Mast cells contribute to the resolution of allergic inflammation by releasing resolvin D1

Abstract

Background: Mast cells are initiators and main effectors of allergic inflammation, together with eosinophils, with whom they can interact in a physical and soluble cross-talk with marked pro-inflammatory features, the Allergic Effector Unit. The pro-resolution role of mast cells, alone or in co-culture with eosinophils, has not been characterized yet.

Objectives: We aimed to investigate select pro-resolution pathways in mast cells in vitro and in vivo in allergic inflammation.

Methods: In vitro, we employed human and murine mast cells and analyzed release of resolvin D1 and expression of 15-lipoxygenase after IgE-mediated activation. We performed co-culture of IgE-activated mast cells with peripheral blood eosinophils and investigated 15-lipoxygenase expression and Resolvin D1 release. In vivo, we performed Ovalbumin/Alum and Ovalbumin/S. aureus enterotoxin B allergic peritonitis model in Wild Type mice following a MC "overshoot" protocol.

Results: We found that IgE-activated mast cells release significant amounts of resolvin D1 30 min after activation, while 15-lipoxygenase expression remained unchanged. Resolvin D1 release was found to be decreased in IgE-activated mast cells co-cultured with peripheral blood eosinophils for 30 min In vivo, mast cell-overshoot mice exhibited a trend of reduced inflammation, together with increased peritoneal resolvin D1 release.

Conclusions: Mast cells can actively contribute to resolution of allergic inflammation by releasing resolvin D1.

Keywords: Allergic inflammation; Allergic peritonitis; Eosinophils; Mast cells; Resolution; Resolvin D1.

Figure 1.

Human mast cells release resolvin D1. Resolvin D1 (RvD1) production from preincubated with 20 μM docosahexaenoic acid cord blood-derived mast cells (CBMCs) (A), laboratory of allergic diseases 2 (LAD-2) cells (B), foreskin-derived mast cells (FSMCs) (C) and nasal polyp-derived mast cells (NPMCs) (D) was determined 30 min (30’) or 1h after immunoglobulin E-mediated activation (aIgE) and in non-activated (NA) cells (***p<0.01). Data are expressed as mean ± SEM of three independent experiments.

Figure 2.. 15-lipoxygenase expression in mast cells is not modulated after cell activation.

RT-PCR analysis of Alox15 (A) and Alox5 (B) expression in immunoglobulin E-activated (αIgE) and not-activated (NA) laboratory of allergic diseases 2 (LAD-2) cells, preincubated with 20 μM docosahexaenoic acid (DHA), 30 min, 1.5 and 24 h after activation. (*p<0.05, ***p<0.001). (C) Representative histogram of 15-lipoxygenase protein expression in cord blood-derived mast cells (CBMCs) preincubated with 20 μM docosahexaenoic acid (DHA) 30 min after IgE-mediated activation. RT-PCR analysis of Alox15 (D) and Alox5 (E) expression in IgE-activated and not-activated foreskin-derived mast cells (FSMCs), preincubated with 20 μM docosahexaenoic acid (DHA), 1h after activation. Data are expressed as mean ± SEM of three independent experiments.

Figure 3.. Mast cell-eosinophil co-culture modulates resolvin D1 production but not 15-lipoxygenase expression in mast cells.

(A) Resolvin D1 (RvD1) production (n=3), (B) Intracellular flow cytometry staining of 15-lipoxygenase, (C) tryptase release (n=3), and (D) eosinophil peroxidase (EPX) release from immunoglobulin E-activated (aIgE) cord blood-derived mast cells (CBMCs) and their respective non activated (NA) counterparts, alone and in co-culture with NA peripheral blood eosinophils (pbEos). (E) Eosinophil peroxidase (EPX) release from peripheral blood eosinophils (Eos) after 1h incubation with 1000 nM resolvin D1 (RvD1). Activation of pbEos was assessed following incubation in the presence of 10⁻⁶ M platelet activating factor (PAF). All measurements were performed 30 min after cell activation. Data are expressed as mean ± SEM of three independent experiments. (*p<0.05, **p<0.01; ***p<0.001).

Figure 4.. Release of resolvin D1 from bone marrow-derived mast cells is increased by FcεRI cross-linking, while expression of 15-lipoxygenase is not modulated by cell activation.

Resolvin D1 (RvD1) release (A), 15-lipoxygenase (Alox15) expression (B) and tryptase release (C) from IgE-activated (DNP-BSA) and not-activated (NA) bone marrow-derived mast cells 1h after activation (BMMCs) (*p<0.05; **p<0.01) Data are expressed as mean ± SEM of three independent experiments.

Figure 5.. Bone marrow-derived mast cells release tryptase and resolvin D1 1h after intraperitoneal injection in ovalbumin/Staphylococcus aureus enterotoxin B-challenged mice.

(A) Schematic representation of the model. Tryptase (B) and resolvin D1 (RvD1) (C) levels in the peritoneal lavages of wild type (WT) mice challenged with either PBS, ovalbumin and Staphylococcus aureus enterotoxin B (SEB) (OVA/SEB) or OVA/SEB plus bone marrow-derived mast cells (OVA/SEB-BMMCs) 1h after injection of BMMCs and 3d after OVA/SEB challenge (n=4 mice/group, ***p<0.001). Data are expressed as mean ± SEM.

Figure 6.. Mast cell overshoot mice show a trend of reduced inflammation in an ovalbumin/Staphylococcus aureus enterotoxin B-induced allergic peritonitis model.

(A) Schematic representation of the model. Total cells (B), eosinophils (Eos) (C), mast cells (MCs) numbers (D) and resolvin D1 (RvD1) levels (E) in the peritoneal lavages of wild type (WT) mice challenged with either PBS, ovalbumin and Staphylococcus aureus enterotoxin B (SEB) (OVA/SEB) or OVA/SEB plus bone marrow-derived mast cells (OVA/SEB-BMMCs)). Data are expressed as mean ± SEM of three independent experiments (3-6 mice/group/experiment, **p<0.01, ***p<0.001).