Structural requirements for the inhibition of calcium mobilization and mast cell activation by the pyrazole derivative BTP2

Abstract

Mast cells play a critical role in the development of the allergic response. Upon activation by allergens and IgE via the high affinity receptor for IgE (FcɛRI), these cells release histamine and other functional mediators that initiate and propagate immediate hypersensitivity reactions. Mast cells also secrete cytokines that can regulate immune activity. These processes are controlled, in whole or part, by increases in intracellular Ca(2+) induced by the FcɛRI. We show here that N-(4-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2), a pyrazole derivative, inhibits activation-induced Ca(2+) influx in the rat basophil cell line RBL-2H3 and in bone marrow-derived mast cells (BMMCs), without affecting global tyrosine phosphorylation of cellular proteins or phosphorylation of the mitogen-activated protein kinases Erk1/2, JNK and p38. BTP2 also inhibits activation-induced degranulation and secretion of interleukin (IL)-2, IL-3, IL-4, IL-6, IL-13, tumor necrosis factor (TNF)-α, and granulocyte macrophage-colony stimulating factor (GM-CSF) by BMMCs, which correlates with the inhibition of Nuclear Factor of Activated T cells (NFAT) translocation. In vivo, BTP2 inhibits antigen-induced histamine release. Structure-activity relationship analysis indicates that substitution at the C3 or C5 position of the pyrazole moiety on BTP2 (5-trifluoromethyl-3-methyl-pyrazole or 3-trifluoromethyl-5-methyl-pyrazole, respectively) affected its activity, with the trifluoromethyl group at the C3 position being critical to its activity. We conclude that BTP2 and related compounds may be potent modulators of mast cell responses and potentially useful for the treatment of symptoms of allergic inflammation.

Figure 1. BTP2 blocks intracellular calcium mobilization in RBL-2H3 cells and BMMCs

(A) RBL-2H3 cells were pre-treated with BTP2 (1 μM) or vehicle (DMSO) for 30 min, then analyzed for calcium responses as described in the materials and methods following stimulation with 500 nM ionomycin in the presence of CaCl₂ for the indicated time period. [Ca²⁺]_i was expressed as ratiometric Fura-2AM, and representative data of 3–5 independent experiments is shown (left panel). Quantification of mean calcium increase (middle panel); peak calcium increase (right panel), with data expressed as the mean ± SEM of 3–5 independent experiments. *p<0.05 vs. Vehicle. (B) BMMCs were pre-treated with BTP2 (1 μM) or vehicle for 30 min, then stimulated with mouse anti-DNP IgE and rat anti-mouse IgE (10 μg/mL), and analyzed as in (A), except that [Ca²⁺]_i was expressed as Fluo-4 relative fluorescence with representative data of 3–5 independent experiments shown (left panel). Quantification of mean calcium increase (middle panel); peak calcium increase (right panel) as in (A). Data are expressed as the mean ± SEM of 3–5 independent experiments. *p<0.05 vs. Vehicle. (C) BMMCs were pre-treated with BTP2 (1 μM) or vehicle for 30 min, then stimulated with 100 ng DNP-HSA and analyzed as in (A), except that [Ca²⁺]_i was expressed as Fluo-4 relative fluorescence with representative data of 3–5 independent experiments shown (left panel). Quantification of mean calcium increase (middle panel); peak calcium increase (right panel) as in (A). Data are expressed as the mean ± SEM of 3–5 independent experiments. *p<0.05 vs. Vehicle.

Figure 2. BTP2 does not affect tyrosine kinase nor MAP kinase activation following FcεRI triggering

BMMCs were treated with either BTP2 (1 μM) or vehicle (DMSO) for 30 min and then stimulated with mouse IgE anti-DNP and DNP-HSA for the indicated time periods, lysed, and analyzed by western blot for (A) total phosphotyrosine; (B) phospho-ERK; (C) phospho-JNK; (D) phospho-p38; or (E) c-Fos expression. Blots were stripped and reprobed with control antibodies for β-actin. Numbers indicate fold increase corrected for expression levels.

