Activation Of Human Mast Cells Research Articles

A novel human mast cell activation test for peanut allergy

A novel human mast cell activation test for peanut allergy

Issue Highlights-May 2018 (94B3).

Issue Highlights-May 2018 (94B3).

MP29-02 reduces nasal hyperreactivity and nasal mediators in patients with house dust mite-allergic rhinitis.

Nasal hyperreactivity (NHR) is an important clinical feature of allergic rhinitis (AR). The efficacy of MP29-02 (azelastine hydrochloride (AZE) and fluticasone propionate [FP]) nasal spray on local inflammatory mediators and NHR in AR is unknown. We tested if MP29-02 decreases inflammatory mediators and NHR in AR and if this effect is due to restoration of nasal epithelial barrier function. A 4-week double-blinded placebo-controlled trial with MP29-02 treatment was conducted in 28 patients with house dust mite (HDM) AR. The presence of NHR was evaluated by measuring reduction in nasal flow upon cold dry air exposure. The effects of AZE ± FP on barrier integrity and airway inflammation were studied in a murine model of HDM-induced NHR and on reduced activation of murine sensory neurons and human mast cells. MP29-02 but not placebo reduced NHR (P < .0001 vs P = .21), levels of substance P (P = .026 vs P = .941), and β-hexosaminidase (P = .036 vs P = .632) in human nasal secretions. In wild-type C57BL6 mice, the reduction in β-hexosaminidase levels (P < .0001) by AZE + FP treatment upon HDM challenge was found in parallel with a decreased transmucosal passage (P = .0012) and completely reversed eosinophilic inflammation (P = .0013). In vitro, repeated applications of AZE + FP desensitized sensory neurons expressing the transient receptor potential channels TRPA1 and TRPV1. AZE + FP reduced MC degranulation to the same extent as AZE alone. MP29-02 treatment reduces inflammatory mediators and NHR in AR. The effects of AZE + FP on MC degranulation, nasal epithelial barrier integrity, and TRP channels provide novel insights into the pathophysiology of allergic rhinitis.

Influence of IL-6, IL-33, and TNF-α on human mast cell activation: Lessons from single cell analysis by flow cytometry.

Mechanisms that govern priming and degranulation of human mast cells (MCs) remain elusive. Besides, most of our knowledge is based on experiments from which data only reflect an average of all stimulated cells. This study aims at investigating the effects of proinflammatory cytokines IL-6, IL-33, and TNF-α on IgE-dependent and IgE-independent activation of individual MCs. MCs were derived from CD34+ progenitors isolated from 50 mL whole blood from 4 healthy controls and 5 birch pollen allergic patients. Passively sensitized MCs were preincubated with IL-6, IL-33, or TNF-α and stimulated with anti-IgE/birch pollen allergen or substance P, the latter being a ligand for the G-protein-coupled MRGPRX2-receptor. Activation-i.e., upregulation of CD203c-and anaphylactic degranulation-i.e., appearance of CD63-were measured using flow cytometry. Preincubation with IL-33 demonstrated upregulated CD203c density without degranulation. Subsequent IgE-dependent stimulation (anti-IgE/birch pollen allergen) resulted in higher appearance of CD63 as compared to cells without preincubation, indicating IL-33 to exert a priming effect (P = 0.04). IL-6 only increased allergen-specific responses but to a lesser extent than IL-33. Combination of IL-33/IL-6 had a synergistic effect, demonstrating more degranulation in response to allergen. TNF-α had no effect on IgE-mediated activation, nor synergistic effects with IL-33. Stimulation with substance P resulted in degranulation that could not be enhanced by preincubation. In conclusion, IL-33, and in a lesser extent IL-6, prime individual MCs for subsequent IgE-mediated activation. Moreover, this priming effect is synergistic. In contrast, none of the cytokines had a priming effect on MRGPRX2-mediated activation of MCs. © 2017 International Clinical Cytometry Society.

TGF-β1 Suppresses IL-33-Induced Mast Cell Function.

