Activation Of Human Mast Cells Research Articles

Differential MRGPRX2-dependent activation of human mast cells by polymyxins and octapeptins

The emergence of multi-drug resistant Gram-negative bacteria has led to renewed interest in the antimicrobial activity of polymyxins and novel polymyxin analogues (e.g. nonapeptides and octapeptin). In some individuals, clinically used polymyxins can cause acute hypersensitivity reactions through mast cell activation, with a recent study attributing this effect to activation of the MAS-related G protein-coupled receptor X2 (MRGPRX2). In the present study, HEK293 cells expressing human MRGPRX2 and the human mast cell line LAD2 were used to characterize the activity of the broader family of polymyxins. Octapeptin C4, polymyxin B and colistin produced concentration-dependent calcium mobilization, degranulation, and CCL-2 (MCP-1) release in LAD2 mast cells, with the former being highly potent. CRISPR-Cas9 knockdown of MRGPRX2 in LAD2 cells and a MRGPRX2 inverse agonist caused a significant reduction in calcium mobilization, degranulation, and CCL-2 release, demonstrating dependency on MRGPRX2 expression. In contrast, polymyxin nonapeptides were far less potent calcium mobilisers and failed to induce functional degranulation in LAD2 cells. Our results confirm that activation of mast cells induced by polymyxin-related antibiotics is MRGPRX2-dependent and reveal that octapeptin C4 might be more liable, whilst nonapeptides are less liable, to trigger immediate hypersensitivity reactions clinically. The mechanism underpinning the difference in MRGPRX2 activation between polymyxin-related antibiotics is important to better understand as it may help design new, safer polymyxins and guide the optimal clinical use of existing polymyxin drugs.

Rocuronium-specific antibodies drive perioperative anaphylaxis but can also function as reversal agents in preclinical models.

Neuromuscular blocking agents (NMBAs) relax skeletal muscles to facilitate surgeries and ease intubation but can lead to adverse reactions, including complications because of postoperative residual neuromuscular blockade (rNMB) and, in rare cases, anaphylaxis. Both adverse reactions vary between types of NMBAs, with rocuronium, a widely used nondepolarizing NMBA, inducing one of the longest rNMB durations and highest anaphylaxis incidences. rNMB induced by rocuronium can be reversed by the synthetic γ-cyclodextrin sugammadex. However, in rare cases, sugammadex can provoke anaphylaxis. Thus, additional therapeutic options are needed. Rocuronium-induced anaphylaxis is proposed to rely on preexisting rocuronium-binding antibodies. To understand the pathogenesis of rocuronium-induced anaphylaxis and to identify potential therapeutics, we investigated the memory B cell antibody repertoire of patients with suspected hypersensitivity to rocuronium. We identified polyclonal antibody repertoires with a high diversity among V(D)J genes without evidence of clonal groups. When recombinantly expressed, these antibodies demonstrated specificity and low affinity for rocuronium without cross-reactivity for other NMBAs. Moreover, when these antibodies were expressed as human immunoglobulin E (IgE), they triggered human mast cell activation and passive systemic anaphylaxis in transgenic mice, although their affinities were insufficient to serve as reversal agents. Rocuronium-specific, high-affinity antibodies were thus isolated from rocuronium-immunized mice. The highest-affinity antibody was able to reverse rocuronium-induced neuromuscular blockade in nonhuman primates with kinetics comparable to that of sugammadex. Together, these data support the hypothesis that antibodies cause anaphylactic reactions to rocuronium and pave the way for improved diagnostics and neuromuscular blockade reversal agents.

Combinatorial Genomic Biomarkers Associated with High Response in IgE-Dependent Degranulation in Human Mast Cells.

