Protein kinase C alpha, betaI, and betaII isozymes regulate cytokine production in mast cells through MEKK2/ERK5-dependent and -independent pathways

Regulation of MAPK pathways by PKC isoforms was examined in murine bone marrow-derived mast cells (BMMCs). The PKCalpha, betaI, and betaII isoforms showed the most robust activation after FcepsilonR1-mediated stimulation by anti-ovalbumin specific IgE and ovalbumin (IgE-ova). PKCalpha, betaI, and betaII were all involved in activation of JNK, MEKK2, and ERK5, with differential relative contributions of each isoform to specific MAPK pathway components. BMMCs from mice lacking MEKK2 showed reduced production (50-60%) of IL-6, IL-13, and TNF-alpha after stimulation, demonstrating MEKK2-dependent and -independent pathways for cytokine production. Cytokine production was stimulated by over-expression of PKC in cells from MEKK2-deficient and wild-type mice. Activation of ERK5 did not occur in BMMCs lacking MEKK2, indicating that MEKK2-independent cytokine production was also ERK5-independent. Since MAPK modules differentially regulate mast cell functions, including degranulation and cytokine production, it is suggested that specific functions could be targeted by inhibiting specific PKC isoforms.     

Pseudomonas aeruginosa activates human mast cells to induce neutrophil transendothelial migration via mast cell-derived IL-1 alpha and beta

The mechanisms of neutrophil (PMN) recruitment to Pseudomonas aeruginosa infection remain incompletely defined. Mast cells (MC) involvement in this process has not been studied previously. In this study, we demonstrate that human cord blood-derived MC phagocytose P. aeruginosa and release mediators that activate HUVEC monolayers for supporting PMN transmigration. Pretreatment of supernatants from P. aeruginosa-MC cocultures with neutralizing anti-IL-1alpha plus anti-IL-1beta Abs, or IL-1R antagonist before addition to HUVEC for stimulation completely abrogated MC-induced PMN transmigration, while anti-TNF-alpha treatment had no effect. The expression of E-selectin and ICAM-1 on HUVEC, the latter a ligand for PMN CD11/CD18, was significantly up-regulated by P. aeruginosa-induced MC mediators. Pretreatment of human PMN with anti-CD18 mAb or pretreatment of HUVEC with a combination of three mAbs (against ICAM-1, ICAM-2, and E-selectin) inhibited by 85% the MC-dependent PMN transmigration. Moreover, P. aeruginosa-induced production of IL-1alpha and IL-1beta was down-regulated by IL-10 and dexamethasone. This study demonstrates for the first time that MC may mediate P. aeruginosa-induced PMN recruitment via production of IL-1alpha and beta. These findings have important implications for diseases involving P. aeruginosa infection and suggest novel targets for modulating P. aeruginosa-induced inflammation.     

Feedlot dust stimulation of interleukin-6 and -8 requires protein kinase Cepsilon in human bronchial epithelial cells

Individuals exposed to dusts from concentrated animal feeding operations report increased numbers of respiratory tract symptoms, and bronchoalveolar lavage samples from such individuals demonstrate elevated lung inflammatory mediators, including interleukin (IL)-8 and IL-6. We previously found that exposure of bronchial epithelial cells to hog barn dusts resulted in a protein kinase C (PKC)-dependent increase in IL-6 and IL-8 release. We hypothesized that cattle feedlot dusts would also generate bronchial epithelial interleukin release in vitro. To test this, we used interleukin ELISAs and direct PKC isoform assays. We found that a dust extract from cattle feedlots [feedlot dust extract (FLDE)] augments PKC activity of human bronchial epithelial cells in vitro. A 5-10% dilution of FLDE stimulated a significant release of IL-6 and IL-8 at 6-24 h in a PKC-dependent manner vs. control medium-treated cells. An increase in PKCalpha activity was observed with 1 h of FLDE treatment, and PKCepsilon activity was elevated at 6 h of FLDE exposure. The PKCalpha inhibitor, G?-6976, did not inhibit FLDE-stimulated IL-8 and IL-6 release. However, the PKCepsilon inhibitor, Ro 31-8220, effectively inhibited FLDE-stimulated IL-8 and IL-6 release. Inhibition of FLDE-stimulated IL-6 and IL-8 was confirmed in a dominant-negative PKCepsilon-expressing BEAS-2B cell line but not observed in a PKCalpha dominant negative BEAS-2B cell line. These data support the hypothesis that FLDE exposure stimulates bronchial epithelial IL-8 and IL-6 release via a PKCepsilon-dependent pathway.     

Extracellular ATP induces cytokine expression and apoptosis through P2X7 receptor in murine mast cells

Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.     

Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed α, β, and γ subunits of high-affinity immunoglobulin E (IgE) receptor (FcεRI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-α, and cyclooxygenase 2, and production of prostaglandin D(2) and leukotriene C(4) in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-α expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on FcεRI- and TLR-mediated effector functions of mast cells.     

A crucial role of Flagellin in the induction of airway mucus production by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis.     

Mast cell-dependent B and T lymphocyte activation is mediated by the secretion of immunologically active exosomes

Mitogenic activity of bone marrow-derived mouse mast cells and mast cell lines P815 and MC/9 on B and T lymphocytes is present in their culture supernatants. To identify this activity, mast cells were incubated in serum-free medium and the supernatant was subjected to differential centrifugation, which resulted in two fractions, the hypodense and dense fraction (pellet). When analyzed for their mitogenic activity on spleen cells, all activity was found to be associated with the dense fraction. Electron microscopy studies revealed the presence in this fraction of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induced blast formation, proliferation, as well as IL-2 and IFN-gamma production, but no detectable IL-4. Similar data were obtained by injecting exosomes into naive mice. In contrast to mast cell lines, a pretreatment with IL-4 is required for bone marrow-derived mast cells to secrete active exosomes. Structurally, exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1, and ICAM-1. These findings indicate that mast cells can represent a critical component of the immunoregulatory network through secreted exosomes that display mitogenic activity on B and T lymphocytes both in vitro and in vivo.     

Prostaglandin E2 selectively enhances the IgE-mediated production of IL-6 and granulocyte-macrophage colony-stimulating factor by mast cells through an EP1/EP3-dependent mechanism

PGE(2) is an endogenously synthesized inflammatory mediator that is over-produced in chronic inflammatory disorders such as allergic asthma. In this study, we investigated the regulatory effects of PGE(2) on mast cell degranulation and the production of cytokines relevant to allergic disease. Murine bone marrow-derived mast cells (BMMC) were treated with PGE(2) alone or in the context of IgE-mediated activation. PGE(2) treatment alone specifically enhanced IL-6 production, and neither induced nor inhibited degranulation and the release of other mast cell cytokines, including IL-4, IL-10, IFN-gamma, and GM-CSF. IgE/Ag-mediated activation of BMMC induced the secretion of IL-4, IL-6, and GM-CSF, and concurrent PGE(2) stimulation synergistically increased mast cell degranulation and IL-6 and GM-CSF, but not IL-4, production. A similar potentiation of degranulation and IL-6 production by PGE(2), in the context of IgE-directed activation, was observed in the well-established IL-3-dependent murine mast cell line, MC/9. RT-PCR analysis of unstimulated MC/9 cells revealed the expression of EP(1), EP(3), and EP(4) PGE receptor subtypes, including a novel splice variant of the EP(1) receptor. Pharmacological studies using PGE receptor subtype-selective analogs showed that the potentiation of IgE/Ag-induced degranulation and IL-6 production by PGE(2) is mediated through EP(1) and/or EP(3) receptors. Our results suggest that PGE(2) may profoundly alter the nature of the mast cell degranulation and cytokine responses at sites of allergic inflammation through an EP(1)/EP(3)-dependent mechanism.     

Negative regulation of phosphatidylinositol 3-kinase and Akt signalling pathway by PKC

Although substantial studies have begun to explore the regulation of phosphatidylinositol 3-kinase/Akt cascade by different signalling pathways, whether protein kinase C (PKC) activity plays a crucial role remains as yet unclear. In this study, we found that in A549 and HEK293 cells non-selective PKC inhibitors Ro 31-8220 and bisindolylmaleimide VIII, and PKCbeta inhibitor LY 379196, caused Akt/PKB phosphorylation at Ser 473 and increased the upstream activator, integrin-linked kinase (ILK) activity. The increased Akt phosphorylation was blocked by phosphatidylinositol 3-kinase inhibitor wortmannin and the newly identified PIP(3)-dependent kinases (PDK) inhibitor SB 203580. In contrast to the Akt stimulation caused by PKC inhibitors, PMA attenuated Akt/PKB phosphorylation. We also found that this stimulating effect on Akt phosphorylation by PKC inhibitors was not the result of phosphatase inhibition, since treatment with PP2A, PP2B and tyrosine phosphatase inhibitors (okadaic acid, FK506 and sodium orthovanadate, respectively) had no effect. We conclude that phosphatidylinositol 3-kinase/Akt signalling pathway is regulated by PKC in a negative manner.     