TGF-β1 is involved in many pathological conditions, including autoimmune disorders, cancer, and cardiovascular and allergic diseases. We have previously found that TGF-β1 can suppress IgE-mediated mast cell activation of human and mouse mast cells. IL-33 is a member of the IL-1 family capable of inducing mast cell responses and enhancing IgE-mediated activation. In this study, we investigated the effects of TGF-β on IL-33-mediated mast cell activation. Bone marrow-derived mast cells cultured in TGF-β1, β2, or β3 showed reduced IL-33-mediated production of TNF, IL-6, IL-13, and MCP-1 in a concentration-dependent manner. TGF-β1 inhibited IL-33-mediated Akt and ERK phosphorylation as well as NF-κB- and AP-1-mediated transcription. These effects were functionally important, as TGF-β1 injection suppressed IL-33-induced systemic cytokines in vivo and inhibited IL-33-mediated cytokine release from human mast cells. TGF-β1 also suppressed the combined effects of IL-33 and IgE-mediated activation on mouse and human mast cells. The role of IL-33 in the pathogenesis of allergic diseases is incompletely understood. These findings, consistent with our previously reported effects of TGF-β1 on IgE-mediated activation, demonstrate that TGF-β1 can provide broad inhibitory signals to activated mast cells.

LL-37-induced human mast cell activation through G protein-coupled receptor MrgX2

Human LL-37 is an important class of cationic antimicrobial peptide (CAP) that is known to stimulate mast cell activation. While many studies have been conducted on LL-37, to date little is known about the functional receptors for LL-37-induced human mast cell activation, in particular in terms of the release of de novo synthesized mediators. Thus, the aim of the present study is to identify the functional receptors for LL-37-induced human mast cell activation in terms of the degranulation and release of de novo synthesized mediators and investigate the downstream signalling pathways involved in mast cell activation. Overall, our study importantly demonstrates that LL-37-induced human mast cell degranulation and release of de novo synthesized mediators function primarily through the activation of MrgX2. We furthermore show that LL-37-induced human mast cell line LAD2 cells are involved in the degranulation and release of IL-8, and that FPRL1 and P2X7 have only a partial effect on IL-8 release, and no effect on mast cell degranulation triggered by LL-37. Instead, we find that silencing the expression of MrgX2 in human mast cell significantly inhibits the LL-37-induced degranulation and release of IL-8. Overall, this effect is associated with the activation of the Gi protein, PLC/PKC/Calcium/NFAT, PI3K/Akt and MAPKs signalling pathways.

Fluvastatin enhances IL-33-mediated inflammatory functions in mast cells

Abstract Statins are commonly used HMG-CoA reductase (HMGCR) inhibitors prescribed for treating cardiovascular diseases by lowering cholesterol levels. Independent of lowering cholesterol, statins have been shown to inhibit proinflammatory functions in macrophages and T cells. Our lab has previously found that fluvastatin suppresses IgE activation of mouse and human mast cells in vitro, while also protecting C57BL/6 mice from passive systemic anaphylaxis. However, a recent study found that statins can induce proinflammatory cytokines in LPS stimulated macrophages. Due to the conflicting nature of statins, we extended our studies to IL-33, a strong mast cell activator with signaling similar to LPS. In contrast to our findings with IgE signaling, we found that fluvastatin augmented TNF and IL-6 production induced by IL-33 in primary mouse and human mast cells. These effects were dependent on the mitogen SCF, since in its absence the enhancing effects were absent. Mevalonic acid, the product of the HMGCR reaction, reversed fluvastatin effects. Additionally, fluvastatin effects required loss of isoprenoids downstream of HMGCR, since geranylgeranyl pyrophosphate and farnesyl pyrophosphate reduced fluvastatin enhancement of TNF and IL-6 secretion. Fluvastatin enhanced NFκB transcriptional activity in IL-33 stimulated BMMC, and by using an NFκB inhibitor, we found that fluvastatin enhancement of IL-6 and TNF induced by IL-33 is NFκB dependent. This study demonstrates that HMGCR blockade can have either anti or proinflammatory effects on mast cell function, dependent upon the stimulus. These data further reveal the divergent roles for isoprenoid lipids in mast cell function, which may offer new clinical targets for inflammation.