Mast cells are the major effector cells that mediate IgE-dependent allergic reactions. We sought to use integrated network analysis to identify genomic biomarkers associated with high response in IgE-mediated activation of primary human mast cells. Primary human mast cell cultures derived from 262 normal donors were categorized into High, Average and Low responder groups according to their activation response profiles. Transcriptome analysis was used to identify genes that were differentially expressed in different responder cultures in their baseline conditions, and the data were analyzed by constructing a personalized perturbed profile (PEEP). For upregulated genes, the construction of PEEP for each individual sample of all three responder groups revealed that High responders exhibited a higher percentage of "perturbed" samples whose PEEP values lay outside the normal range of expression. Moreover, the integration of PEEP of four selected upregulated genes into distinct sets of combinatorial profiles demonstrated that the specific pattern of upregulated expression of these four genes, in a tandem combination, was observed exclusively among the High responders. In conclusion, this combinatorial approach was useful in identifying a set of genomic biomarkers that are associated with high degranulation response in human mast cell cultures derived from the blood of a cohort of normal donors.

Mast cells: a novel therapeutic avenue for cardiovascular diseases?

Mast cells are tissue-resident immune cells strategically located in different compartments of the normal human heart (the myocardium, pericardium, aortic valve, and close to nerves) as well as in atherosclerotic plaques. Cardiac mast cells produce a broad spectrum of vasoactive and proinflammatory mediators, which have potential roles in inflammation, angiogenesis, lymphangiogenesis, tissue remodelling, and fibrosis. Mast cells release preformed mediators (e.g. histamine, tryptase, and chymase) and de novo synthesized mediators (e.g. cysteinyl leukotriene C4 and prostaglandin D2), as well as cytokines and chemokines, which can activate different resident immune cells (e.g. macrophages) and structural cells (e.g. fibroblasts and endothelial cells) in the human heart and aorta. The transcriptional profiles of various mast cell populations highlight their potential heterogeneity and distinct gene and proteome expression. Mast cell plasticity and heterogeneity enable these cells the potential for performing different, even opposite, functions in response to changing tissue contexts. Human cardiac mast cells display significant differences compared with mast cells isolated from other organs. These characteristics make cardiac mast cells intriguing, given their dichotomous potential roles of inducing or protecting against cardiovascular diseases. Identification of cardiac mast cell subpopulations represents a prerequisite for understanding their potential multifaceted roles in health and disease. Several new drugs specifically targeting human mast cell activation are under development or in clinical trials. Mast cells and/or their subpopulations can potentially represent novel therapeutic targets for cardiovascular disorders.

Functional human skin explants as tools for assessing mast cell activation and inhibition.

Mast cells are activated through a variety of different receptors to release preformed granules and mediators synthesized de novo. However, the physiology and function of mast cells are not fully understood. Traditional studies of mast cell activation in humans have utilized cultures of tissue-derived mast cells including CD34+ progenitor cells or well-characterized commercially available cell lines. One limitation of these methods is that mast cells are no longer in a natural state. Therefore, their applicability to human skin disorders may be limited. Human skin explant models have been utilized to investigate the short-term effects of cell mediators, drugs, and irritants on skin while avoiding the ethical concerns surrounding in vivo stimulation studies with non-approved agents. Nonetheless, few studies have utilized intact human tissue to study mast cell degranulation. This "Methods" paper describes the development and application of an intact skin explant model to study human mast cell activation. In this manuscript, we share our protocol for setting up ex vivo human skin explants and describe the results of stimulation experiments and techniques to minimize trauma-induced histamine release. Skin explants were generated using de-identified, full-thickness, non-diseased skin specimens from plastic and reconstructive surgeries. Results were reproducible and demonstrated FcɛRI- and MRGPRX2-induced mediator release which was inhibited with the use of a BTK inhibitor and QWF, respectively. Thus, this explant model provides a quick and accessible method of assessing human skin mast cell activation and inhibition.

An optimized method for IgE-mediated degranulation of human lung mast cells.

Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.