Figure 3. BTP2 inhibits mast cell degranulation in vitro and FcεRI-mediated histamine release in vivo

(A) RBL-2H3 cells were pretreated with BTP2 or vehicle (DMSO) at 1 μM and then stimulated with ionomycin (500 nM) for 30 minutes. (B) RBL-2H3 cells were pretreated with vehicle (DMSO) or BTP2 at the indicated concentrations for 30 minutes then stimulated as in (A). (C) RBL-2H3 cells were pretreated with BTP2 (1 μM) or vehicle for 30 min, then stimulated with mouse IgE anti-OVA and rat anti-mouse IgE for 30 min. All cells were analyzed for degranulation via an annexin-V binding assay. Data are expressed as the mean ± SEM of 3 independent experiments. *p<0.05 vs. Vehicle. (D) C57BL/6 mice were sensitized with mouse IgE anti-DNP overnight, then treated with BTP2 (BTP2, 10 mg/kg) or vehicle (DMSO) for 1 hour. Mice, treated with BTP2 or vehicle, were then challenged with DNP-HSA or PBS (as a control) for 3 min. Serum histamine concentration levels were then determined and data are expressed as the mean ± SEM, n=8 except for control mice treated with BTP and no antigen, n=3. *p<0.05 vs. Vehicle.

Figure 4. BTP2 inhibits cytokine secretion of PMA/ionomycin-activated BMMCs

BMMCs were pretreated with BTP2 (1 μM) or vehicle (DMSO) for 30 min, then stimulated with PMA and ionomycin. 8 and 24 hours after stimulation, the concentration of the indicated cytokines were determined. Data are expressed as the mean ± SEM of technical non-independent replicates, representative of 3 independent experiments. *p<0.05 vs. Vehicle.

Figure 5. BTP2 inhibits cytokine secretion of IgE/anti-IgE-activated mast cells

BMMCs were sensitized with mouse anti-DNP IgE, pretreated with BTP2 (1 μM) or vehicle (DMSO) for 30 min, and then stimulated rat anti-mouse IgE. 8 and 24 hours after stimulation, the concentration of the indicated cytokines were determined. Data are expressed as the mean ± SEM of technical non-independent replicates, representative of 3 independent experiments. *p<0.05 vs. Vehicle.

Figure 6. BTP2 inhibits cytokine secretion of IgE/antigen-mediated activation of mast cells

BMMCs were sensitized with mouse anti-DNP IgE, pretreated with BTP2 (1 μM) or vehicle (DMSO) for 30 min, and then stimulated with DNP-HSA (100 ng/mL). 8 and 24 hours after stimulation, the concentration of the indicated cytokines were determined. Data are expressed as the mean ± SEM of technical non-independent replicates, representative of 3 independent experiments. *p<0.05 vs. Vehicle.

Figure 7. BTP2 inhibits stimulus-induced NFAT nuclear translocation

(A) RBL-2H3 cells were pretreated with BTP2 (1 μM) or vehicle (DMSO) for 30 min prior to stimulation for 45 min with ionomycin or (B) mouse anti-DNP IgE and DNP-HSA (100 ng/mL). Cells were fixed and permeabilized, and NFATc1 localization determined by confocal microscopy. The nucleus was identified using TO-PRO-3 staining, and nuclear localization of NFAT is evident by the yellow nuclear staining (red plus green). White bars indicate 40 μm. Representative data of 3–5 independent experiments is shown.

Figure 8. BTP2 does not affect preformed cytokine mRNA expression

BMMCs were pretreated with BTP2 (1 μM) or vehicle (DMSO) for 2 days and levels of IL-4, IL-6, and TNF-α were quantified. Data are expressed as the mean ± SEM of duplicate experiments.

Figure 9. The 3-trifluoromethyl group is critical for BTP2 activity

(A) Structures of BTP2 and derivatives (BTP1; BTP2; Pyrazole: Pyr; 3-trifluoromethyl-5-methyl-pyrazole: 3T5M-P; 3-trifluoromethyl-pyrazole: 3T-P; 5-trifluoromethyl-3-methyl-pyrazole: 5T3MP; 5-trifluoromethyl-methyl-pyrazole: 5T-P; 3-methyl-5-methyl-pyrazole: 3M5M-P). (B) BMMCs were pretreated with vehicle, 2-APB (at 50 μM), or BTP analogs (at 1 μM) and then stimulated with ionomycin. Degranulation was determined as indicated in materials and methods. (C) RBL-2H3 cells were pretreated with vehicle or the indicated BTP analogs at 1 μM (left panel) or 3T5M-P at the indicated concentrations (right panel). Cells were then stimulated with ionomycin and degranulation determined as in figure 3. (D) BMMCs were pretreated with 3T5M-P (1 μM) or vehicle for 30 min, then stimulated with mouse IgE anti-DNP and DNP-HSA (100 ng/mL). Calcium responses were determined as in figure 1. Representative data of 3–6 experiments is shown. (E) Mean calcium increase. Data are expressed as the mean ± SEM of 3 independent experiments. Data are expressed as the mean ± SEM of 3 independent experiments. *,**p< 0.05 vs. vehicle.