Alpha7-nicotinic acetylcholine receptors involve the imidacloprid-induced inhibition of IgE-mediated rat and human mast cell activation

Although our recent study indicated that imidacloprid, a widely used neonicotinoid insecticide, inhibited IgE-mediated mast cell activation, the inhibition mechanism still remains unclear.

Individual strains of Lactobacillus paracasei differentially inhibit human basophil and mouse mast cell activation.

IntroductionThe microbiota controls a variety of biological functions, including immunity, and alterations of the microbiota in early life are associated with a higher risk of developing allergies later in life. Several probiotic bacteria, and particularly lactic acid bacteria, were described to reduce both the induction of allergic responses and allergic manifestations. Although specific probiotic strains were used in these studies, their protective effects on allergic responses also might be common for all lactobacilli.MethodsTo determine whether allergic effector cells inhibition is a common feature of lactobacilli or whether it varies among lactobacilli strains, we compared the ability of 40 strains of the same Lactobacillus paracasei species to inhibit IgE‐dependent mouse mast cell and human basophil activation.ResultsWe uncovered a marked heterogeneity in the inhibitory properties of the 40 Lactobacillus strains tested. These segregated into three to four clusters depending on the intensity of inhibition. Some strains inhibited both mouse mast cell and human basophil activation, others strains inhibited only one cell type and another group induced no inhibition of activation for either cell type.ConclusionsIndividual Lactobacillus strains of the same species differentially inhibit IgE‐dependent activation of mouse mast cells and human basophils, two cell types that are critical in the onset of allergic manifestations. Although we failed to identify specific bacterial genes associated with inhibition by gene‐trait matching analysis, our findings demonstrate the complexity of the interactions between the microbiota and the host. These results suggest that some L. paracasei strains might be more beneficial in allergies than others strains and provide the bases for a rational screening of lactic acid bacteria strains as next‐generation probiotics in the field of allergy.

Lactic acid inhibits IL-33-mediated mast cell inflammatory responses via HIF-1α suppressing miR-155 expression

Abstract Lactic acid (LA) is elevated in tumors, asthma, and wound healing, environments that also include mast cells and IL-33. However, how LA affects mast cell function is unknown. Therefore we evaluated how LA modifies the IL-33-mediated mast cell response. When bone marrow derived mast cells were cultured with LA, we noted reduced IL-33-mediated cytokine production, an effect that was both monocarboxylate transporter (MCT)1- and pH-dependent. LA selectively decreased IL-33-induced TAK1, JNK, ERK, and NFkB phosphorylation, but not p38 activation. Additionally, LA increased HIF-1α expression. Since HIF-1α has been shown to regulate the pro-inflammatory microRNA miR-155, we examined miR-155 expression. miR-155-5p was reduced by LA, an effect that was reversed by HIF-1α antagonism. More importantly, miR-155-5p overexpression abolished the suppressive effects of LA. Additionally, the negative regulator SOCS1, a known miR-155 target, was elevated by LA addition. This suggests that LA employs HIF-1a to suppress miR-155, indirectly increasing SOCS-1 and limiting cytokine production. These data were recapitulated in vivo, since C57BL/6 mice injected with LA showed less IL-33-induced plasma cytokine levels than control mice. Lastly, LA suppressed IL-33-mediated human skin mast cell activation, an effect that was also MCT1-dependent. Our data demonstrate that lactic acid, present in inflammatory and malignant microenvironments, alters mast cell function to suppress inflammation.

Carboxyl-Terminal Cleavage of Apolipoprotein A-I by Human Mast Cell Chymase Impairs Its Anti-Inflammatory Properties.

Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I. Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α-activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB-dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)- and M-CSF (macrophage colony-stimulating factor)-differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)-activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils. The findings identify C-terminal cleavage of apoA-I by human mast cell chymase as a novel mechanism leading to loss of its anti-inflammatory functions. When targeting inflamed protease-rich atherosclerotic lesions with apoA-I, infusions of protease-resistant apoA-I might be the appropriate approach.

