The Step Further to Understand the Role of Cytosolic Phospholipase A2 Alpha and Group X Secretory Phospholipase A2 in Allergic Inflammation: Pilot Study

Allergens, viral, and bacterial infections are responsible for asthma exacerbations that occur with progression of airway inflammation. cPLA2 α and sPLA2X are responsible for delivery of arachidonic acid for production of eicosanoids—one of the key mediators of airway inflammation. However, cPLA2 α and sPLA2X role in allergic inflammation has not been fully elucidated. The aim of this study was to analyze the influence of rDer p1 and rFel d1 and lipopolysaccharide (LPS) on cPLA2 α expression and sPLA2X secretion in PBMC of asthmatics and in A549 cell line. PBMC isolated from 14 subjects, as well as A549 cells, were stimulated with rDer p1, rFel d1, and LPS. Immunoblotting technique was used to study the changes in cPLA2 α protein expression and ELISA was used to analyze the release of sPLA2X. PBMC of asthmatics released more sPLA2X than those from healthy controls in the steady state. rDer p1 induced more sPLA2X secretion than cPLA2 α protein expression. rFel d1 caused decrease in cPLA2 α relative expression in PBMC of asthmatics and in A549 cells. Summarizing, Der p1 and Fel d1 involve phospholipase A2 enzymes in their action. sPLA2X seems to be one of important PLA2 isoform in allergic inflammation, especially caused by house dust mite allergens.


Introduction
Despite intensive studies on asthma pathogenesis and seeking the effective treatment, the number of new cases is increasing globally. Asthma is a very heterogeneous disease whose etiology has not been fully understood. Allergens, drugs, viral and bacterial infections, and stress are the most common factors that initiate and exacerbate asthma. About 70% of asthmatics are atopic [1]. The proposed mechanism of allergic asthma development suggests that allergen exposure causes sensitization, and continued exposure leads to airway hyperresponsiveness and inflammation. Airway inflammation primarily initiated as a defense process aiming to eliminate the damaging factor, which evolves to chronic state causing airway remodeling and impaired lung functions. In general population of Lodz province (Poland) the most common sensitizing indoor allergens are house dust mites and cat [2]. Sensitivity to house dust mite and cat dander are risk factors associated with the development of asthma [3]. Many studies indicate that allergen exposure causes the exacerbation of asthma that occurs with impaired lung function and increases the need for hospitalization [4,5].
Group X secretory phospholipase A 2 (sPLA 2 X) has recently been investigated as one of the most important members of secretory PLA 2 in the inflammatory process [6]. Except its enzymatic activity sPLA 2 can act through the membrane receptors causing cell degranulation and initiating chemokines and cytokines production [7,8]. Moreover, sPLA 2 can influence cytosolic PLA 2 (cPLA 2 ) action [9,10]. In human airways a lot of resident cells (mast cells, macrophages, endothelial cells, epithelial cells, and bronchial smooth muscle cells (SMC)) and haematopoetic cells (basophils, eosinophils, neurophils, lymphocytes, and monocytes) are potential source of secretory phospholipases [11]. In asthmatics expression of sPLA 2 -X predominates in airway epithelium. Moreover, both sPLA 2 -X and sPLA 2 -IIA are the main phospholipases detected in BAL fluid [6,12]. sPLA 2 -X and sPLA 2 -XII are elevated in induced sputum cells of patients with asthma [13]. The studies with knockout mice showed that deficiency of sPLA 2 -X reduced allergen-induced features of airway inflammation [14].
Cytosolic phospholipase A 2 group IVA (cPLA 2 ) is the most potent enzyme in phospholipase A 2 superfamily catalyzing liberation of arachidonic acid (AA) from membrane phospholipids [15]. Our previous studies revealed that cPLA 2 participates in asthma pathogenesis [16]. What is more, rDer p1 caused overexpression of PLA2G4A in PBMC of asthmatics (unpublished data). Whalen et al. showed that PBMC of asthmatic patients stimulated with allergens in the presence of cPLA 2 inhibitor exhibited decreased production of proinflammatory cytokines [17]. cPLA 2 actions are mainly regulated by Ca 2+ concentration and serine residue phosphorylation [18,19]. LPS can modulate activity of cPLA 2 by phosphorylation [20]. Der p1 can activate MAPKs in different types of cells [21,22]. Despite the abovementioned facts that prove the Der p1-cPLA 2 interactions, other mechanisms of allergens impact on lipid mediators remains not fully understood. Thus, we investigated whether allergens or LPS can directly stimulate the expression and/or phosphorylation of cPLA 2 protein in PBMC of severe asthmatics with atopic origin.

