Staphylococcus aureus δ-Toxin Induces Ca²⁺ Influx-Independent Degranulation of Murine Cultured Mast Cells

Abstract

Cutaneous colonization with Staphylococcus aureus (SA) is frequently observed in patients with atopic dermatitis. SA produces a wide variety of bacterial toxins, among which δ-toxin was found to induce degranulation of mast cells. Degranulation of mast cells could enhance bacterial clearance and protection from future SA infection but lead to exacerbation of atopic dermatitis. Because it remains to be determined how δ-toxin triggers degranulation, we investigated δ-toxin-induced changes in murine bone marrow-derived cultured mast cells in this study. We found that δ-toxin-induced degranulation could be classified into two phases, an early Ca²⁺-independent and a late Ca²⁺-dependent phase. Recent studies suggest that NOD-like receptor family, pyrin domain containing 3 is involved in the degranulation of mast cells, raising a possibility that leakage of K⁺ induced by δ-toxin is involved in the Ca²⁺-independent phase. However, Ca²⁺-independent degranulation remains unchanged although Ca²⁺-influx and degranulation induced by δ-toxin were significantly suppressed in the presence of high concentrations of K⁺. Because actin depolymerization was reported to induce degranulation in the absence of Ca²⁺ in the permeabilized rat peritoneal mast cells, a slow but steady decrease in the amount of filamentous actin observed here may be involved in Ca²⁺-independent degranulation induced by δ-toxin. Although Mas-related G protein-coupled receptor (MRGPR) X2 in humans and Mrgprb2 in mice are regarded as the receptors responsible for immunoglobulin E-independent degranulation, δ-toxin-induced degranulation remained unchanged in Mrgprb2^−/− mast cells. Our findings pave the way for identification of the target receptors of δ-toxin.

INTRODUCTION

Staphylococcus aureus (SA) causes various diseases, such as manageable skin infections and fatal necrotizing pneumonia.¹⁾ The infection with SA is frequently found in patients with atopic dermatitis, and the disease severity is correlated with the surface density of SA.^2–4) Accumulating evidence suggests that cutaneous mast cells should be closely associated with atopic dermatitis.⁵⁾ SA was found to release a wide variety of bacterial peptide toxins, including phenol-soluble modulins (PSMs), which play critical roles in the pathogenesis of SA. Nakamura et al. demonstrated that one of PSMs, δ-toxin, stimulated cutaneous mast cells to induce degranulation.⁶⁾ The following studies indicated that δ-toxin-induced activation of cutaneous mast cells and keratinocytes should induce T-helper 2 (Th2)-biased responses and immunoglobulin E (IgE) production, which enhances systemic protection against SA infection.^7–9) It remains unknown how δ-toxin could induce degranulation of cutaneous mast cells. Human neutrophils were found to undergo chemotaxis in response to δ-toxin, of which actions were mediated by N-formyl peptide receptor 2 (FPR2),¹⁰⁾ although δ-toxin could induce degranulation of murine bone marrow-derived cultured mast cells lacking FPR2.⁶⁾

Degranulation of mast cells can be classified into two categories, IgE-dependent and IgE-independent degranulation. The latter category of degranulation has been characterized as pertussis toxin-sensitive responses, which are specific to tissue connective type mast cells.¹¹⁾ Tatemoto et al. demonstrated that Mas-related G protein-coupled receptor X2 (MRGPRX2) should be a potent candidate responsible for IgE-independent degranulation.¹²⁾ Although more than 20 genes are regarded as murine orthologs of MRGPRX2, McNeil et al. proposed that Mrgprb2 should be the major candidate using the gene-targeted mice.¹³⁾

Our purpose in this study is to further characterize the δ-toxin-induced degranulation of mast cells. Identification of the target molecules of δ-toxin will contribute to the development of novel therapeutic approaches for SA infection. We could not determine the targets of δ-toxin here, but unexpectedly, our findings raised the possibility that a novel Ca²⁺-influx-independent machinery is involved in δ-toxin-induced degranulation.

