCs) were maintained in either SCF (50 ng/ml) or 20% Concanavalin A-stimulated spleen conditioned medium (CM) for 4-6 weeks. Western blot analysis of G-protein expression was then performed on BMCMCs in SCF or CM. BMCMCs in SCF or CM were incubated with3 H-5-hydroxytryptamine (5-HT) in the presence or absence of the PKC inhibitors calphostin C (CC; 1 μM) or bisindolylmaleimide I (BIM; 10μM), and then challenged with either SP (10-4 M) or medium alone. Radioactive counts in the supernatants and cell pellets were measured and specific 5-HT release calculated. Results: SP induced significant 5-HT release from BMCMCs in SCF compared with medium alone (12.5 ± 1.5% vs. 1.6 ± 0.2%, respectively, p < 0.05), while no 5-HT release occurred from BMCMCs in CM. Western blot analysis demonstrated a marked increase in the expression of the G-proteins Gαi-2 (by 5 fold) and Gαi-3 (by 20 fold) in BMCMCs in SCF compared with BMCMCs in CM. Furthermore, the PKC inhibitors CC or BIM inhibited SP-induced 5-HT release from BMCMCs in SCF (by 74% and 100%, respectively). Conclusions: We found that mast cells maintained in SCF acquire the ability to release serotonin upon stimulation with SP and express increased levels of Gαi-2 and Gαi-3 proteins. Furthermore, our data demonstrate that SP-induced mast cell pre-formed mediator release involves a protein kinase C-dependent pathway.