Leukotriene A4 Hydrolase Activation and Leukotriene B4 Production by Eosinophils in Severe Asthma

Abstract

Asthma is associated with the overproduction of leukotrienes (LTs), including LTB4. Patients with severe asthma can be highly responsive to 5-lipoxygenase (5-LO) inhibition, which blocks production of both the cysteinyl LTs and LTB4. Production of LTB4 has traditionally been ascribed to neutrophils, mononuclear phagocytes, and epithelial cells, and acts as a chemoattractant for inflammatory cells associated with asthma. The source of LTB4 is unclear, especially in eosinophilic asthma. We speculated that the benefit of 5-LO inhibition could be mediated in part by inhibition of eosinophil-derived LTB4. LTB4 concentrations were assayed in BAL fluid from patients with severe asthma characterized by isolated neutrophilic, eosinophilic, and paucigranulocytic inflammation. Expression of LTA4 hydrolase (LTA4H) by airway eosinophils was determined by immunohistochemistry (IHC). Subsequently, peripheral blood eosinophils were activated and secreted LTB4 was quantified by enzyme immunoassay. Blood eosinophil LTA4H expression was determined by flow cytometry, qPCR, and IHC. LTB4 concentrations were elevated in BAL fluid from patients with severe asthma, including those with isolated eosinophilic inflammation, and these eosinophils displayed LTA4H via IHC. LTA4H expression by blood eosinophils was confirmed by flow cytometry, IHC, and qPCR. Robust LTB4 production by blood eosinophils was observed in response to some, but not all, stimuli. We demonstrated that eosinophils express LTA4H transcripts and protein, and can be stimulated to secrete LTB4. We speculate that in many patients with asthma, eosinophil-derived LTB4 is increased, and this may contribute to the efficacy of 5-LO inhibition.

Keywords: asthma; eosinophils; leukotrienes; neutrophils.

Figure 1.

Leukotriene B4 (LTB4) levels in BAL fluid from patients with severe asthma and different granulocyte patterns. BAL was collected during bronchoscopy of individuals with severe treatment-resistant asthma, and enzyme immunoassays were run to measure LTB4 concentrations. Samples were analyzed based on BAL granulocyte patterns and LTB4 levels (presented as pg/ml). Data are presented as individual data points along with the mean ± SEM for each group. *P < 0.005 as compared with neutrophilic.

Figure 2.

Immunofluorescence for LTA4 hydrolase (LTA4H) in BAL eosinophils. LTA4H staining of BAL eosinophils using a primary antibody directed against LTA4H and an APC-labeled secondary antibody (red) with DAPI nuclear stain (yellow). The inset (i) shows a close-up view, with white arrows indicating eosinophils.

Figure 3.

Flow cytometry for eosinophils and LTA4H. (A) Purified eosinophils were analyzed for neutrophil contamination by measuring CD16b expression by flow cytometry; results are shown in a representative histogram plot. (B) Eosinophil expression of LTA4H and LTC4 synthase (LTC4S) was determined by examining Siglec 8⁺ eosinophils for 5-lipoxygenase (5-LO) expression, and within that population the percentage of cells that expressed these molecules. Circles approximate the Siglec 8⁺ population that was gated upon to identify cells that were also positive for 5-LO and similarly the 5-LO⁺ cells that were analyzed for percent positive for LTA4H or LTC4S. Eos = eosinophil; Iso = isotype; PMN = polymorphonuclear neutrophil; SSC = side scatter.

Figure 4.

Immunofluorescence for LTA4H in purified blood eosinophils. Eosinophils were purified from blood by Ficoll-Hypaque density centrifugation and magnetic bead affinity column purification. Staining of LTC4S was performed using an Alexa Fluor 647–labeled antibody directed against LTC4S (green), LTA4H staining was performed using an Alexa Fluor 555–labeled antibody directed against LTA4H (red), and DAPI was used as a nuclear stain (blue). (A and B) Note that all eosinophils are positive for expression of LTC4S (A) and most, but not all, are positive for LTA4H (B). (C and D) A merged image is shown (C), and the isotype control is included to show background staining (D). Arrows represent dual positive staining eosinophils for LTC4S and LTA4H. Scale bars: 10 μM.

Figure 5.

LTA4H mRNA expression in eosinophils. After separation of blood using Ficoll-Hypaque density centrifugation and dextran sedimentation, eosinophils were enriched using magnetic affinity column purification. Transcript levels of LTA4H were quantified using PCR with SYBR Green detection. Data (mean ± SEM) reflect the relative expression of each gene in comparison with the housekeeping gene β-actin (∆C_T). Control samples (n = 12) are depicted by solid triangles, and asthma samples are indicated (n = 17) by open circles.

Figure 6.

LTB4 release from aspirin-activated eosinophils. Eosinophils isolated from peripheral blood of control subjects (open bars: n = 12) and patients with asthma (solid bars: n = 11) were activated with lysine aspirin (LysASA; 10 mM), the combination of ionomycin (2 μg/ml) with low (L; 33 ng/ml) or high (H; 100 ng/ml) dose phorbol 12-myristate 13-acetate (PMA), trypsin 1 μM, or platelet-activating factor (PAF) 1 μM for 30 minutes. Supernatants were collected and LTB4 levels were quantified by enzyme immunoassay (pg/10⁵ cells). Data are presented as mean ± SEM with *P < 0.001 or **P < 0.02 in comparison with unstimulated cells.