MATERIALS AND METHODS

Animals

Specific-pathogen-free, 8 to 10-week-old male BALB/cCrSlc mice, and 8 to 10-week-old C57BL/6NJcl were obtained from Japan SLC (Hamamatsu, Japan) and CLEA Japan (Tokyo, Japan). The mutant strain that lacks functional Mrgprb2 was generated by electroporation of two CRISPR RNAs (crRNAs), tracrRNA, and Alt-R Cas9 endonuclease (Integrated DNA Technologies, Coralville, IA, U.S.A.) in fertilized eggs of a C57BL/6NJcl mouse using NEPA21 (NEPA GENE, Ichikawa, Japan). crRNAs were designed to target the following sequences; 5′-CAT TTA GTC CCA TCC CAA CC-3′ and 5′-ACG TTT ACA GCG ATA CCA AA-3′. The established strain was found to have a truncated Mrgprb2 gene, which encodes the amino-terminal 49 residues of Mrgprb2 and the segment generated by a frame-shift (NH₂-DGT KCH SAV VPG HPY AHE CLH CLH SQP GYG-COOH). Animal use was approved by the Animal Care and Use Committee, Okayama University (OKU-2018086) and by the Committee on the Ethics of Animal Research of Kyoto Pharmaceutical University (A23-014) conforming to the Guidelines for the Proper Conduct of Animal Experiments of Science Council of Japan and the Policy on the Care and Use of Laboratory Animal of Okayama University and Kyoto Pharmaceutical University.

Materials

The following materials were commercially obtained from the sources indicated: SA δ-toxin from AnaSpec (Fremont, CA, U.S.A.), compound 48/80 (mast cell secretagogue), mitomycin C, piceatannol (Syk inhibitor), probenecid, digitonin, and p-nitrophenyl-β-D-acetoamide-2-deoxyglucopyranoside from Sigma-Aldrich (St. Louis, MO, U.S.A.), pertussis toxin from Bordetella pertussis from List Biological Laboratories, an anti-trinitrophenyl (TNP) IgE antibody (clone IgE-3) from BD Biosciences (San Diego, CA, U.S.A.), LY294002 (phosphatidylinositol 3-kinase inhibitor) from Cayman Chemical (Ann Arbor, MI, U.S.A.), TNP-conjugated bovine serum albumin (TNP-BSA) from LSL (Tokyo, Japan), thapsigargin (sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor), and BAPTA-AM (Ca²⁺ chelator) from Merck Millipore (Billeria, MA, U.S.A.), recombinant mouse stem cell factor, and Hoechst 33342 from Nakalai Tesque (Kyoto, Japan), Fura-2AM (1-[6-Amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N’,N’-tetraacetic acid, pentaacetoxymethyl ester) from Dojindo (Kumamoto, Japan), phalloidin-iFluor 488 from Abcam (Cambridge, U.K.), and recombinant mouse interleukin (IL)-3 from R&D Systems (Minneapolis, MN, U.S.A.). All other chemicals were commercial products of reagent grade.

Preparation of Bone Marrow-Derived Cultured Mast Cells

IL-3-dependent murine bone marrow-derived cultured mast cells (BMMCs) and connective tissue type mast cell-like cultured mast cells (CTMCs) were prepared as previously described.¹⁴⁾ Briefly, murine bone marrow cells were cultured in the presence of 10 ng/mL IL-3 for approx. 30 d. On Day-30, greater than 95% of the cells exhibited metachromasy by acidic toluidine blue staining and were FcεRI⁺c-kit⁺ on the flow cytometry. CTMCs were obtained by co-culturing BMMCs with mitomycin C-treated Swiss 3T3 fibroblasts in the presence of 100 ng/mL recombinant murine stem cell factor (SCF) for 16 d. Greater than 90% of the cells were confirmed as mature mast cells by Safranin-O staining.

