To investigate why responses of mast cells to antigen-induced IgE receptor (Fc?RI) aggregation depend nonlinearly on antigen dose, we characterized a new artificial ligand, DF3, through complementary modeling and experimentation. This ligand is a stable trimer of peptides derived from bacteriophage T4 fibritin, each conjugated to a hapten (DNP). We found low and high doses of DF3 at which degranulation of mast cells sensitized with DNP-specific IgE is minimal, but ligand-induced receptor aggregation is comparable to aggregation at an intermediate dose, optimal for degranulation. This finding makes DF3 an ideal reagent for studying the balance of negative and positive signaling in the Fc?RI pathway. We find that the lipid phosphatase SHIP and the protein tyrosine phosphatase SHP-1 negatively regulate mast cell degranulation over all doses considered. In contrast, SHP-2 promotes degranulation. With high DF3 doses, relatively rapid recruitment of SHIP to the plasma membrane may explain the reduced degranulation response. Our results demonstrate that optimal secretory responses of mast cells depend on the formation of receptor aggregates that promote sufficient positive signaling by Syk to override phosphatase-mediated negative regulatory signals.
PKA phosphorylates multiple molecules involved in calcium (Ca2+) handling in cardiac myocytes and is considered to be the predominant regulator of ?-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca2+ storage and the magnitude of Ca2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKC?. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.
Factors that can induce the release of histamine from basophils have been studied for more than 30 years. A protein termed histamine-releasing factor (HRF) was purified and molecularly cloned in 1995. HRF can stimulate histamine release and IL-4 and IL-13 production from IgE-sensitized basophils and mast cells. HRF-like activities were found in bodily fluids during the late phase of allergic reactions, implicating HRF in allergic diseases. However, definitive evidence for the role of HRF in allergic diseases has remained elusive. On the other hand, we found effects of monomeric IgE on the survival and activation of mast cells without the involvement of a specific antigen, as well as heterogeneity of IgEs in their ability to cause such effects. The latter property of IgE molecules seemed to be similar to the heterogeneity of IgEs in their ability to prime basophils in response to HRF. This similarity led to our recent finding that ~30% of IgE molecules can bind to HRF via their Fab interactions with two binding sites within the HRF molecule. The use of peptide inhibitors that block HRF-IgE interactions revealed an essential role of HRF to promote skin hypersensitivity and airway inflammation. This review summarizes this and more recent findings and provides a perspective on how they impact our understanding of allergy pathogenesis and potentially change the treatment of allergic diseases.
Atopic dermatitis (AD) is a chronic inflammatory skin disease. Here, we show that phospholipase C-?3 (PLC-?3)-deficient mice spontaneously develop AD-like skin lesions and more severe allergen-induced dermatitis than wild-type mice. Mast cells were required for both AD models and remarkably increased in the skin of Plcb3(-/-) mice because of the increased Stat5 and reduced SHP-1 activities. Mast cell-specific deletion of Stat5 gene ameliorated allergen-induced dermatitis, whereas that of Shp1 gene encoding Stat5-inactivating SHP-1 exacerbated it. PLC-?3 regulates the expression of periostin in fibroblasts and TSLP in keratinocytes, two proteins critically involved in AD pathogenesis. Furthermore, polymorphisms in PLCB3, SHP1, STAT5A, and STAT5B genes were associated with human AD. Mast cell expression of PLC-?3 was inversely correlated with that of phospho-STAT5, and increased mast cells with high levels of phospho-STAT5 were found in lesional skin of some AD patients. Therefore, STAT5 regulatory mechanisms in mast cells are important for AD pathogenesis.
Rapid progress has recently been made regarding how phospholipase C (PLC)-? functions downstream of G protein-coupled receptors and how PLC-? functions in the nucleus. PLC-? has also been shown to interplay with tyrosine kinase-based signaling pathways, specifically to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1. In this regard, a new multimolecular signaling platform, named SPS complex, has been identified. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation. Furthermore, a growing body of work suggests that PLC-? also participates in the differentiation and activation of immune cells that control both the innate and adaptive immune systems.