Siglec-9 is an inhibitory receptor on human mast cells in vitro

Mast cell activation is critical for the development of allergic diseases. Ligation of sialic acid-binding immunoglobin-like lectins (Siglecs), such as Siglec-6, -7, and -8 as well as CD33, have been shown to inhibit mast cell activation. Recent studies showed that human mast cells express Siglec-9, an inhibitory receptor also expressed by neutrophils, monocytes, macrophages, and dendritic cells. We aimed to characterize Siglec-9 expression and function in human mast cells invitro. We assessed the expression of Siglec-9 and Siglec-9 ligands on human mast cell lines and human primary mast cells by real-time quantitative PCR, flow cytometry, and confocal microscopy. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt the SIGLEC9 gene. We evaluated Siglec-9 inhibitory activity on mast cell function by using native Siglec-9 ligands, glycophorin A(GlycA), and high-molecular-weight hyaluronic acid, a monoclonal antibody against Siglec-9, and coengagement of Siglec-9 with the high-affinity receptor for IgE (FcεRI). Human mast cells express Siglec-9 and Siglec-9 ligands. SIGLEC9 gene disruption resulted in increased expression of activation markers at baseline and increased responsiveness to IgE-dependent and IgE-independent stimulation. Pretreatment with GlycA or high-molecular-weight hyaluronic acid followed by IgE-dependent or -independent stimulation had an inhibitory effect on mast cell degranulation. Coengagement of Siglec-9 with FcεRI in human mast cells resulted in reduced degranulation, arachidonic acid production, and chemokine release. Siglec-9 and its ligands play an important role in limiting human mast cell activation invitro.

Remibrutinib inhibits hives effector cells stimulated by serum from chronic urticaria patients independently of FcεR1 expression level and omalizumab clinical response.

Despite advances in the treatment of chronic urticaria, in a significant percentage of the patients symptoms are not fully controlled with conventional approaches. New strategies under development include blocking intracellular mediators of mast cell and basophil activation. We aim to investigate the effects of the Bruton's tyrosine kinase (BTK) inhibitor remibrutinib on human blood basophils and CD34+ -derived mast cells activation induced by serum obtained from chronic urticaria patients. Twenty-two patients with chronic spontaneous urticaria (mean age 52years, 27% women) and 22 patients with chronic inducible urticaria (46years, 27% women) were included in the study together with a sex-matched control group. Patients were classified as responders or non-responders to anti-IgE therapy on the basis of their clinical data, FcεR1a expression on blood basophils and total IgE levels. Changes on CD63 expression-as an activation marker-, were used to evaluate in vitro the response of basophils and mast cells to serum exposure and the inhibitory effects of remibrutinib. Remibrutinib inhibits degranulation induced by IgE cross-linking in mast cells and basophils and also the activation triggered by factors present in the sera of spontaneous and inducible chronic urticaria patients. Patient's serum induces a greater degranulation of effector cells than controls. Activation of mast cells and basophils by patient sera and remibrutinib effects were not related to omalizumab responsiveness. Remibrutinib inhibits activation of human basophils and mast cells induced in vitro by exposure to the serum of chronic urticaria patients independently of their response to omalizumab.

Differential effects of PEGylated Cd-free CuInS2/ZnS quantum dot (QDs) on substance P and LL-37 induced human mast cell activation

CuInS2/ZnS-PEG quantum dots (QDs) are among the most widely used near infrared non-cadmium QDs and are favored because of their non-cadmium content and strong tissue penetration. However, with their increasing use, there is great concern about whether exposure to QDs is potentially risky to the environment and humans. Furthermore, toxicological data related to CuInS2/ZnS-PEG QDs are scarce. In the study, we found that CuInS2/ZnS-PEG QDs (0–100 μg/mL) could internalize into human LAD2 mast cells without affecting their survival rate, nor did it cause degranulation or release of IL-8 and TNF-α. However, CuInS2/ZnS-PEG QDs significantly inhibited Substance P (SP) and LL-37-induced degranulation and chemotaxis of LAD2 cells by inhibiting calcium mobilization. Lower concentrations of CuInS2/ZnS-PEG QDs promoted the release of TNF-α and IL-8 stimulated by SP, but higher concentrations of CuInS2/ZnS-PEG QDs significantly inhibited the release of TNF-α and IL-8. On the other hand, CuInS2/ZnS-PEG QDs promoted LL-37-mediated TNF-α release from LAD2 cells in a dose-dependent manner from 6.25 to 100 μg/mL, while release of IL-8 triggered by LL-37 was dose-dependently inhibited within a dose concentration of 12.5–100 μg/mL. Collectively, our data demonstrated that CuInS2/ZnS-PEG QDs differentially mediated human mast cell activation induced by SP and LL-37.