Measurement of Degranulation

The cultured mast cells were suspended in PIPES buffer (25 mM PIPES–NaOH, pH 7.4 containing 125 mM NaCl, 2.7 mM KCl, 1 mM CaCl₂, 5.6 mM glucose, and 0.1% bovine serum albumin), and then stimulated for 30 min at 37 °C. They were centrifuged at 800 × g at 4 °C for 5 min to obtain the supernatants (extracellular fractions, E). The resultant pellets were resuspended in PIPES buffer containing 0.5% Triton X-100 and were centrifuged at 10000 × g for 10 min to obtain the supernatants (cell-associated fractions, C). Degranulation was evaluated by measuring the enzyme activity of a lysosomal enzyme, β-hexosaminidase, in each fraction, using the specific substrate, p-nitrophenyl-β-D-2-acetoamide-2-deoxyglucopyranoside (3.4 mM). The reactions were performed in 67 mM citrate, pH 4.5. The amounts of p-nitrophenol were determined by measuring OD₄₀₅. The percentages of degranulation were calculated; E/(C + E) × 100 (%). PIPES buffer was replaced with N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer (10 mM HEPES-NaOH, pH 7.3 containing 125 mM NaCl, 2.7 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, and 0.1% bovine serum albumin) in the experiments performed under Ca²⁺ free-conditions.

Antigen Stimulation

The cultured mast cells were sensitized with 1 µg/mL an anti-TNP IgE (clone IgE-3) for 3 h in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 50 µM 2-mercaptoethanol, and 0.1 mM non-essential amino acid for 3 h and then twice washed in PIPES buffer to remove unbound free IgE. The cells were then stimulated with 100 ng/mL TNP-BSA in PIPES buffer for 30 min at 37 °C.

Measurement of Cytosolic Ca²⁺ Concentrations

BMMCs were twice washed in Tyrode-HEPES buffer (10 mM HEPES-NaOH, pH 7.4, containing 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, and 0.1% BSA) and incubated for 45 min at room temperature in Tyrode-HEPES buffer containing 2 µM Fura-2AM and 2.5 mM probenecid. The cells were twice washed in Tyrode-HEPES buffer containing 2.5 mM probenecid. The cells were resuspended in Tyrode-HEPES buffer or Ca²⁺-free Tyrode-HEPES buffer (10 mM HEPES-NaOH, pH 7.4, containing 130 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5.6 mM glucose, 0.3 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 0.1% BSA), and the fluorescence values at 510 nm upon alternatively excited at 340 and 360 nm were measured using CAF110 (JASCO, Tokyo, Japan). The relative concentrations of Ca²⁺ were presented as the fold increased.

Immunofluorescence Study

The control and stimulated cells were collected and gently stabilized onto poly L-lysine-coated slide glasses (Matsunami Glass Ind., Kishiwada, Japan) using Cytospin 4 cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, U.S.A.). The cells were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde, 1 mM EGTA, and 2.5 mM MgCl₂ for 30 min and permeabilized with PBS containing 0.1% Triton X-100. The cells were then stained with Phalloidin-iFluor 488 (1 : 500). The fluorescence intensity was measured and calculated using the software, Image J.¹⁵⁾

Statistical Analysis

Statistical significance for comparisons was determined using one-way ANOVA. Additional comparisons were made with Dunnett multiple comparison test for comparison with the control groups or Tukey–Kramer multiple comparison test for all pairs of column comparison. Two-tailed unpaired Student’s t-test was used for comparison between two populations.

RESULTS

Characterization of δ-Toxin-Induced Degranulation in Murine Bone Marrow-Derived Cultured Mast Cells

