Acute activation of SERCA with CDN1163 attenuates IgE ‐mediated mast cell activation through selective impairment of ROS and p38 signaling
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Abstract:
Mast cells are granulocytic immune sentinels present in vascularized tissues that drive chronic inflammatory mechanisms characteristic of allergic pathologies. IgE-mediated mast cell activation leads to a rapid mobilization of Ca2+ from intracellular stores, which is essential for the release of preformed mediators via degranulation and de novo synthesized proinflammatory cytokines and chemokines. Given its potent signaling capacity, the dynamics of Ca2+ localization are highly regulated by various pumps and channels controlling cytosolic Ca2+ concentrations. Among these is sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), which functions to maintain low cytosolic Ca2+ concentrations by actively transporting cytosolic Ca2+ ions into the endoplasmic reticulum. In this study, we characterized the role of SERCA in allergen-activated mast cells using IgE-sensitized bone marrow-derived mast cells (BMMCs) treated with the SERCA activating compound, CDN1163, and simultaneously stimulated with allergen through FcεRI under stem cell factor (SCF) potentiation. Acute treatment with CDN1163 was found to attenuate early phase mast cell degranulation along with reactive oxygen species (ROS) production. Additionally, treatment with CDN1163 significantly reduced secretion of IL-6, IL-13, and CCL3, suggesting a role for SERCA in the late phase mast cell response. The protective effects of SERCA activation via CDN1163 treatment on the early and late phase mast cell response may be driven by the selective suppression of p38 MAPK signaling. Together, these findings implicate SERCA as an important regulator of the mast cell response to allergen and suggest SERCA activity may offer therapeutic potential targeting allergic pathologies, warranting further investigation.Keywords:
SERCA
Proinflammatory cytokine
The relations between human IgE and mouse peritoneal mast cells were studied in vitro. Non-heated human IgE sensitizes the mouse mast cells for degranulation on challenge with anti-human IgE. This capacity is lost after heating of human IgE at 56 degrees C. The degranulation only occurs in determined quantitative IgE-anti-IgE relationships, an excess of one or the other reagent inhibiting the reaction. Sensitization is practically instanteneous, and the degranulation is independent on the order of addition of the human IgE and anti-IgE. The human IgE can be removed from mouse peritoneal mast cells by a single washing. The results show that human IgE is unable to bind firmly to mouse peritoneal mast cells in vitro. It seems to induce the formation of biologically active human IgE-anti-human IgE complexes, which act on mouse mast cells and induce their degranulation.
Mast (botany)
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The correlations among the potentiating activity of various PS analogs on concanavalin A (Con A)-induced rat mast cell degranulation, the hemolytic activity and the incorporation into the mast cell membrane were studied. The following results were obtained. Lysophosphatidylserine (LysoPS) caused rat mast cell activation (degranulation) in the presence of Con A. The order of the activity was as follows: 1-stearoyl lysoPS = 1-palmitoyl lysoPS greater than 1-myristoyl lysoPS greater than 1-lauroyl lysoPS. The relative hemolytic activity of these compounds was similar to that observed in the mast cell activation. Dilauroyl PS, which shows similar hemolytic activity to 1-myristoyl lysoPS, did not activate mast cells appreciably. The relative activity of these phospholipids in the binding to mast cells was 1-stearoyl lysoPS greater than dilauroyl PS greater than 1-lauroyl lysoPS. Hemolytic activity, as well as activity on mast cells, of lysoPS analogs was well correlated to mast cell membrane incorporation, whereas such a correlation was not found with PS analogs. Dilauroyl PS could be accumulated in the mast cell membrane and showed hemolytic activity, but did not activate histamine secretion.
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Abstract Mast cells are key effectors of allergic inflammation. IgE-mediated mast cell activation induces degranulation and inflammatory mediator release. Omalizumab (Xolair; Genentech Inc.) is a recombinant humanized monoclonal anti-IgE antibody that prevents IgE binding to its receptor, FcϵRI. Objective: We investigated the effects of omalizumab on IgE pre-sensitized human LAD2 mast cells. Methods: LAD2 degranulation was determined by β-hexosaminidase assay. Chemokine expression and prostaglandin synthesis was measured by quantitative PCR analysis and ELISA, respectively. IgE binding and FcϵRI expression was determined by flow cytometry. Results: Omalizumab pretreatment inhibited IgE binding to LAD2 cells and entirely prevented IgE-dependent upregulation of FcϵRI expression. In addition, omalizumab removed FcϵRI-prebound IgE as early as 24 hrs after treatment. After 5 days, bound IgE was reduced by 92%. Furthermore, omalizumab concomitantly reversed IgE-dependent FcϵRI upregulation by 49% 48 hrs post treatment and by 93% 5 days post treatment. Consequently, omalizumab attenuated ongoing IgE-mediated responses, reducing degranulation by 34%, chemokine expression up to 79% and prostaglandin synthesis by 34% after 7 days of omalizumab treatment. Conclusions: Omalizumab is able to remove pre-bound IgE from sensitized mast cells thereby reducing ongoing response to FcϵRI-dependent signals. This data suggests that omalizumab is an effective inhibitor of sensitized human mast cells.