Human mast cells induce osteoclastogenesis through cell surface RANKL.

We employed the co-culture of CD34+ stem cell-derived human mast cells (HMC) and human monocyte-derived osteoclast precursors to evaluate if mast cells contribute to the pathogenesis of osteoporosis through regulation of osteoclast proliferation and activation. Mature HMC and osteoclast precursors were cultured from monocytes isolated from human buffy coat. The osteoclast precursors were incubated with HMC or receptor activator of nuclear factor kappa-B ligand (RANKL) for a week prior to determination of osteoclast maturation through characterization by their morphology and tartrate resistant acid phosphatase (TRAP) expression. The bone absorption activity was determined by pit formation on osteo-assay plate. Mature osteoclasts were identified following co-culture of osteoclast precursors with HMC for one week in the absence of RANKL and they were capable of bone resorption. These actions of HMC on osteoclasts were not affected by mast cell activators such anti-IgE or substance P but could be reversed by osteoprotegerin (OPG) in the co-culture system suggesting the involvement of RANKL. The expression of RANKL on the cell surface of HMC was confirmed by flow cytometry and the density was not affected by activation of HMC. Our study provided direct evidence confirming the initiation of osteoclast proliferation and activation by mast cells through cell surface RANKL suggesting that mast cells may contribute to bone destruction in pathological conditions such as osteoporosis.

The effect of substance P and its common in vivo-formed metabolites on MRGPRX2 and human mast cell activation.

The tachykinin neuropeptide substance P (SP) is the canonical agonist peptide for the neurokinin 1 receptor (NK1R). More recently, it has also been shown to activate the Mas‐related G protein‐coupled receptor X2 (MRGPRX2) receptor on mast cells (MCs), triggering degranulation and release of inflammatory mediators. SP undergoes rapid C‐terminal truncation in vivo by a number of proteases to generate the metabolites SP(1–9)‐COOH and in particular SP(1–7)‐COOH. While the C terminus of SP is critical for NK1R activation, studies have shown that the peptide polycationic N terminus is key for MRGPRX2 and mast cell activation. The study thus aimed to determine if the C‐terminally truncated metabolites of SP, SP(1–9)‐COOH, and SP(1–7)‐COOH retained stimulatory activity at MRGPRX2. SP, SP(1–9)‐COOH, and SP(1–7)‐COOH were synthesized and tested on HEK293 cells expressing NK1R or MRGPRX2, and LAD2 human mast cells, to determine the activity of SP and its metabolites in Ca2+ mobilization, degranulation, and cytokine assays. As expected from prior studies, both C‐terminally truncated SP metabolites had essentially no activity at NK1R, even at very high concentrations. In contrast, the in vivo metabolite of SP, SP(1–9)‐COOH retained ability to activate MRGPRX2 across all parameters tested, albeit with reduced potency compared to intact SP. SP(1–7)‐COOH did not produce any significant MRGRPX2 activation. Our results suggest that the SP metabolite, SP(1–9)‐COOH, may play a regulatory role through the activation of MRGPRX2. However, given the relatively low potency of both SP and SP(1–9)‐COOH at MRGPRX2, additional work is needed to better understand the biological importance of this expanded SP/MRGPRX2 pathway.

Bacillus Calmette-Guérin-Induced Human Mast Cell Activation Relies on IL-33 Priming.

Bacillus Calmette–Guérin (BCG) vaccine is an attenuated strain of Mycobacterium bovis that provides weak protection against tuberculosis (TB). Mast cells (MCs) are tissue-resident immune cells strategically that serve as the first line of defence against pathogenic threats. In this study, we investigated the response of human MCs (hMCs) to BCG. We found that naïve hMCs exposed to BCG did not secrete cytokines, degranulate, or support the uptake and intracellular growth of bacteria. Since we could show that in hMCs IL-33 promotes the transcription of host-pathogen interaction, cell adhesion and activation genes, we used IL-33 for cell priming. The treatment of hMCs with IL-33, but not IFN-γ, before BCG stimulation increased IL-8, MCP-1 and IL-13 secretion, and induced an enhanced expression of the mycobacteria-binding receptor CD48. These effects were comparable to those caused by the recombinant Mycobacterium tuberculosis (Mtb) 19-KDa lipoprotein. Finally, stimulation of hMCs with IL-33 incremented MC-BCG interactions. Thus, we propose that IL-33 may improve the immunogenicity of BCG vaccine by sensitising hMCs.