Material
2.1. Patients. Patients ( = 7) with severe asthma, who were allergic to house dust mite (Der p1) and cat (Fel d1) allergens, and healthy controls ( = 7) were enrolled to the study. The project was approved by the local ethics committee and an informed consent was obtained from every subject prior to the study. Patients were recruited from the Department of Internal Diseases, Asthma and Allergy of Medical University of Lodz. Asthma was recognized at least 6 months prior to the study and met the criteria of GINA Guidelines [23]. The severity of the disease was assessed according to the American Thoracic Society Workshop on Refractory Asthma 2000 Report [24]. All patients were classified as severe asthmatics.
Patients were asked to not administer antihistamine drugs, oral gluccocorticoids and leukotriene receptors antagonists 24 hours, and inhaled glucocorticoids and long-acting beta agonists 12 hours before blood drawing. Healthy volunteers had no known history of asthma or seasonal allergies. Table 1 presents characteristics of subjects enrolled to the study.

3.1.
Immunoblotting. Total protein from PBMC of patients with asthma, healthy subjects, and A549 cells was extracted in RIPA protein extraction buffer (Sigma-Aldrich, St. Louis, MO), supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The lysate was centrifuged at 14,000 RPM and 4 ∘ C for 20 min, and the pellet discarded. Protein concentrations were determined by the BCA Protein Assay Kit (Pierce Thermo Scientific, Rockford, IL) according to the manufacturer's protocol and using bovine serum albumin as a standard. 20 g of total protein was mixed with NuPAGE LDS Sample Buffer (Life Technologies, Carlsbad, CA) and in a 1 : 10 ratio with NuPAGE Reducing Agent (10x), heated for 10 min at 70 ∘ C. Protein samples were subjected to electrophoresis in 4-12% SDS-NuPAGE Gels (Life Technologies, Carlsbad, CA) at 200 V and electrophoretically transferred to a nitrocellulose membrane at 30 V for one hour. The membrane was blocked in 5% nonfat milk in TBST (20 mM Tris-HCL, 500 mM NaCl, 0.05% Tween 20, and pH 7.5) for 1 hour at room temperature. Then, the membranes were incubated for 12 h at 4 ∘ C with the one of the following antibody: polyclonal rabbit anti-cPLA 2 and anti-phospho-cPLA 2 (Ser505) and anti--actin antibodies (Cell Signaling, Danvers, MA). At the end of the overnight incubation, the membrane was washed with TBST and incubated for one hour in TBST containing the goat anti-rabbit IgG secondary antibodies conjugated with alkaline phosphatase (Sigma-Aldrich, St. Louis, MO). After incubation with secondary antibodies, the membrane was washed three times (3 × 5 mins) in TBST buffer. The band was developed using BCIP/NBT alkaline phosphatase substrate (Merck Millipore, Darmstadt, Germany). Densitometric analysis of bands was performed with Image J 1.34s software (Wayne Rasband, National Institutes of Health, Bethesda, MD) and the results are presented as fold change of optical density (OD).

ELISA.
The sPLA 2 X protein in supernatants form PBMC and A549 cells was measured by enzyme-linked immunoabsorbent assay (ELISA) using commercially available kit (Cloud-Clone Corp., Houston, TX) according to the manufacturer's protocol. The limit of detection for sPLA 2 X protein was 7.813 pg/mL.

Statistical Analysis.
The data were analyzed using Statistica (v. 10.0; StatSoft, Tulsa, OK). Comparisons between groups were performed by using Mann-Whitney U tests or ANOVA followed by the Tukey's post hoc test when appropriate. Values of < 0.05 were considered statistically significant.