Omalizumab
Prostaglandin D2
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Compound 48/80
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Background: Although allergen‐specific IgE content in serum can be determined immunochemically, little is known about the relationship between this parameter and the strength of the degranulation response upon allergen triggering. Objectives: Analyse the degranulation capacity of immunochemically defined purified and serum IgE after challenge with anti‐IgE or allergen using a rat mast cell line (RBL) transfected with the α ‐chain of the human high‐affinity IgE receptor (Fc ɛ RI). Methods: Purified IgE specific for 4‐hydroxy‐3nitrophenylacetyl, purified IgE of unknown specificity, and sera from allergic patients sensitive to Dermatophagoides pteronyssinus and Dactylis glomerata were assessed. Degranulation was measured by a β ‐hexosaminidase release assay after anti‐IgE or allergen‐specific challenge. Results: For purified monoclonal IgE a significant correlation ( r = 0.97) was found between the proportion of bound allergen‐specific IgE and the strength of the degranulation response. In contrast, no correlation ( r = 0.27) was detected after sensitization with serum IgE. Conclusion: Our studies demonstrate that mast cell activation mediated through IgE from allergic patients is a result of complex relationships that are not only dependent on allergen‐specific IgE content but also relate to the capacity to efficiently sensitize and trigger the signalling responses that lead to degranulation.
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Compound 48/80
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Ketotifen
Compound 48/80
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As tissue-resident immune cells, mast cells are frequently found in close proximity to afferent neurons and are subjected to immunoactive mediators secreted by these neurons, including substance P (SP) and calcitonin gene-related peptide (CGRP). Neurogenic inflammation is thought to play an important role in the pathophysiology of many diseases. Unraveling the cellular mechanisms at the interface between the immune response and the peripheral nervous system is important for understanding how these diseases arise and progress. In this work, mast cell degranulation following direct exposure to CGRP and SP was studied both at the bulk and single-cell levels to characterize the mouse peritoneal mast cell response to neuropeptides and compare this response to well-studied mast cell activation pathways. Results show that mast cells secrete fewer chemical messenger-filled granules with increased IgE preincubation concentrations. The biophysical characteristics of mast cell degranulation in response to SP and CGRP is in many ways similar to calcium ionophore-induced mast cell degranulation; however, neuropeptide-stimulated mast cells secrete reduced chemical messenger content per secretion event, resulting in an overall relative decrease in secreted chemical messengers.
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Mouse and rat IgE fix firmly to the peritoneal mast cells from the other species, sensitizing them for anaphylactic reaction. Sensitization with IgE can be demonstrated by inducing degranulation either with specific antigens or with corresponding anti-IgE. Sensitization of rat mast cells by mouse IgE antibodies is more easily obtained than that of mouse mast cells by rat IgE antibodies. In this case, anti-IgE-induced degranulation is higher than antigen-induced degranulation. Heterologous sensitization by IgE is time requiring and temperature-dependent. Its kinetics depend upon IgE concentration. Cross-reactions between IgE from one species and anti-IgE from another species have been observed: anti-IgE for one species is able to neutralize PCA reaginic activity of sera from the other species; anti-rat IgE induces degranulation of mouse actively sensitized mast cells. The results suggest strongly that there exists a structural and functional similarity between the IgE molecules from the two species.
Heterologous
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Traditional analyses of calcium homeostasis have separately quantified either calcium accumulation or release mechanisms. To define the system as a whole, however, requires multiple experimental techniques to examine both accumulation and release. Here we describe a technique that couples the simultaneous quantification of radio-labeled calcium accumulation in endoplasmic reticulum (ER) microsomes with the release of inorganic phosphate (Pi) by the hydrolytic activity of sarco-endoplasmic reticulum calcium ATPase (SERCA) all in the convenience of a 96-well format.
SERCA
Calcium ATPase
Calcium Signaling
Homeostasis
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