6-O-angeloylplenolin inhibits anti-IgE-stimulated human mast cell activation via suppressing calcium influx and ERK phosphorylation.

Objective(s):Mast cells are important immune cells that primarily localize in the interface between the host and external environment, and protect us from pathogen infection. However, they are also involved in the pathology of allergic diseases such as asthma and atopic dermatitis. A novel S phase kinase-associated protein 1 (SKP1) inhibitor 6-O-angeloylplenolin (6-OAP), was studied with its potential ability to alleviate the anti-IgE-induced inflammatory responses of primary human cultured mast cells (HCMCs) and LAD2 cell line. Materials and Methods:We isolated the HCMCs from the buffy coat of voluntary blood donors. The effects of 6-OAP on mast cell activation were evaluated by measuring degranulation, cytokine release, migration, calcium influx, and ERK phosphorylation using spectro-fluorescence assay, multiplex cytometric bead assay/ELISA, migration assay, Fluo-4 calcium flux assay, and western blot, respectively. Results:It was found that 6-OAP exerted anti-inflammatory effects on human mast cells by dose-dependently suppressing the anti-IgE-mediated degranulation and release of cytokines such as proinflammatory cytokines (IL-8 and TNF-α), growth factors (GM-CSF, VEGF, and FGF), and chemokines (CCL2 and CCL3) in HCMC and LAD2 cells. It also suppressed the migration of immature HCMCs induced by CXCL12. Moreover, the process of calcium influx and ERK phosphorylation in activated HCMC cells were inhibited by 6-OAP administration. Conclusion:Our results showed that 6-OAP inhibited anti-IgE-induced inflammatory responses of human mast cells via suppressing calcium influx and ERK phosphorylation.

Mast Cells in Atopic Diseases: More Than Just Histamine

Mast cells are present in all tissues and are able to release multiple mediators in response to allergic, autoimmune, environmental, neurohormonal and pathogenic triggers. Histamine has received most of the attention in terms of pathophysiology and drug development, while tryptase remains to this date with no clear function and no known inhibitor. Mast cells can also release pro-inflammatory and pruritogenic molecules, such as IL-6 and IL-31, selectively without degranulation. One such critical molecule is platelet activating factor (PAF), which is vasoactive, can cause wheal and flare on ita own, but can also stimulate eosinophils and mast cells that are critical in the pathogenesis of chronic spontaneous urticaria (CSU) and rhinitis. Mast cell-derived cytokines and PAF have also been implicated in inflammatory processes including COVID-19. Among the second generation histamine-1 receptor antagonists, rupatadine is more effective overall, it has potent anti-PAF activity, and also inhibits activation of human mast cells and eosinophils. Rupatadine could, therefore, serve as a first-line drug for CSU and rhinitis, but may also be used, especially for patients resistant to antihistamines.

P192 DEVELOPMENT OF A FUNCTIONAL HUMAN SKIN EXPLANT MODEL TO STUDY MAST CELL ACTIVATION

The human skin explant model has been utilized to determine the short-term effects of cytokines and irritants on human skin while avoiding the ethical problems of in vivo stimulation studies with non-approved agents. Therefore, we sought to develop an explant model to study human skin mast cell activation.

Novel Insights on MRGPRX2-Mediated Hypersensitivity to Neuromuscular Blocking Agents And Fluoroquinolones.

