Lovastatin decreases acute mucosal inflammation via 15-epi-lipoxin A4

Abstract

The widespread use of statins for hypercholesterolemia has uncovered pleiotropic anti-inflammatory properties that were unexpected based on the drugs' original design; yet, mechanisms for these protective actions remain uncertain. In this study lovastatin triggered biosynthesis of the anti-inflammatory and pro-resolving mediator 15-epi-lipoxin A(4) (15-epi-LXA(4)). During interactions between human neutrophils and airway epithelial cells, the statin-induced increase in 15-epi-LXA(4) was associated with increased 14,15-epoxyeicosatrienoic acid (14,15-EET) generation. When added to activated neutrophils, 14,15-EET enhanced 15-epi-LXA(4) biosynthesis. In a murine model of airway mucosal injury and inflammation, lovastatin increased 15-epi-LXA(4) formation in vivo and markedly decreased acute lung inflammation. Administration of 15-epi-LXA(4) also inhibited lung inflammation in an additive manner with lovastatin. Together, these results indicate that statin-triggered 15-epi-LXA(4) generation during human leukocyte-airway epithelial cell interactions is an endogenous mechanism for statin-mediated tissue protection at mucosal surfaces that may also be relevant in the statins' ability to stimulate the resolution of inflammation.

Figure 1. Lovastatin triggers 15-epi-LXA₄ biosynthesis by activated human airway epithelial cells and PMNs

(A) 15-epi-LXA₄ levels were determined after Calu-3 cells were exposed to TNF-α (1 ng/ml, 24h, 37°C) and then to human PMNs in the presence or absence of lovastatin (1 μM, 30 min, 37°C) (see Methods). (B) Dose dependent relationship between PMNs relative to Calu-3 cells was determined for 15-epi-LXA₄ generation by lovastatin exposed cells. (C) Select incubations with PMNs and Calu-3 cells (5:1 ratio) were carried out in the presence of lovastatin (1 μM), mevalonate (100 μM), simvastatin (10 μM), NDGA (LO inhibitor, 5 μM) or vehicle (0.1% ethanol). (D) Representative RT-PCR for pivotal lipoxin biosynthetic genes in Calu-3 cells (without and with exposure to TNF-α), freshly isolated human PMNs, and NHBE exposed to IL-13 (10 ng/ml, 96h, 37°C). Results are expressed as the mean ± SEM for n ≥ 3 independent experiments. *P < 0.05 by ANOVA, n.s. = not significant.

Figure 2. Lovastatin and the sEH inhibitor AUDA increase 14,15-EET and 15-epi-LXA₄ formation

Neutrophils (PMNs) were incubated with TNF-α-exposed Calu-3 cells (5:1, PMNs:Calu-3) in the presence of lovastatin (1 μM) and in some cases AUDA (1 μM) for 30 min (37°C). Lipids were extracted and analyzed by RP-HPLC using (A) absorbance at 301 nm (Inset, UV spectrum for (i.) non-specific materials at 13.3 min and (ii.) for 15-epi-LXA₄ at 13.7 min) or (B) charged aerosol detector (CAD) (see Methods). Authentic materials are shown in the upper panels. (C,D) Concentration response to AUDA (0.01 – 1 μM) on statin-triggered 15-epi-LXA₄ formation relative to (C) vehicle and (D) 14,15-EET by TNF-α-exposed Calu-3 cells and PMNs. 15-epi-LXA₄ levels were determined by ELISA. Results are expressed as mean ± SEM (n = 3 independent experiments). *P < 0.05 relative to no AUDA and **P < 0.05 compared to 14,15-EET by ANOVA.

Figure 3. Regiospecific influence of 14,15-EET on 15-epi-LXA₄ biosynthesis by activated human PMNs

Freshly isolated human PMNs were activated with A23187 in the presence of EETs (1 μM) or vehicle (0.1% ethanol). After extraction, 15-epi-LXA₄ levels were measured by (A) RP-HPLC (Authentic 15-epi-LXA₄ is shown in the upper panel) or (B) ELISA (see Methods). Concentration response for 14,15-EET (10 – 10,000 pmoles) on (C) 15-epi-LXA₄ and (D) LTB₄ formation. Values represent the mean ± SEM for n ≥ 3. *P < 0.05 by Student’s t-test.

Figure 4. Lovastatin promotes 15-epi-LXA₄ formation in vivo and ALI resolution

Lovastatin (0.2 or 2 mg/kg Statin) or vehicle (0.9% saline) were given i.v. 15 min before HCl-initiated ALI and BALFs were obtained 18h later. (A) Total BALF cells were enumerated, and (B) the number of BALF macrophages (Mϕ) and neutrophils (PMN) were determined (see Methods). (C) Immunostaining for Ly-6G (1:50 dilution) in murine lung tissue obtained 18h after acid-induced ALI in the absence (upper panels) or presence of lovastatin (lower panels). PMNs are highlighted by arrows and original magnifications are indicated. (D) 15-epi-LXA₄ levels were determined by ELISA in BALFs. For purposes of direct comparison, resolution of lung leukocyte infiltration at 18h was determined by monitoring (E) total BALF leukocytes and (F) BALF PMNs in mice administered (2 mg/kg, i.v., 100 μl) lovastatin, 15-epi-LXA₄, 14,15-EET, the combination of 15-epi-LXA₄ and lovastatin (2 mg/kg each) or vehicle (1% ethanol) 15 min prior to intratracheal acid. Values represent the mean ± SEM (n ≥ 4 from at least 3 independent experiments). *P < 0.05 compared to no acid or statin, **P < 0.05 compared to vehicle and #P < 0.05 compared to 15-epi-LXA₄ alone.