Degranulation induced by δ-toxin was initially demonstrated using murine bone marrow-derived cultured mast cells and fetal skin-derived mast cells (FSMCs).⁶⁾ We here compared the sensitivity to δ-toxin between IL-3-dependent murine bone BMMCs and CTMCs, which were obtained by co-culturing BMMCs with murine fibroblasts in the presence of SCF. BMMCs were insensitive to compound 48/80 whereas CTMCs underwent degranulation in the presence of compound 48/80 (Fig. 1A). An inhibitor of endoplasmic reticulum Ca²⁺-ATPase, thapsigargin, which was known to induce Ca²⁺ influx through depletion of endoplasmic Ca²⁺, induced degranulation both in BMMCs and CTMCs. A comparable profile of concentration dependency was found in CTMCs stimulated with δ-toxin (Figs. 1B, 1C). In murine FSMCs, an inhibitor of PI3 kinase, LY294002, significantly suppressed δ-toxin-induced degranulation whereas the absence of Syk had no significant effects on it.⁶⁾ As previously reported, LY294002 and an inhibitor of Syk, piceatannol, significantly suppressed antigen-induced degranulation of the sensitized BMMCs and CTMCs. However, neither LY294002 nor piceatannol suppressed δ-toxin-induced degranulation (Figs. 2A–2D). Piceatannol was found rather to enhance δ-toxin-induced degranulation. Pertussis toxin, which has the potential to inactivate a subunit of the trimeric G proteins, G_i/o, significantly suppressed degranulation induced by compound 48/80 but had no effects on that caused by δ-toxin (Figs. 2E, 2F).

Fig. 1. Degranulation of Murine Bone Marrow-Derived Cultured Mast Cells Induced by δ-Toxin

A. BMMCs (open circles) and CTMCs (closed circles) were stimulated with 300 nM thaspsigargin (Thg), or 10 µg/mL compound 48/80 (CP), and the levels of degranulation were determined by measuring β-hexosaminidase activity. B, C. BMMCs (B) or CTMCs (C) were stimulated with the indicated concentrations of δ-toxin (DT) and the levels of degranulation were determined as described above. Bars in A indicate the means.

Fig. 2. Distinct Profiles of the Sensitivity to Inhibitors of Degranulation

The effects of 50 µM LY294002 (A, B), 100 µM piceatannol (C, D), and 100 ng/mL pertussis toxin (E, F) on degranulation induced by 100 µg/mL δ-toxin (DT), IgE-mediated antigen stimulation (IgE/Ag), or 10 µg/mL compound 48/80 (CP) were investigated using BMMCs (A, C, E) or CTMCs (B, D, F). The levels of degranulation were measured without (open circles) or with each treatment. In case of antigen stimulation, the cells were sensitized with an anti-TNP IgE (clone IgE-3, 1 µg/mL) for 3 h and then stimulated with 100 ng/mL TNP-BSA. LY294002 and piceatannol were pretreated 1 h before the stimulation. Pertussis toxin was pretreated 3 h before the stimulation. The values * p < 0.05 and ** p < 0.01 were regarded as significant. Bars indicate the means.

Characterization of Ca²⁺ Mobilization Induced by δ-Toxin

We then investigated the changes in cytosolic Ca²⁺ concentrations in BMMCs and CTMCs stimulated with δ-toxin. Consistent with the previous reports, δ-toxin induced a transient cytosolic Ca²⁺ increase in both kinds of cultured mast cells (Figs. 3A, 3B) as well as thapsigargin in both kinds of cultured mast cells and compound 48/80 in CTMCs. In the absence of extracellular Ca²⁺, δ-toxin did not induce any detectable increases in the cytosolic Ca²⁺, and adding CaCl₂ induced a drastic Ca²⁺ increase (Fig. 3C), indicating that δ-toxin should activate a Ca²⁺ channel on the surface of BMMCs. Ca²⁺ mobilization induced by δ-toxin was sensitive to an inhibitor of phospholipase C, U73122, but not to an inactive analog, U73343 (Figs. 3D, 3E), as well as that induced upon IgE-mediated antigen stimulation.

