Key Role of Group V Secreted Phospholipase A2 in Th2 Cytokine and Dendritic Cell-Driven Airway Hyperresponsiveness and Remodeling

Background Previous work has shown that disruption of the gene for group X secreted phospholipase A2 (sPLA2-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA2s in the mammalian genome, the involvement of other sPLA2s in the asthma model is possible – in particular, the group V sPLA2 (sPLA2-V) that like sPLA2-X is highly active at hydrolyzing membranes of mammalian cells. Methodology and Principal Findings The allergen-driven asthma phenotype was significantly reduced in sPLA2-V-deficient mice but to a lesser extent than observed previously in sPLA2-X-deficient mice. The most striking difference observed between the sPLA2-V and sPLA2-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA2-V−/− mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V−/− mice diminishes Th2 cytokine responses in the airways. Conclusions This paper illustrates the diverse roles of sPLA2s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA2-V as a potential new therapy to treat asthma and other allergic disorders.


Introduction
Leukotriene B 4 (LTB 4 ) and the cysteinyl leukotriene (CysLT)s C 4 , D 4 , and E 4 (LTC 4 , LTD 4 , and LTE 4 ) are biologically potent 5-lipoxygenase (5-LO) products of arachidonic acid metabolism [1]. The leukotrienes are important mediators of allergen-induced airway inflammation and remodeling in asthma. They mobilize CD34 + pluripotent hematopoeitic stem-cell progenitors from the bone marrow to the bloodstream where they promote adhesion to the endothelium, transmigration into sites of inflammation, and increased survival and activation of leukocytes [1]. Through crosstalk with type 2 helper T cell (Th2) cytokines IL-4, IL-5, and IL-13, the actions of both the Th2 cytokines and leukotrienes are amplified leading to dendritic cell (DC) activation, goblet cell mucus hypersecretion, endothelial cell increased vascular permeability, augmented collagen synthesis by fibroblasts and myofibroblasts, and smooth muscle cell proliferation in the airways [1]. In asthma, other eicosanoids such as the cyclooxygenase (COX) arachidonate product prostaglandin D 2 (PGD 2 ) also contribute to this Th2-driven inflammatory process.
The biosynthesis of eicosanoids is controlled in part by the availability of arachidonic acid, which is thought to be liberated from membrane phospholipids via the action of one or more lipolytic enzymes, most notably phospholipases A 2 (PLA 2 )s. Mammalian cells contain multiple types of PLA 2 s [2], but it is generally accepted that cytosolic PLA 2 -a (cPLA 2 -a, also known as group IVA PLA 2 ) plays a pivotal role in agonist-mediated arachidonate release for the biosynthesis of the eicosanoids. This is based on studies with cPLA 2 -a inhibitors [3][4][5][6] and studies with cPLA 2 -a-deficient mice [7][8][9]. The mammalian genome also encodes 10 secreted PLA 2 s (sPLA 2 )s. The role of these enzymes in eicosanoid biosynthesis is much less clear. A systematic investigation of the interfacial kinetic and binding properties of the full set of mouse and human sPLA 2 s shows that the group X sPLA 2 (sPLA 2 -X) stands out as having the highest specific phospholipolysis activity when added to cultured cells [10,11]. We have recently demonstrated that mice that lack group X sPLA 2 show a dramatic reduction in parameters of Th2-driven airway inflammation and remodeling [12]. Immunohistochemical studies demonstrate that group X sPLA 2 is expressed in airway epithelial cells and macrophages in bronchoalveolar lavage (BAL) fluid [12]. Airway hyperreactivity to methacholine challenge, a hallmark asthmatic phenotype, is largely suppressed in the group X sPLA 2 knockout after ovalbumin (OVA) allergen challenge. Markers of airway remodeling such as occlusion of the airways by mucus and subepithelial deposition of collagen were reduced significantly when sPLA 2 -X was deleted. Although T cell function was unimpaired, sPLA 2 -X-deficiency was characterized by a marked reduction in trafficking of T cells to the allergen-challenged airways in the mouse asthma model [12]. OVA-induced CysLT and PGD 2 production were near fully blocked in the sPLA 2 -X mouse indicating an important mechanism for the effect of group X sPLA 2 -deficiency. Human group X sPLA 2 is also found in induced sputum samples in patients with exercise-induced asthma and its levels in BAL fluid correlated with asthma severity [13], supporting a role of this PLA 2 in human airway inflammation [14].
Group V sPLA 2 also displays relatively high specific activity when added to mammalian cells in culture that is second to group X sPLA 2 but well above that of the other mammalian sPLA 2 s [10,15]. Exogenous addition of nanomolar concentrations of group V sPLA 2 to neutrophils and eosinophils leads to augmentation of arachidonic acid release and eicosanoid formation [16,17]. In the case of neutrophils, exogenously added group V sPLA 2 leads to an activation of cPLA 2 -asuggesting that these two enzymes work together to maximize arachidonic acid release [16]. In eosinophils, exogenously added group V sPLA 2 acts without the involvement of the cytosolic PLA 2 [17]. Disruption of the mouse group V sPLA 2 leads to a ,50% reduction in LTC 4 and prostaglandin E 2 (PGE 2 ) production in peritoneal macrophages that have been stimulated with the fungal-derived agonist opsonized zymosan [18]. In these cells there is also crosstalk between group V sPLA 2 and cPLA 2 -a. The mechanistic basis for this crosstalk between secreted and cytosolic PLA 2 s remains to be determined. These studies point to the possible role of group V sPLA 2 in promoting eicosanoid biosynthesis related to inflammation. It should be mentioned that, in general, the study of secreted enzymes with single types of primary cells or cell lines in culture is very different than the study of these enzymes in a whole animal disease model. Secreted enzymes including sPLA 2 can obviously act on cells different than those that produce them.
Based on these early actions of group V sPLA 2 and the need to carry out whole animal studies of sPLA 2 s, we investigated the possible role of sPLA 2 -V in mouse asthma models by using sPLA 2 -V-deficient mouse for studies of allergen-induced airway inflammation, hyperresponsiveness, and remodeling.