PBMC of Asthmatics Overproduced sPLA 2 X in the Steady
State. Hallstrand et al. [13] reported that sputum cells from asthmatics contained more sPLA 2 X mRNA (qPCR) and protein (immunostaining) in comparison to controls, so we hypothesized that similar observation in PBMC is very possible. Taking into account the fact that sPLA 2 X is produced as a zymogen and its cellular amount may not be relevant to its biological function we concluded that secretion of sPLA 2 X will be the best approach to discover its potential involvement in asthma pathogenesis. The levels of sPLA 2 X released by PBMC were compared in asthmatics and controls. The steady state concentration of sPLA 2 X was significantly higher in asthmatics (411.09 pg/mL ± 129.2) than in healthy subjects (91.96 pg/mL ± 16.37) (Figure 1).

rDer p1 Stimulation
Results in Different sPLA 2 X and cPLA 2 Production Patterns in Asthmatics and Controls. PBMC of asthmatics and healthy subjects were stimulated with rDer p1 for 24 hours in three different concentrations: 1, 5, and 10 g/mL. There were no differences in sPLA 2 X secretion between asthmatics and controls in any dose of rDer p1 whereas dust mite allergen in concentration of 10 g/mL significantly induced release of sPLA 2 X (1.54 ± 0.24) when compared with relative protein expression of cPLA 2 (0.79 ± 0.17) in asthmatics (Figure 2(a)). On the contrary, in healthy subjects, we did not observe similar fluctuation (Figure 2(b)).

Regulation of Relative cPLA 2 Protein Expression by LPS, rFel d1, and rDer p1 in PBMC in Time-Dependent Manner.
Relative expression of cPLA 2 protein was compared between healthy and asthmatic patients after stimulation with rDer p1 (5 g/mL), rFel d1 (5 g/mL), and LPS (100 ng/mL). cPLA 2 basal expression was significantly lower in asthmatics as compared to healthy subjects. While we did not observe differences in cPLA 2 protein synthesis between patients and controls after stimulation, there was a statistically significant increase of cPLA 2 protein expression in PBMC of severe asthmatics in all tested time points after stimulation with rDer p1 when compared to steady state level. Also rFel d1 induced expression of cPLA 2 protein after 0.5 h and 6 h of stimulation in patients. 24-hour incubation with LPS results in induction of cPLA 2 in asthmatics when compared with level before stimulation. In healthy subjects cPLA 2 protein expression did not change significantly over time (Figures 3  and 4-blot).

cPLA 2 Protein Synthesis Is Diminished by rFel d1 in
PBMC of Asthmatics. PBMC from asthmatics and healthy subjects were stimulated with rDer p1, rFel d1, and LPS in three different concentrations for 24 hours. While being not significant (as compared to control), there was a trend of increased relative expression of cPLA 2 in healthy subjects and decreased protein content in asthmatics after stimulation with rFel d1. However PBMC from asthmatics produced significantly less cPLA 2 (0.65 ± 0.15) than those from healthy subjects (1.61 ± 0.28) after stimulation with rFel d1 in concentration of 10 g/mL (Figure 4).

LPS Induced cPLA 2 Phosphorylation in PBMC of Healthy
Subjects. Phosphorylation of cPLA 2 was analyzed in PBMC stimulated with rDer p1 (5 g/mL), rFel d1 (5 g/mL), and LPS (100 ng/mL). The cells from controls (1.38 ± 0.22) contained more phosphorylated form of cPLA 2 than patients' PBMC (0.87 ± 0.1) after 2 h of incubation with LPS. The rapid change in phosphorylation of cPLA 2 was observed after 6 h stimulation with LPS ( Figure 5). The allergens did not change the phosphorylation of cPLA 2 .

4.6.
Regulation of cPLA 2 Protein Synthesis by LPS, rFel d1, and rDer p1 in A549 Cells. In A549 culture the rFel d1 in concentration 10 g/mL significantly decreased synthesis of cPLA 2 (0.61 ± 0.01) ( Figure 6). Any other stimulators did not change cPLA 2 expression in short time of incubation.

Recombinant Der p1 Induces Morphological Changes in A549
Cells. rDer p1 dose-and time-dependently caused morphological changes in A549 cells. Low concentrations and short incubation did not induce visible changes whereas higher concentrations and longer incubations led to cells shrinking and desquamation (Figure 7).