Fig. 3. Characterization of Ca²⁺ Mobilization Induced by δ-Toxin

Changes of cytosolic Ca²⁺ concentrations were monitored by the fluorescent Ca²⁺ indicator, Fura-2 AM. A, B. The peak levels of cytosolic Ca²⁺ concentrations are presented in BMMCs (A) or CTMCs (B). BMMCs or CTMCs were stimulated with 100 µg/mL δ-toxin (DT), 300 nM thapsigargin (Thg), or 10 µg/mL compound 48/80 (CP). The levels of the baseline fluorescence were presented (C). C. BMMCs were stimulated with vehicle (C), 100 µg/mL δ-toxin (DT), or 100 ng/mL TNP-BSA (Ag) under Ca²⁺-free conditions, and then Ca²⁺ (final concentration: 2 mM) was added. In the case of antigen stimulation, BMMCs were pretreated with an anti-TNP IgE (clone IgE-3, 1 µg/mL) for 3 h. D, E. BMMCs were pretreated with U73122 (5 µM, 12) or U73343 (5 µM, 43) for 15 min and then stimulated with 100 µg/mL δ-toxin or 100 ng/mL TNP-BSA. In the case of antigen stimulation, BMMCs were pretreated with an anti-TNP IgE (clone IgE-3, 1 µg/mL) for 3 h. The results of repeated measurements are presented. The values ** p < 0.01 were regarded as significant. Bars indicate the means.

Ca²⁺-Influx Independent Degranulation Induced by δ-Toxin

We noticed that detectable levels of degranulation were found in BMMCs stimulated with δ-toxin even in the absence of extracellular Ca²⁺, and further investigated this response. Time course studies raised the possibility that the release of β-hexosaminidase induced by δ-toxin could be classified into two phases. The early phase occurred even under the extracellular Ca²⁺-depleted condition. In contrast, the late phase was observed only in the presence of extracellular Ca²⁺ (Fig. 4A). Elevation of extracellular Ca²⁺ concentrations increasingly augmented the levels of degranulation, whereas detectable levels of degranulation were confirmed in the absence of extracellular Ca²⁺ (Fig. 4B). A cytosolic Ca²⁺ chelator, BAPTA, significantly suppressed δ-toxin-induced degranulation in the presence of extracellular Ca²⁺ as well as thapsigargin-induced degranulation but failed to suppress that under Ca²⁺-free condition (Fig. 4C).

Fig. 4. Ca²⁺-Independent Degranulation Induced by δ-Toxin

A. BMMCs were stimulated with 100 µg/mL δ-toxin under Ca²⁺-sufficient (open circles) or Ca²⁺-depleted (closed circles) conditions. BMMCs were sensitized with an anti-TNP IgE (IgE-3, 1 µg/mL) for 3 h and then stimulated with 100 ng/mL TNP-BSA under Ca²⁺-sufficient (open squares) or Ca²⁺-depleted (a closed square) conditions. The values are presented as the means ± standard error of the mean (S.E.M.) (n = 3). The values ** p < 0.01 were regarded as significant. B. BMMCs were stimulated without (open circles) or with 100 µg/mL δ-toxin (closed circles) for 30 min in the presence of indicated concentrations of extracellular Ca²⁺. EGTA (2 mM) was added to exclude the effects of residual Ca²⁺ on the cell surface. Bars indicate the means. C. BMMCs were pretreated with BAPTA-AM (10 µM) for 15 min and then stimulated without (Control), 100 µg/mL δ-toxin (closed circles, DT), or 300 nM thapsigargin (TG) under Ca²⁺-sufficient or Ca²⁺-depleted conditions. The values ** p < 0.01 were regarded as significant. Bars indicate the means.

Effects of Extracellular K⁺ on Degranulation Induced by δ-Toxin

It was reported that δ-toxin has the potential to damage the plasma membrane integrity, leading to the leakage of K⁺.¹⁶⁾ We then investigated the effects of high concentration (140 mM) of extracellular K⁺ on δ-toxin-induced Ca²⁺ mobilization and degranulation. Increases in cytosolic Ca²⁺ concentrations induced by δ-toxin or those induced by thapsigargin were significantly suppressed under high extracellular K⁺ conditions (Figs. 5A, 5B). The levels of δ-toxin-induced degranulation were also significantly decreased under high extracellular K⁺ conditions, whereas δ-toxin-induced degranulation under Ca²⁺-free conditions remained unchanged in the presence of high extracellular K⁺ (Fig. 5C).