Developmental changes in excitation-contraction mechanisms were examined in the ventricular myocardium from fetal, neonatal, and 1-, 2-, and 4-week-old mice. In isolated tissue, the negative inotropic effect of nifedipine decreased, while that of ryanodine increased with age. Action potential duration decreased with age, especially during the late fetal period. In ventricular cardiomyocytes, fluorescence imaging revealed that the sarcoplasmic reticulum increases progressively during pre- and postnatal development. t-Tubules were absent in the fetus and neonate, were observed only in the subsarcolemmal region at 1 week after birth, and were present throughout the cytoplasm at 2 and 4 weeks after birth. The amplitude of Ca(2+) transients, as well as its ryanodine-sensitive component, increased with age. In the neonate and 1-week-old mice, Ca(2+) at the cell center showed slower rise than the subsarcolemmal region, but in 2- and 4-week-old mice, Ca(2+) increased simultaneously across the entire width of the cell. These results suggest that in the mouse ventricular myocardium, the shortening of the action potential during the late fetal period and the development of t-tubule-sarcoplasmic reticulum coupling during the second postnatal week largely contribute to the developmental increase in the dependence of contraction on sarcoplasmic reticulum function.
We report the case of a 64-year old man who presented memory disturbance, low-grade fever, weight loss, and bilateral hand tremors for three months. He was diagnosed with non-herpetic acute limbic encephalitis (NHALE). Follow-up magnetic resonance imaging (MRI) revealed new lesions after symptomatic improvement following steroid pulse therapy. This may indicate that there is a time lag between the disturbance or recovery of neurons and astrocytes. Thus, other lesions might occasionally appear during convalescence in patients with NHALE, even if only minimal lesions were found on the initial MRI.
Atopic dermatitis (AD) is a chronic pruritic inflammatory skin disease. We recently described an animal model in which repeated epicutaneous applications of a house dust mite extract and Staphylococcal enterotoxin B induced eczematous skin lesions. In this study we showed that global gene expression patterns are very similar between human AD skin and allergen/staphylococcal enterotoxin B-induced mouse skin lesions, particularly in the expression of genes related to epidermal growth/differentiation, skin barrier, lipid/energy metabolism, immune response, or extracellular matrix. In this model, mast cells and T cells, but not B cells or eosinophils, were shown to be required for the full expression of dermatitis, as revealed by reduced skin inflammation and reduced serum IgE levels in mice lacking mast cells or T cells (TCR?(-/-) or Rag1(-/-)). The clinical severity of dermatitis correlated with the numbers of mast cells, but not eosinophils. Consistent with the idea that T helper type 2 (Th2) cells play a predominant role in allergic diseases, the receptor for the Th2-promoting cytokine thymic stromal lymphopoietin and the high-affinity IgE receptor, Fc?RI, were required to attain maximal clinical scores. Therefore, this clinically relevant model provides mechanistic insights into the pathogenic mechanism of human AD.
Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN). Our recent study found that phospholipase C (PLC)-?3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-?3-dependent manner. PLC-?3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-?3 (PLC-?3-CT) accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-?3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998) suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-?3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-?3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies.
IgE-mediated activation of mast cells and basophils underlies allergic diseases such as asthma. Histamine-releasing factor (HRF; also known as translationally controlled tumor protein [TCTP] and fortilin) has been implicated in late-phase allergic reactions (LPRs) and chronic allergic inflammation, but its functions during asthma are not well understood. Here, we identified a subset of IgE and IgG antibodies as HRF-interacting molecules in vitro. HRF was able to dimerize and bind to Igs via interactions of its N-terminal and internal regions with the Fab region of Igs. Therefore, HRF together with HRF-reactive IgE was able to activate mast cells in vitro. In mouse models of asthma and allergy, Ig-interacting HRF peptides that were shown to block HRF/Ig interactions in vitro inhibited IgE/HRF-induced mast cell activation and in vivo cutaneous anaphylaxis and airway inflammation. Intranasally administered HRF recruited inflammatory immune cells to the lung in naive mice in a mast cell- and Fc receptor-dependent manner. These results indicate that HRF has a proinflammatory role in asthma and skin immediate hypersensitivity, leading us to suggest HRF as a potential therapeutic target.
Eczema vaccinatum is the most common severe pathology associated with smallpox vaccination (vaccinia virus), occurring at high rates among individuals with a previous history of atopic dermatitis (atopic eczema).