Discussion
We observed that asthmatics' PBMC released more sPLA 2 X than control cells in the steady state. This observation is supported by previous reports, showing that asthmatics have increased content of sPLA 2 X in airway epithelium, BALF, and sputum. The novelty of our study relates to regulation of different PLA 2 isoforms in response to rDer p1 stimulation. Interestingly rDer p1 in highest dose (10 g/mL) significantly upregulated release of sPLA 2 X protein when compared to relative cPLA 2 protein expression in asthmatics whereas in healthy subjects we did not observe this tendency. This observation suggests that sPLA 2 X may be one of the important isoforms of PLA 2 in allergic response. Despite evidence that both enzymes cooperate in liberation of AA, sPLA 2 X is also able to release AA independently to cPLA 2 [25] 6 BioMed Research International and therefore may alone promote allergen-induced inflammation. Misso et al. suggested that increased activity of sPLA 2 may be associated with atopic status [26]. Some data related to the role of sPLA 2 X in airway inflammation come also from animal studies. Knock-in of human sPLA 2 X to msPLA 2 X −/− mice restored allergen-induced inflammatory cell recruitment into airways as well as hyperresponsiveness to methacholine [27]. PBMC stimulated with rDer p1, rFel d1, or LPS showed increased production of cPLA 2 in comparison to steady state level in asthmatics but not in healthy subjects. The most rapid changes were observed after rDer p1 action, whereas 24hour incubation with LPS was needed to induce significant increase of cPLA 2 content. The mechanism of Der p1 action is still not fully determined. Der p1 acts by PAR-2 receptor as well as in PAR-2-independent manner through activation of NF-B and ERK1/2 [22,28]. Activation of NF-B pathway is prerequisite for cPLA 2 expression in many cell types [29][30][31], so this pathway can partially be involved in the induction of cPLA 2 expression. In our study we did not observe the changes in production of cPLA 2 between asthmatics and controls after stimulation with allergens and LPS (timeresponse scheme).
Dose-response scheme of our experiment showed that rFel d1 in highest dose (10 g/mL) significantly decreased expression of cPLA 2 in asthmatics when compared to healthy subjects. The similar significant decrease of cPLA 2 synthesis was observed in A549 cells after stimulation with rFel d1. Fel d1 has been shown to may have enzymatic activity [32]. The structural analysis of Fel d1 revealed homology of the allergen with -subunit of mouse salivary androgen binding protein [33], uteroglobin and with the related Clara cell phospholipid binding protein, CC10 [34]. Uteroglobin is anti-inflammatory protein and can inhibit PLA 2 activity [35]. The similarity between rFel d1 and uteroglobin may suggest that the allergen has cytokine-like properties; thus it may be capable of inflecting the immune response [34]. Although Der p1 and Fel d1 are able to increase activity and phosphorylation of cPLA 2 in eosinophils [36], in our study we did not observe the significant changes in cPLA 2 phosphorylation after allergen stimulation. Only stimulation with LPS resulted in elevated level of phosphorylated cPLA 2 form in healthy subjects. PBMC of healthy volunteers respond better to LPS treatment. The diverse effects of LPS action have been observed earlier [37,38]. Moreover it has been proved that LPS-induced cPLA 2 activity is TLR-4dependent [39]. Different experiment systems and doses of LPS used in experiments seem to condition the results of LPS stimulation. In U937 cell line and macrophages LPS significantly increased the expression of cPLA 2 protein after 8 hours but not after 24 hours of stimulation whereas in tracheal smooth muscle cells the effective time points were 16 and 24 hours [30,40]. rDer p1 in higher doses induces the desquamation of A549 cells. This effect was observed earlier and is result of enzymatic activity of the allergen [28]. Der p1 is a cysteine protease able to degrade the occludin protein and ZO-1 protein in tight junction between epithelial cells [28,41].

Conclusions
Results of the study showed that Der p1 and Fel d1 involve phospholipase A 2 enzymes in their action. sPLA 2 X seems to be the more important PLA 2 isoform in airway inflammation, especially caused by house dust mite allergens. Der p1 has protease activity and can actively degrade and penetrate the epithelium barrier in airways. Moreover stimulation of sPLA 2 X production whose activity is connected with further cysteinyl leukotrienes synthesis may sustain inflammatory process . This phenomenon might be supported additionally by decreased synthesis of cPLA 2 and subsequent diminished PGE 2 synthesis, which in respiratory track may also play protective role. Fel d1 seems to act rather by decreasing the cPLA 2 expression than induction of sPLA 2 X. Further studies focusing on expression of different PLA 2 isoforms in different timepoints after inflammatory stimulus exposition should be analyzed to better understand the molecular mechanism of allergic inflammation.