Nerve growth factor modifies the expression of inflammatory cytokines by mast cells via a prostanoid-dependent mechanism

Nerve growth factor (NGF) is well recognized to have a number of potent effects on mast cells, including increasing mast cell numbers in vivo and inducing mast cell degranulation in vitro. More recently, NGF has been demonstrated to induce PGD2 production by mast cells through the induction of mast cell cyclooxygenase expression. We have observed that NGF at doses as low as 10 ng/ml will induce IL-6 production and inhibit TNF-alpha release from rat peritoneal mast cells in the presence of lysophosphatidylserine as a cofactor. NGF synergizes with LPS treatment of peritoneal mast cells (PMC) for the induction of IL-6. Examination of the mechanism of this phenomenon has revealed that NGF can induce both rat PMC and mouse bone marrow-derived cultured mast cells to produce substantial levels of PGE2. This response is maximal at later time points 18-24 h after NGF activation. The ability of NGF to induce PGE2 is not dependent on mast cell degranulation. Other stimuli capable of inducing IL-6, such as LPS, do not induce production of this prostanoid. Inhibition of cyclooxygenase activity by PMC using either flurbiprofen or indomethacin inhibited both the NGF-induced PGE2 synthesis and the NGF-induced alterations in TNF-alpha and IL-6 production. These results suggest a role for mast cell-derived prostanoids in the regulation of local inflammatory responses and neuronal degeneration after tissue injury involving induction of NGF production.     

Protein Phosphatase 2A Enables Expression of Interleukin 17 (IL-17) through Chromatin Remodeling

Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase involved in essential cellular functions. T cells from patients with systemic lupus erythematosus (SLE) express high levels of the catalytic subunit of PP2A (PP2Ac). A mouse overexpressing PP2Ac in T cells develops glomerulonephritis in an IL-17-dependent manner. Here, using microarray analyses, we demonstrate that increased expression of PP2Ac grants T cells the capacity to produce an array of proinflammatory effector molecules. Because IL-17 is important in the expression of glomerulonephritis, we studied the mechanism through which PP2Ac dysregulation facilitates its production. We report that PP2Ac is involved in the regulation of the Il17 locus by enhancing histone 3 acetylation through a mechanism that involves activation of interferon regulatory factor 4. Increased histone 3 acetylation of the Il17 locus is shared between T cells of PP2Ac transgenic mice and patients with SLE. We propose that, by promoting the inflammatory capacity of T cells, PP2Ac dysregulation contributes to the pathogenesis of SLE.     

Protein phosphatase 2A and neutral sphingomyelinase 2 regulate IRAK-1 protein ubiquitination and degradation in response to interleukin-1beta

The IL-1β signaling cascade is initiated by the phosphorylation of IL-1β receptor-associated kinase-1 (IRAK-1), followed by its ubiquitination and degradation. This paper investigates the regulation of IRAK-1 degradation in primary hepatocytes and in HEK cells overexpressing the IL-1β receptor. We provide evidence that protein phosphatase 2A (PP2A) is a negative regulator of the phosphorylation, Lys(48)-linked ubiquitination, and degradation of IRAK-1. PP2A catalytic activity increased within 30 min of stimulation with IL-1β. siRNA against PP2A catalytic subunit (PP2Ac) or treatment with pharmacological inhibitor, okadaic acid, enhanced IRAK-1 Lys(48)-linked ubiquitination and degradation. Direct interaction between PP2Ac and IRAK-1 was observed, suggesting that IRAK-1 might be a PP2A substrate. The mechanisms of PP2A activation by IL-1β involved neutral sphingomyelinase-2 (NSMase-2) and an accumulation of ceramide. Overexpression of NSMase-2 delayed IRAK-1 degradation in a PP2A-dependent manner, whereas NSMase-2 silencing had the opposite effect. The addition of sphingomyelinase, ceramide, or a proteasome inhibitor all led to retention of IRAK-1 at the cell membrane and to increased JNK phosphorylation. This study suggests that NSMase-2- and PP2A-dependent regulation of IRAK-1 degradation is a novel mechanism to fine tune the magnitude of IL-1β response.     

Interleukin (IL)-33 induces the release of pro-inflammatory mediators by mast cells

IL-33 (or IL-1F11) was recently identified as a ligand for the previously orphaned IL-1 family receptor T1/ST2. Previous studies have established that IL-33 and T1/ST2 exert key functions in Th2 responses. In this study, we demonstrate that IL-33 induces the production of pro-inflammatory mediators in mast cells. IL-33 dose and time-dependently stimulated IL-6 secretion by P815 mastocytoma cells and primary mouse bone marrow-derived mast cells (BMMC). This effect was dependent on T1/ST2 binding. In addition, IL-33 also induced IL-1β, TNF-α, MCP-1, and PGD2 production in BMMC. By RNase protection assay, we demonstrated that IL-33 increased IL-6 and IL-1β mRNA expression. These effects of IL-33 appeared to occur independently of mast cell degranulation, The results of this study show for the first time that IL-33, a novel member of the IL-1 family of cytokines, stimulates the production of pro-inflammatory mediators by mast cells in addition to its effect on T helper 2 responses. These findings open new perspectives for the treatment of inflammatory diseases by targeting IL-33.