The modulatory effect of TLR2 on LL-37-induced human mast cells activation

The sole and endogenous anti-microbial peptide LL-37 is a significant effector molecule in the innate host defense system. Apart from its broadly direct anti-microbial activity, the peptide also activates mast cell in respect of allergic diseases and inflammation. On the other hand, mast cell can be activated by Toll-like receptors (TLRs) which are at the center of innate immunity. It was the aim of the study to illustrate the modulatory effect of TLR2 ligands peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4) on LL-37 induced LAD2 cells (a human mast cell line) activation. LL-37 induced LAD2 cells degranulation and the release of IL-8. TLR2 ligands didn't induce LAD2 cells degranulation, but triggered the release of IL-8. Incubation with PGN or Pam3CSK4 significantly suppressed LL-37-induced degranulation through inhibition of calcium mobilization from LAD2 cells. Similarly, the release of IL-8 was inhibited when LAD2 cells were co-stimulated with TLR2 ligands and LL-37. Studies with inhibitors of key enzymes involved in mast cell signaling indicated that the release of IL-8 induced by TLR2 ligands and LL-37 involved the activation of the PI3K, ERK, JNK and calcineurin signaling pathways. In contrast, p38 activation down-regulated the release of IL-8 induced by TLR2 ligands and LL-37. Taken together, these observations suggest that activation of human mast cells by LL-37 could be modified by TLR2 ligands and the function of human mast cells could be switched from allergic reactions to innate immune response.

An antimicrobial peptide with angiogenic properties, AG-30/5C, activates human mast cells through the MAPK and NF-κB pathways.

Apart from their direct antimicrobial activities against invading pathogens, antimicrobial peptides exhibit additional protective functions that have led to their being named host defense peptides (HDPs). These functions include the stimulation of the production of cytokines/chemokines, the promotion of chemotaxis and cell proliferation and the induction of angiogenesis and wound healing. AG-30/5C is a novel angiogenic HDP that in addition to its antimicrobial activity also activates fibroblasts and endothelial cells and promotes angiogenesis and wound healing. Given that mast cells are found primarily in the vicinity of vessels, where they are intimately involved in wound healing, we hypothesized that AG-30/5C may activate mast cells. We demonstrated that AG-30/5C activated LAD2 human mast cells to degranulate and produce lipid mediators including leukotriene C4, prostaglandin D2 and E2. Moreover, AG-30/5C increased mast cell chemotaxis and induced the production of the cytokines GM-CSF and TNF-α and various chemokines, such as IL-8, MCP-1, MCP-3, MIP-1α and MIP-1β. The chemotaxis and cytokine/chemokine production induced by AG-30/5C were suppressed by both pertussis toxin and U-73122, suggesting the involvement of the G protein and phospholipase C pathways in AG-30/5C-induced mast cell activation. Furthermore, these pathways were activated downstream of the MAPK and NF-κB signaling molecules, as demonstrated by the inhibitory effects of ERK-, JNK-, p38- and NF-κB-specific inhibitors on cytokine/chemokine production. Interestingly, AG-30/5C caused the phosphorylation of MAPKs and IκB. We suggest that the angiogenic and antimicrobial peptide AG-30/5C plays a key role in the recruitment and activation of human mast cells at inflammation and wound sites.

Modulation of host defense peptide-mediated human mast cell activation by LPS.

Human β-defensin3 (hBD3) and the cathelicidin LL-37 are host defense peptides (HDPs) that directly kill microbes and display immunomodulatory/wound-healing properties via the activation of chemokine, formylpeptide and epidermal growth factor receptors on leukocytes and epithelial cells. A C-terminal 14 amino acid hBD3 peptide with all Cys residues replaced with Ser (CHRG01) and an LL-37 peptide consisting of residues 17-29 (FK-13) display antimicrobial activity but lack immunomodulatory property. Surprisingly, we found that CHRG01 and FK-13 caused Ca(2+) mobilization and degranulation in human mast cells via a novel G protein-coupled receptor known as Mas-related gene-X2 (MrgX2). At local sites of bacterial infection, the negatively charged LPS likely interacts with cationic HDPs to inhibit their activity and thus providing a mechanism for pathogens to escape host defense mechanisms. We found that LPS caused almost complete inhibition of hBD3 and LL-37-induced Ca(2+) mobilization and mast cell degranulation. In contrast, it had no effect on CHRG01 and FK-13-induced mast cell responses. These findings suggest that HDP derivatives that kill microbes, harness mast cell's host defense and wound-healing properties via the activation of MrgX2 but are resistant to inhibition by LPS could be utilized for the treatment of antibiotic-resistant microbial infections.