Fig. 5. Effects of Extracellular K⁺ on δ-Toxin-Induced Degranulation

A, B. BMMCs were stimulated with 100 µg/mL δ-toxin (DT) or 300 nM thapsigargin (Thg) in the presence of 5 mM (Control, open circles) or 140 mM KCl (High K⁺, closed circles). The peak levels of cytosolic Ca²⁺ concentrations are presented. C. BMMCs were stimulated without or with 100 µg/mL δ-toxin (DT) or 300 nM thapsigargin (Thg) under Ca²⁺-sufficient or Ca²⁺-depleted conditions in the presence of 5 mM (open circles) or 140 mM KCl (closed circles). The values * p < 0.05 and ** p < 0.01 were regarded as significant. Bars indicate the means.

Changes in the Cortical Actin Network

A transient decrease in the amount of filamentous actin (F-actin) was observed in BMMCs during degranulation upon IgE-mediated antigen stimulation, as previously described.¹⁷⁾ In BMMCs treated with δ-toxin, the amount of F-actin was not changed similarly to that in the cells stimulated with the antigens (Figs. 6A, 6B). The cortical actin network was slowly reorganized in the cells stimulated with δ-toxin.

Fig. 6. Reorganization of the Cortical Actin Network

A, B. BMMCs were stimulated with 100 µg/mL δ-toxin (DT, open circles) under Ca²⁺-depleted conditions for the indicated periods. In the case of antigen stimulation, BMMCs were sensitized with an anti-TNP IgE (IgE-3, 1 µg/mL) and then stimulated with 100 ng/mL TNP-BSA (IgE/Ag, closed circles) for the indicated periods. Filamentous actin was visualized using phalloidin-iFluor 488 (A). The mean fluorescence intensity (MFI) was calculated from the obtained images. The values are presented as the means ± S.E.M. (B, n = 3).

Mrgprb2 Is Dispensable for δ-Toxin-Induced Degranulation

An in vitro expression study using HEK293 cells demonstrated that δ-toxin could induce Ca²⁺ mobilization through MRGPRX2.¹⁸⁾ Because Mrgprb2 is regarded as the potent candidate for the murine counterpart of MRGPRX2, we then investigated whether Mrgprb2 should be involved in δ-toxin-mediated degranulation. The lack of degranulation in Mrgprb2^−/− CTMCs stimulated with compound 48/80 indicated the functional deficiency of Mrgprb2 (Fig. 7A). Degranulation induced by δ-toxin remains unchanged in both BMMCs and CTMCs derived from the Mrgprb2^−/− bone marrow cells (Fig. 7B).

Fig. 7. Mrgprb2 Is Not Involved in δ-Toxin-Induced Degranulation

BMMCs and CTMCs generated from the bone marrow cells of the wild type (+/+) and Mrgprb2^−/− mice (−/−) were stimulated with 3 µg/mL compound 48/80 (A, CP) or 100 µg/mL δ-toxin (B, DT) under Ca²⁺-sufficient conditions. Bars indicate the means.

DISCUSSION

It remains unknown how δ-toxin induces degranulation of mast cells. We observed a biphasic degranulation in murine cultured mast cells stimulated with δ-toxin. The early phase occurred even in the absence of the extracellular Ca²⁺ and in the presence of a Ca²⁺-chelator, BAPTA. The levels of degranulation during the late phase were dependent on extracellular Ca²⁺. Nishida et al. demonstrated that the translocation of secretory granules towards the plasma membrane was dependent on microtubules and was independent of Ca²⁺ in BMMCs upon IgE-mediated antigen stimulation,¹⁹⁾ but it is generally accepted that a cytosolic increase in Ca²⁺ concentrations is essential for the granule fusion. Koffer et al. demonstrated that cytochalasin E, an inhibitor of actin polymerization, could induce degranulation in the presence of EGTA in streptolysin-O-permeabilized rat peritoneal mast cells.²⁰⁾ This result raised the possibility that actin depolymerization could trigger degranulation in the absence of Ca²⁺. In the absence of extracellular Ca²⁺, a slow but steady collapse of filamentous actin was observed in BMMCs stimulated with δ-toxin in this study. This actin reorganization may be involved in Ca²⁺-independent degranulation.