Mast cells are major effectors in high-affinity IgE receptor (Fc?RI)-dependent allergic reactions. Here we show that phospholipase C (PLC)-?3 is crucial for Fc?RI-mediated mast cell activation. Plcb3(-/-) mice showed blunted Fc?RI-dependent late-phase, but not acute, anaphylactic responses and airway inflammation. Accordingly, Fc?RI stimulation of Plcb3(-/-) mast cells exhibited reduced cytokine production but normal degranulation. Reduced cytokine production in Plcb3(-/-) cells could be accounted for by increased activity of the negative regulatory Src family kinase Lyn and reduced activities of the positive regulatory protein kinases MAPKs. Mechanistically, PLC-?3 constitutively interacts with Fc?RI, Lyn, and SHP-1 (protein phosphatase). SHP-1 probably recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM. Consistent with PLC-?3- and SHP-1-mediated repression of Lyn activity by dephosphorylation at Tyr396, Fc?RI-mediated phenotypes were similar in Plcb3(-/-) and SHP-1 mutant mast cells. Thus, we have defined a PLC-?3- and SHP-1-mediated signaling pathway for Fc?RI-mediated cytokine production.
Rheumatoid arthritis (RA) is a chronic inflammatory disease marked by bone and cartilage destruction. Current biologic therapies are beneficial in only a portion of patients; hence small molecules targeting key pathogenic signaling cascades represent alternative therapeutic strategies. Here we show that c-Jun N-terminal kinase (JNK) 1, but not JNK2, is critical for joint swelling and destruction in a serum transfer model of arthritis. The proinflammatory function of JNK1 requires bone marrow-derived cells, particularly mast cells. Without JNK1, mast cells fail to degranulate efficiently and release less IL-1? after stimulation via Fc? receptors (Fc?Rs). Pharmacologic JNK inhibition effectively prevents arthritis onset and abrogates joint swelling in established disease. Hence, JNK1 controls mast cell degranulation and Fc?R-triggered IL-1? production, in addition to regulating cytokine and matrix metalloproteinase biosynthesis, and is an attractive therapeutic target in inflammatory arthritis.
Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)-?3-dependent manner. Thus, PLC-?3-deficient mice develop myeloproliferative neoplasm, like Lyn (Src family kinase)- deficient mice. Here we show that Lyn/PLC-?3 doubly deficient lyn(-/-);PLC-?3(-/-) mice develop a Stat5-dependent, fatal myelodysplastic/myeloproliferative neoplasm, similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn(-/-);PLC-?3(-/-) mice that cause the CMML-like disease, phosphorylation of SHP-1 at Tyr(536) and Tyr(564) is abrogated, resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore, SHP-1 phosphorylation at Tyr(564) by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand, Tyr(536) in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore, we identify a novel Lyn/PLC-?3-mediated regulatory mechanism of SHP-1 and Stat5 activities.
Src family kinases (SFK) are critical for initiating and regulating the response of mast cells activated by engagement of the high-affinity IgE receptor, FcepsilonRI. Lyn is the predominant SFK in mast cells and has been ascribed both positive and negative roles in regulating mast cell activation. We analyzed the mast cell phenotype of WeeB, a recently described mouse mutant that expresses a Lyn protein with profoundly reduced catalytic activity. Surprisingly, we found that this residual activity is sufficient for wild-type levels of cytokine production and degranulation in bone marrow-derived mast cells after low-intensity stimulation with anti-IgE. High-intensity stimulation of lyn(-/-) bone marrow-derived mast cells with highly multivalent Ag resulted in enhanced cytokine production as previously reported, and WeeB cells displayed an intermediate phenotype. Under this latter condition, SFK inhibition using PP2 increased cytokine production in wild-type and WeeB but not lyn(-/-) cells, resulting in substantially higher levels in the PP2-treated WeeB than in lyn(-/-) cells. Restoration of wild-type and WeeB lyn alleles in lyn(-/-) cells generated activation phenotypes similar to those in nontransduced wild-type and WeeB cells, respectively, whereas a kinase-dead allele resulted in a phenotype similar to that of empty-vector-transduced cells. These data indicate that inhibition of Lyn and/or SFK activity can result in higher levels of mast cell activation than simple deletion of lyn and that only near-complete inhibition of Lyn can impair its positive regulatory functions. Furthermore, the data suggest that both positive and negative regulatory functions of Lyn are predominantly carried out by its catalytic activity and not an adaptor function.