Activation of human mast cells by retrocyclin and protegrin highlight their immunomodulatory and antimicrobial properties.

Preclinical evaluation of Retrocyclins (RC-100, RC-101) and Protegrin-1 (PG-1) antimicrobial peptides (AMPs) is important because of their therapeutic potential against bacterial, fungal and viral infections. Human mast cells (HMCs) play important roles in host defense and wound healing but the abilities of retrocyclins and protegrin-1 to harness these functions have not been investigated. Here, we report that chemically synthesized RC-100 and PG-1 caused calcium mobilization and degranulation in HMCs but these responses were not blocked by an inhibitor of formyl peptide receptor-like 1 (FPRL1), a known receptor for AMPs. However, RC-100 and PG-1 induced degranulation in rat basophilic leukemia (RBL-2H3) cells stably expressing Mas related G protein coupled receptor X2 (MrgX2). Chemical synthesis of these AMPs is prohibitively expensive and post-synthesis modifications (cyclization, disulfide bonds, folding) are inadequate for optimal antimicrobial activity. Indeed, we found that synthetic RC-100, which caused mast cell degranulation via MrgX2, did not display any antimicrobial activity. Green-fluorescent protein (GFP)-tagged RC-101 (analog of RC-100) and GFP-tagged PG-1 purified from transgenic plant chloroplasts killed bacteria and induced mast cell degranulation. Furthermore, GFP-PG1 bound specifically to RBL-2H3 cells expressing MrgX2. These findings suggest that retrocyclins and protegrins activate HMCs independently of FPRL1 but via MrgX2. Harnessing this novel feature of AMPs to activate mast cell's host defense/wound healing properties in addition to their antimicrobial activities expands their clinical potential. Low cost production of AMPs in plants should facilitate their advancement to the clinic overcoming major hurdles in current production systems.

Ability of Interleukin-33- and Immune Complex-Triggered Activation of Human Mast Cells to Down-Regulate Monocyte-Mediated Immune Responses.

Mast cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). In particular, their activation by interleukin-33 (IL-33) has been linked to the development of arthritis in animal models. The aim of this study was to evaluate the functional responses of human mast cells to IL-33 in the context of RA. Human mast cells were stimulated with IL-33 combined with plate-bound IgG or IgG anti-citrullinated protein antibodies (ACPAs), and their effects on monocyte activation were evaluated. Cellular interactions of mast cells in RA synovium were assessed by immunofluorescence analysis, and the expression of messenger RNA (mRNA) for mast cell-specific genes was evaluated in synovial biopsy tissue from patients with early RA who were naive to treatment with disease-modifying antirheumatic drugs. IL-33 induced the up-regulation of Fcγ receptor type IIa and enhanced the activation of mast cells by IgG, including IgG ACPAs, as indicated by the production of CXCL8/IL-8. Intriguingly, mast cell activation triggered with IL-33 and IgG led to the release of mediators such as histamine and IL-10, which inhibited monocyte activation. Synovial mast cells were found in contact with CD14+ monocyte/macrophages. Finally, mRNA levels of mast cell-specific genes were inversely associated with disease severity, and IL-33 mRNA levels showed an inverse correlation with the levels of proinflammatory markers. When human mast cells are activated by IL-33, an immunomodulatory phenotype develops, with human mast cells gaining the ability to suppress monocyte activation via the release of IL-10 and histamine. These findings, together with the presence of synovial mast cell-monocyte interactions and the inverse association between the expression of mast cell genes at the synovial level and disease activity, suggest that these newly described mast cell-mediated inhibitory pathways might have a functional relevance in the pathogenesis of RA.

Mechanism of the antigen-independent cytokinergic SPE-7 IgE activation of human mast cells in vitro.