Neuromuscular blocking agents (NMBAs) like atracurium and rocuronium as well as fluoroquinolones (FQs) cause mast cell-mediated anaphylaxis by activating Mas-related G protein-coupled receptor X2 (MRGPRX2), but many questions remain unanswered. Here, we address three of them, namely whether primary human mast cells show similar activation by these drugs as murine mast cells and mast cell lines, how sugammadex protects from atracurium-induced MRGPRX2-mediated mast cell activation, and why some but not all patients treated with rocuronium develop anaphylaxis. We used peripheral blood-derived cultured mast cells from healthy donors and patients, assessed mast cell activation and degranulation by quantifying intracellular calcium and CD63 expression, respectively, and made use of MRGPRX2-silencing, via electroporation with Dicer-substrate small interfering RNAs, and single cell flow cytometric analyses. Atracurium, ciprofloxacin, and levofloxacin activated and degranulated primary human mast cells, but only MRGPRX2-positive and not MRGPRX2-negative or -silenced mast cells. Sugammadex attenuated the atracurium-induced and MRGPRX2-mediated activation and degranulation of human mast cells by reducing free atracurium levels. The mast cells of patients with IgE-independent anaphylaxis to rocuronium were similar, in their MRGPRX2 expression and function, to those of patients with IgE-mediated anaphylaxis. These findings further improve our understanding of the role and relevance of MRGPRX2-driven mast cell activation in anaphylactic reactions to NMBAs and FQs and may help to improve their prediction, prevention, and treatment.

Resveratrol Is a Natural Inhibitor of Human Intestinal Mast Cell Activation and Phosphorylation of Mitochondrial ERK1/2 and STAT3

Mast cells play a critical role as main effector cells in allergic and other inflammatory diseases. Usage of anti-inflammatory nutraceuticals could be of interest for affected patients. Resveratrol, a natural polyphenol found in red grapes, is known for its positive properties. Here, we analyzed the effects of resveratrol on FcεRI-mediated activation of mature human mast cells isolated from intestinal tissue (hiMC). Resveratrol inhibited degranulation and expression of cytokines and chemokines such as CXCL8, CCL2, CCL3, CCL4, and TNF-α in a dose-dependent manner. Further, resveratrol inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3. ERK1/2 is known to be involved in cytokine expression of hiMC and to directly interact with STAT3. Mitochondrial STAT3 is phosphorylated by ERK1/2 and contributes to mast cell degranulation. We were able to isolate mitochondrial fractions from small hiMC numbers and could show that activation of mitochondrial STAT3 and ERK1/2 in hiMC was also inhibited by resveratrol. Our results indicate that resveratrol inhibits hiMC activation by inhibiting the phosphorylation of mitochondrial and nuclear ERK1/2 and STAT3, and it could be considered as an anti-inflammatory nutraceutical in the treatment of mast cell-associated diseases.

Exosomes From Packed Red Cells Induce Human Mast Cell Activation and the Production of Multiple Inflammatory Mediators.

Most blood transfusion-related adverse reactions involve the immunologic responses of recipients to exogenous blood components. Extracellular vesicles isolated from packed red cells can affect the recipient’s immune system. Mast cells are traditionally known as effector cells for allergic transfusion reactions. However, growing evidence supports the notion that activated mast cells might disturb host innate immunologic responses. Exosomes are a type of extracellular vesicle. To determine the effect of exosomes on mast cells, we enriched exosomes derived from volunteer plasma (EXs-nor) and packed red cells (EXs-RBCs) using ultracentrifugation and incubated them with a human mast cell line (HMC-1). We found that EXs-RBC exposure increased the expression of tryptase-1 and prostaglandin D2, the production of multiple inflammatory mediators, and the levels of Toll-like receptor-3 (TLR-3) and phospho-mitogen-activated protein kinase (MAPK) in HMC-1 cells. MAPK inhibitors (SB203580, PD98059, and SP600125) and a TLR-3/dsRNA complex inhibitor reduced the EXs-RBC-stimulated production of inflammatory mediators in HMC-1 cells, whereas the TLR-3 agonist [poly (A:U)] elevated the production of these mediators. These results indicate that EXs-RBCs activate HMC-1 cells and elicit the production of multiple inflammatory mediators, partly via the TLR-3 and MAPK pathways. Mast cells activated by EXs-RBCs exhibit complex inflammatory properties and might play a potential role in transfusion-related adverse reactions.