Mast cells play as the major effector cells in immediate hypersensitivity through activation via the high-affinity IgE receptor, Fc epsilon RI, although many other functions have recently been discovered for this cell type. Given the broad array of proinflammatory mediators secreted from Fc epsilon RI-activated mast cells, as well as sensitization to allergens, IgE elevation, and increased mast cells in a majority of atopic dermatitis patients, mast cells are believed to be involved in the pathogenesis of atopic dermatitis. Numerous animal models have been used to study this epidemic disease. Here we review the recent progress to synthesize our current understanding of this disease and potential mechanisms for a mast cells role in the disease.
Threats of bioterrorism have renewed efforts to better understand poxvirus pathogenesis and to develop a safer vaccine against smallpox. Individuals with atopic dermatitis are excluded from smallpox vaccination because of their propensity to develop eczema vaccinatum, a disseminated vaccinia virus (VACV) infection. To study the underlying mechanism of the vulnerability of atopic dermatitis patients to VACV infection, we developed a mouse model of eczema vaccinatum. Virus infection of eczematous skin induced severe primary erosive skin lesions, but not in the skin of healthy mice. Eczematous mice exhibited lower natural killer (NK) cell activity but similar cytotoxic T lymphocyte activity and humoral immune responses. The role of NK cells in controlling VACV-induced skin lesions was demonstrated by experiments depleting or transferring NK cells. The proinflammatory cytokine interleukin (IL)-17 reduced NK cell activity in mice with preexisting dermatitis. Given low NK cell activities and increased IL-17 expression in atopic dermatitis patients, these results can explain the increased susceptibility of atopic dermatitis patients to eczema vaccinatum.
Mast cells are bone marrow-derived effector cells that can initiate inflammatory responses to infectious organisms or allergens by releasing a multitude of pro-inflammatory factors including prostaglandin (PG) D(2). We demonstrate that primary murine bone marrow-derived mast cells (BMMCs) express the PGD(2) receptor; chemoattractant receptor-homologous molecule expressed on T(h) class 2 cells (CRT(h)2). Activation of CRT(h)2 on BMMC by PGD(2) or the CRT(h)2-specific agonist, 13,14-dihydro-15-keto-prostaglandin D(2) (DK-PGD(2)), resulted in signaling response including Ca(2+) mobilization and phosphorylation of the p42/p44 extracellular signal-regulated kinases (ERKs) kinases. Phosphorylation of the ERKs could be blocked by pertussis toxin, as well as a small molecule antagonist of CRT(h)2, Compound A. Activation of CRT(h)2 on BMMC also resulted in the up-regulation of CD23 and CD30 on the cell surface, as well as CD62L shedding. Finally, PGD(2) and DK-PGD(2) induced the migration of BMMC in vitro and in vivo in response to an intra-dermal DK-PGD(2) injection. Both these processes were inhibited by the CRT(h)2 antagonist. These results raise the possibility that the functional consequences of the PGD(2)-CRT(h)2 interaction on mast cells may be relevant in allergic inflammation.
Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate, phospholipase C (PLC) is implicated in promoting cell growth. However, we found that PLC-beta3-deficient mice develop myeloproliferative disease, lymphoma, and other tumors. The mutant mice have increased numbers of hematopoietic stem cells with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on Stat5 and can be antagonized by the protein phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-beta3 deficiency was suggested in mouse lymphomas in PLC-beta3(-/-) and in Emicro-myc;PLC-beta3(+/-) mice and human Burkitts lymphoma cells. The same mechanism for malignant transformation seems to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-beta3 is likely a tumor suppressor.