Release of pro-inflammatory mediators by mast cells is a key feature of allergic disease. The ‘dogma’ is that IgE molecules merely sensitise mast cells by binding FcεRI prior to cross-linking by multivalent allergen, receptor aggregation and mast cell activation. However, certain monoclonal IgE antibodies have been shown to elicit mast cell activation in an antigen-independent cytokinergic manner, and DNP-specific murine SPE-7 IgE is the most highly cytokinergic antibody known. We show that both monovalent hapten and recombinant SPE-7 IgE Fab inhibit its cytokinergic activity as measured by mast cell degranulation and TNF-α release. Using SPE-7 IgE, a non-cytokinergic human IgE and a poorly cytokinergic murine IgE, we reveal that interaction of the Fab region of ‘free’ SPE-7 IgE with the Fab of FcεRI-bound SPE-7 IgE is the basis of its cytokinergic activity. We rule out involvement of IgE Fc, Cε1 and Cλ/κ domains, and propose that ‘free’ SPE-7 IgE binds to FcεRI-bound SPE-7 IgE by an Fv-Fv interaction. Initial formation of a tri-molecular complex (one ‘free’ IgE molecule cross-linking two receptor-bound IgE molecules) leads to capture of further ‘free’ and receptor-bound IgEs to form larger clusters that trigger mast cell activation.

Differential TLR-induced cytokine production by human mast cells is amplified by FcɛRI triggering.

Mast cells are mainly present in strategic locations, where they may have a role in defence against parasites and bacteria. These pathogens can be recognized by mast cells via Toll-like receptors (TLR). Allergic symptoms are often increased in the presence of pathogens at the site of allergen exposure, but it is unknown which cytokines can mediate such an effect. To study whether an interaction between IgE- and TLR-mediated activation of human mast cells can contribute to exacerbated inflammatory responses. Peripheral blood-derived mast cells were stimulated with TLR ligands, in the presence or absence of anti-IgE triggering, after which degranulation was measured using flow cytometry and cytokine production was evaluated by multiplex assays, and ELISA. For evaluation of allergen-specific responses, mast cells were sensitized with serum of allergic individuals or controls, after which they were stimulated using allergens in combination with TLR ligands. Simultaneous triggering of mast cells via IgE and TLR ligands greatly enhanced cytokine production but not IgE-induced degranulation. Different TLR ligands specifically enhanced the differential production of cytokines in conjunction with FcεRI triggering. Importantly, only TLR-4 and TLR-6 were able to induce robust production of IL-13, an important molecule in allergic reactions. These results indicate that the simultaneous presence of pathogen- or danger-associated signals and FcεRI triggering via specific IgE can significantly modify mast cell-mediated allergic reactions via synergistic production of cytokines and inflammatory mediators and provide an explanation of augmented allergic symptoms during infection.

IgE-dependent activation of human mast cells and fMLP-mediated activation of human eosinophils is controlled by the circadian clock

Symptoms of allergic attacks frequently exhibit diurnal variations. Accordingly, we could recently demonstrate that mast cells and eosinophils – known as major effector cells of allergic diseases – showed an intact circadian clock. Here, we analyzed the role of the circadian clock in the functionality of mast cells and eosinophils. Human intestinal mast cells (hiMC) were isolated from intestinal mucosa; human eosinophils were isolated from peripheral blood. HiMC and eosinophils were synchronized by dexamethasone before stimulation every 4h around the circadian cycle by FcɛRI crosslinking or fMLP, respectively. Signaling molecule activation was examined using Western blot, mRNA expression by real-time RT-PCR, and mediator release by multiplex analysis. CXCL8 and CCL2 were expressed and released in a circadian manner by both hiMC and eosinophils in response to activation. Moreover, phosphorylation of ERK1/2, known to be involved in activation of hiMC and eosinophils, showed circadian rhythms in both cell types. Interestingly, all clock genes hPer1, hPer2, hCry1, hBmal1, and hClock were expressed in a similar circadian pattern in activated and unstimulated cells indicating that the local clock controls hiMC and eosinophils and subsequently allergic reactions but not vice versa.

Differential effects of the Toll-like receptor 2 agonists, PGN and Pam3CSK4 on anti-IgE induced human mast cell activation.

Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.