Palmitoylethanolamide Supplementation during Sensitization Prevents Airway Allergic Symptoms in the Mouse

Abstract

One important risk factor for the development of asthma is allergen sensitization. Recent increasing evidence suggests a prominent role of mast cells in asthma pathophysiology. Since Palmitoylethanolamide (PEA), an endogenous lipid mediator chemically related to - and co-released with- the endocannabinoid anandamide, behaves as a local autacoid down-regulator of mast cell activation and inflammation, we explored the possible contribution of PEA in allergic sensitization, by using ovalbumin (OVA) as sensitizing agent in the mouse. PEA levels were dramatically reduced in the bronchi of OVA-treated animals. This effect was coupled to a significant up-regulation of CB₂ and GPR55 receptors, two of the proposed molecular PEA targets, in bronchi harvested from allergen-sensitized mice. PEA supplementation (10 mg/kg, 15 min before each allergen exposure) prevented OVA-induced bronchial hyperreactivity, but it did not affect IgE plasma increase. On the other hand, PEA abrogated allergen-induced cell recruitment as well as pulmonary inflammation. Evaluation of pulmonary sections evidenced a significant inhibitory action of PEA on pulmonary mast cell recruitment and degranulation, an effect coupled to a reduction of leukotriene C₄ production. These findings demonstrate that allergen sensitization negatively affects PEA bronchial levels and suggest that its supplementation has the potential to prevent the development of asthma-like features.

Keywords: airway inflammation; allergen sensitization; bronchial hyperreactivity; mast cells; palmitoylethanolamide.

FIGURE 1

PEA levels in OVA sensitized mice. (A) Mice were injected with 0.4 ml s.c. of a suspension containing 100 μg of OVA absorbed to 3.3 mg of aluminium hydroxide gel (OVA) or vehicle (control) on days 1 and 8. Bronchi were harvested and analyzed 15 days after vehicle or OVA administration. PEA levels in the bronchi (B) and in plasma (C) were measured by liquid chromatography-mass spectrometry and expressed as pmol/mg tissue and pmol/ml, respectively. Data are expressed as mean ± SEM, n = 6 animals for each group; ^∗P < 0.05 vs. control; one-tailed Student T-test.

FIGURE 2

Expression of PEA targets in OVA sensitized mice. Mice were injected with 0.4 ml s.c. of a suspension containing 100 μg of OVA absorbed to 3.3 mg of aluminium hydroxide gel (OVA) or vehicle (control) on days 1 and 8. Bronchi were harvested and analyzed 15 days after vehicle or OVA administration. mRNA expression of (A) CB₂, (B) GPR55, (C) CB₁, and (D) PPAR-α was evaluated by RT-PCR. Results are calculated as fold expression (mRNA expression). The lower expression values were 32.42 Cq (Control) background N/A, 32.34 Cq (Control) background N/A, 30.60 Cq (Control) background N/A, 27.96 Cq (OVA) background 36.55 Cq for CB₂, GPR55, CB₁ and PPAR-α, respectively. Data are expressed as mean ± SEM, n = 6 animals for each group; ^∗∗∗P < 0.001 vs. control; two-tailed Student T-test.

FIGURE 3

Effect of PEA on OVA-induced bronchial hyperreactivity. (A) Scheme of sensitization and drug treatment. Mice were injected with 0.4 ml s.c. of a suspension containing 100 μg of OVA absorbed to 3.3 mg of aluminium hydroxide gel or vehicle (control) on days 1 and 8. After 22 days after OVA sensitization mice were sacrificed. PEA (10 mg/kg) was administered i.p. 15 min before each OVA administration. (B) Bronchial reactivity to carbachol was evaluated 22 days after OVA injection. (C) Carbachol-induced contractions of bronchi harvested from both control and OVA sensitized-mice in presence or absence of PEA (10^-5 M). Data are expressed as means ± SEM, n = 6 animals for each group. Concentration-response curves (B,C) have been generated by a non-linear regression analysis.^∗∗∗p < 0.001; two-ways ANOVA plus Bonferroni.

FIGURE 4

Effect of PEA on OVA-induced inflammation in air pouch. (A) Scheme of air pouch model. Animals were injected with 0.4 ml s.c. of a suspension containing 100 μg of OVA absorbed to 3.3 mg of aluminium hydroxide gel (OVA) on days 1 and 8. Then they received at days 9 and 12 on the shaved dorsal surface 2.5 ml s.c. of air. On day 15 mice were challenged by injection into the air-pouch with 0.4 ml of sterile saline alone (control) or containing 10 μg OVA. PEA (10 mg/kg) was administered i.p. 15 min before each OVA administration. (B) Cell recruitment, (C) IL-4 and (D) IL-13 were quantified in lavage fluid of air pouch 24 h (cell recruitment) and 2 h (IL-4 and IL-13) after of OVA challenge, respectively. Data are expressed as means ± SEM, n = 5–6 animals for each group; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; one-way ANOVA plus Bonferroni.

FIGURE 5

Effect of PEA on pulmonary inflammation induced by OVA sensitization. (A) Scheme of sensitization and drug treatment. Mice were injected with 0.4 ml s.c. of a suspension containing 100 μg of OVA absorbed to 3.3 mg of aluminium hydroxide gel or vehicle (control) on days 1 and 8. After 15 days after OVA sensitization mice were sacrificed. PEA (10 mg/kg) was administered i.p. 15 min before each OVA administration. (B) H&E staining of lung tissues. (C) IgE plasma levels as well as (D) IL-13 and (E) IL-4 pulmonary levels were quantified by ELISA. Data are expressed as means ± SEM, n = 6–10 animals for each group; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; one-way ANOVA plus Bonferroni.

FIGURE 6

Effect of PEA on pulmonary mast cell recruitment and degranulation induced by OVA sensitization. PEA (10 mg/kg) was administered i.p. 15 min before each OVA administration. Mast cell recruitment and degranulation was evaluated by Toluidine blue staining in pulmonary sections obtained from OVA sensitized mice (C,D), OVA sensitized mice pre-treated with PEA (E,F) and control mice (A,B). Quiescent mast cells were dark blue stained (black arrows) while degranulated mast cells were red purple/violet stained (red arrows). (B,D,F) Are views at higher magnification (100×) of (A,C,E) (40×).

FIGURE 7

Effect of PEA on pulmonary mast cell activation induced by OVA sensitization. PEA (10 mg/kg) was administered i.p. 15 min before each OVA administration. (A) Quantification of mast cell recruitment 15 days after OVA sensitization. (B) Percentage of mast cell degranulation evaluated as ratio between degranulated and non-degranulated mast cells. (C) LTC₄ levels were quantified in lung tissue by ELISA. Data are expressed as means ± SEM, n = 6; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; one-way ANOVA plus Bonferroni.