Activation of human skin mast cells by vancomycin via MrgX2: Comparison to the effects of brimonidine

Abstract Vancomycin (VCM) is a glycopeptide antibiotic that inhibits bacterial peptidoglycan biosynthesis and is used for the treatment of certain Gram-positive bacterial infections of the eyes. However, the use of VCM is associated with several potential adverse reactions. We examined the mechanism of activation of human mast cells by vancomycin (VCM) and compared to brimonidine, an alpha2-adrenergic receptor agonist used to treat in glaucoma. VCM induced degranulation and cytokine secretion by SMCs, which were suppressed by pertussis toxin (PTX) (similar to MrgX2 ligands). Brimonidine induced less degranulation than VCM that was partially inhibited by PTX. MrgX2 expression on the surface of SMCs was detected by FACS analysis. VCM enhanced FceRI-mediated degranulation and cytokine secretion in a dose-dependent manner, whereas brimonidine failed to induce cytokine production and suppressed FceRI-mediated cytokine secretion. VCM induced maximal ERK-phosphorylation by 5 min similar to compound 48/80, an MrgX2 ligand and inhibited by PTX. VCM- but not brimonidine-induced degranulation of MCs was significantly inhibited by QWF, an MrgX2 antagonist. This study indicates that VCM but not brimonidine activates human SMCs via MrgX2 and that MCs become more responsive to VCM when co-stimulated by aggregating FceRI, which may occur when sensitive patients exposed to aeroallergen and VCM.

Sesquiterpene-Loaded Co-Polymer Hybrid Nanoparticle Effects on Human Mast Cell Surface Receptor Expression, Granule Contents, and Degranulation.

Biodegradable polymeric nanoparticles (NPs) such as poly(lactic-co-glycolic acid) (PLGA) and polyvinyl alcohol (PVA) have been used as drug delivery systems for natural and synthetic compounds and are designed to control the loading and release of biodegradable materials to target cells, tissues, and organs. Eremophilane-type sesquiterpenes have anti-inflammatory properties but are lipophilic, cytotoxic, and not biocompatible with many cells. To determine whether biodegradable PLGA/PVA could improve the biocompatibility of sesquiterpenes, sesquiterpene-loaded NPs were synthesized and their effects on human mast cells (LAD2), the major effector cells of allergic inflammation, were determined. NPs composed of PLGA/PVA and two types of sesquiterpenes (fukinone, PLGA/PVA-21 and 10βH-8α,12-epidioxyeremophil-7(11)-en-8β-ol, PLGA/PVA-22) were produced using a microfluidic synthesis method. The NPs’ size distribution and morphology were evaluated by dynamic light scattering and cryogenic transmission electron microscopy (TEM). PLGA/PVA-21 and PLGA/PVA-22 were 60 to 70 nm and were readily internalized by LAD2 as shown by flow cytometry, fluorescence microscopy, and TEM. While unencapsulated sesquiterpenes decreased LAD2 cell viability by 20%, PLGA/PVA-21 and PLGA/PVA-22 did not alter LAD2 viability, showing that encapsulation improved the biocompatibility of the sesquiterpenes. PLGA/PVA-21 and PLGA/PVA-22 decreased the expression of genes encoding the subunits of the high affinity immunoglobulin E receptor (FcεR1α, FcεR1β, FcεR1γ) and the stem cell factor receptor (Kit,), suggesting that hybrid NPs could alter mast cell responses to antigens and shift their maturation. Similarly, PLGA/PVA-21 and PLGA/PVA-22 inhibited tryptase expression but had no effect on chymase expression, thereby promoting a shift to the tryptase-positive phenotype (MCT). Lastly, PLGA/PVA-21 and PLGA/PVA-22 inhibited mast cell degranulation when the LAD2 cells were activated by IgE crosslinking and FcεRI. Overall, our results suggest that PLGA/PVA-21 and PLGA/PVA-22 alter human mast cell phenotype and activation without modifying viability, making them a more biocompatible approach than treating cells with sesquiterpenes alone.