Because δ-toxin has a moderate membrane-damaging activity,¹⁶⁾ leakage of K⁺ may occur in the presence of high concentrations of δ-toxin. Leakage of K⁺ leads to activation of nucleotide binding and oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, which induces pyroptosis.²¹⁾ Recently, degranulation during pyroptosis was reported in a rat basophilic mast cell line, RBL-2H3.²²⁾ Pyroptosis induced by sodium sulfate brought about degranulation, which was suppressed by an NLRP3 inhibitor, MCC950. Mencarelli et al. demonstrated that the inflammasome components, NLRP3 and apoptosis-associated speck-like protein containing a caspase activating and recruitment domain (ASC), should be required for degranulation induced upon IgE-mediated antigen stimulation.²³⁾ These findings suggest that activation of NLRP3 inflammasome is involved in degranulation of mast cells. In this study, both Ca²⁺ influx and Ca²⁺-dependent degranulation were suppressed in the presence of high concentrations of K⁺, but Ca²⁺-independent degranulation remained unchanged. Because more than 50 mM of K⁺ was found to prevent activation of NLRP3 inflammasome,²⁴⁾ NLRP3 inflammasome might not be involved in Ca²⁺-independent degranulation induced by δ-toxin.

Nakamura et al. showed complicated results regarding the involvement of FPRs; FPR antagonists, such as WRW4 and cyclosporin H, could inhibit the degranulation induced by δ-toxin, whereas the agonists, such as MMK1 and lipoxin A4 did not induce degranulation.⁶⁾ Furthermore, δ-toxin evoked degranulation in BMMCs derived from the FPR2^−/− bone marrow. FPRs are known to be coupled with pertussis toxin-sensitive trimeric G proteins, but we found that δ-toxin-induced degranulation was insensitive to pertussis toxin. Sinniah et al. reported that the effects of mast cell stabilizers, such as nedocromil and ketotifen, were abrogated in the absence of annexin A1, which was found to suppress degranulation in collaboration with FPR2.²⁵⁾ Annexin A1 and FPR2 were found to exert anti-inflammatory responses in the model of the ocular allergy.²⁶⁾ Based on these findings, FPR2 might not be involved in δ-toxin-induced degranulation.

The intracellular signaling pathway activated by δ-toxin remains largely unknown. We observed that a Syk inhibitor, piceatannol, rather enhanced degranulation induced by δ-toxin. Nakamura et al. demonstrated that δ-toxin-induced degranulation occurred in the cultured mast cells derived from the fetal skin of Syk^−/− mice.⁶⁾ An early study using a rat mutant mast cell line lacking Syk reported that histamine release induced by IgE-mediated antigen stimulation was abrogated, but that induced by a calcium ionophore, A23187, was intact.²⁷⁾ Phosphorylation of Akt, extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK), was attenuated in Syk^−/− BMMCs activated upon IgE-mediated antigen stimulation, whereas that induced by adenosine remains unchanged.²⁸⁾ These findings indicate that Syk should not be necessary for IgE-independent degranulation of mast cells. Cox et al. demonstrated that Syk should mediate immunoreceptor tyrosine-based activated motif-mediated actin assembly.²⁹⁾ Inhibition of Syk may enhance actin reorganization induced by δ-toxin.

MRGPRX2 was found to be responsible at least in part for IgE-independent degranulation of human mast cells.¹²⁾ Azimi et al. reported that δ-toxin could induce Ca²⁺ mobilization in HEK293 cells expressing MRGPRX2.¹⁸⁾ One of the potential murine homologs of MRGPRX2 is Mrgprb2. However, δ-toxin-induced degranulation was unchanged in both BMMCs and CTMCs derived from the Mrgprb2^−/− bone marrow. Because MRGPRX2 agonist-mediated degranulation was found to be sensitive to pertussis toxin in most cases, δ-toxin-induced degranulation in murine mast cells may occur independently of Mrgpr subtypes.

We identified here the Ca²⁺-independent phase of degranulation in murine bone marrow-derived cultured mast cells stimulated with δ-toxin, although the molecular targets of δ-toxin remain unknown.

Acknowledgments

This research was funded by Grants from the JSPS KAKENHI Grant Numbers: 26670029 and 16K08231.

Conflict of Interest

The authors declare no conflict of interest.

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