Tetranychus urticae (Acari: Tetranychidae) possesses a sac-like ovary with characteristic oocytes that protrude from the ovarian surface. In nondiapause females, transparent oocytes became opaque with yolk deposition between days 0 and 1 in the adult stage at 20 degrees C. In diapause females, however, ovarian development ceased at a stage having transparent oocytes without yolk deposition; this stage corresponded to the day-0 stage of the nondiapause females. Four partial fragments of the vitellogenin (Vg) genes of T. urticae were isolated. This is the first report on the Vg genes of mites. The deduced amino acid sequences of these four Vg gene fragments contained the von Willebrand factor D domain and the GLCG motif, which were reported to be the common features of Vg sequences in insects and ticks. Northern blot analysis did not detect Vg mRNA in the diapause adult females of T. urticae. It is, therefore, suggested that diapause mites do not synthesize Vg mRNA and that vitellogenesis is regulated at the transcriptional level in diapause.
Ag-dependent activation of IgE-bearing mast cells is a critical first step in immediate hypersensitivity and other allergic responses. Recent studies have revealed Ag-independent effects of monoclonal mouse IgE molecules on mast cell survival and activation. However, no studies have been performed on the effects of polyclonal IgE molecules. Here, we tested whether polyclonal mouse and human IgE molecules affect survival and cytokine production in mast cells.
Monomeric IgE molecules, when bound to the high-affinity receptor, exhibit a vast heterogeneity in their ability to induce survival promotion and cytokine production in mast cells. At one end of this spectrum, highly cytokinergic (HC) IgEs can induce potent survival promotion, degranulation, cytokine production, migration, etc., whereas at the other end, poorly cytokinergic (PC) IgEs can do so inefficiently. In this study, we investigated whether IgEs recognize autoantigens and whether IgEs binding of autoantigens correlates with difference s in HC versus PC properties.
MCs (mast cells) are critical components of the host innate immune defence against bacterial pathogens, providing a variety of intra- and extra-cellular antimicrobial functions. In the present study we show, for the first time, that the transcriptional regulator HIF-1? (hypoxia-inducible factor-1?) mediates the extracellular antimicrobial activity of human and murine MCs by increasing the formation of MCETs (MC extracellular traps).
We present a case of cerebral embolism associated with a left atrial myxoma that was treated with intravenous thrombolytic therapy. A 79-year-old right-handed man with no history of neurological or psychiatric illnesses was referred to our hospital because of confusion. He had been self-supported in the activity of daily living and could enjoy gardening until just before his admission. He had aphasia, left conjugate deviation, right hemiparesis, and right pathological reflexes. His NIHSS score was 24. Cranial DWI showed hyperintense lesions in the left middle cerebral artery territory, and MRA revealed left middle cerebral artery occlusion. We started treatment with the recombinant tissue plasminogen activator alteplase intravenously 3 h after the onset. However, the therapy was ineffective, and the NIHSS score was 25 on the second day. A transthoracic echocardiogram and heart MRI showed a left atrial myxoma. However, surgery was contraindicated because of the patients poor general condition. Although intravenous recombinant tissue plasminogen activator is a reasonable treatment for stroke patients, even with a cardiac myxoma, we cannot always expect good effects, especially if the emboli are parts of the tumor itself. In this case, we could not perform an endovascular mechanical embolectomy; however, we speculate that mechanical embolus retrieval in cerebral ischemia might be effective in such cases.
Previous studies suggested that Protein L (PpL), the bacterial Ig-binding protein, activates mast cells. PpL presumably performs the activation by interacting with membrane-bound IgE?, but the underlying mechanisms behind the process remain unclear. In the current study, we found that cell-surface Fc?RI expression is a critical factor participant in PpL-mediated full activation of murine mast cells, which includes cytokine production, the degranulation response, and leukotriene C(4) (LTC(4)) release, and that engagement of the Fc?RI with IgE? and PpL is enough to induce tyrosine phosphorylation of ITAM in the FcR?- and ?-signaling subunits. Introduction of mutations in two canonical tyrosine residues (Y47F/Y58F) of the FcR?-ITAM completely abolished the above-mentioned mast cell functions, with the exception of LTC(4) release. Importantly, the FcR?-ITAM acts as a signal transducer that is responsible for LTC(4) release independently of the FcR?-ITAM. Taken together, our results suggest crucial and distinct functions for the FcR?- and ?-ITAMs in the Fc?RI-dependent full activation of mast cells induced by IgE? and PpL.
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