Effect of sPLA 2 -V Deficiency on Acute Asthma Phenotype
The effect of sPLA 2 -V-deficiency on allergen-induced inflammatory cell infiltration in the BAL fluid and bronchial hyperresponsiveness was determined in a mouse acute asthma model ( Figure 1). OVA-treated sPLA 2 -V +/+ mice had a marked increase in both total inflammatory cells and eosinophils recovered in BAL fluid compared with the saline group control ( Figure 1A). The number of total inflammatory cells and eosinophils in the BAL fluid of OVA-treated sPLA 2 -V 2/2 mice was reduced by 59% (P = 0.008) and 54% (P = 0.019) respectively compared to sPLA 2 -V +/+ controls ( Figure 1A). The OVA-treated wild-type mice, in comparison to saline controls, had significantly increased responsiveness to aerosolized methacholine as determined by lung resistance (R L ) ( Figure 1B). In contrast, hyperresponsiveness to methacholine after OVA challenge to sPLA 2 -V 2/2 was similar to that measured in saline-treated sPLA 2 -V 2/2 mice ( Figure 1B).
The effect of sPLA 2 -V deficiency on levels of eicosanoids derived from arachidonic acid via the cyclooxygenase pathway leading to PGD 2 and via the 5-LO pathway leading to CysLTs, D 4 , and E 4 was determined ( Figure 2). Since PGD 2 is unstable, it was converted to its stable methoxime (MOX) derivative prior to measurement. PGD 2 and CysLTs were significantly increased in the BAL fluid of OVA-treated sPLA 2 -V +/+ mice on d 23 in comparison to saline controls ( Figure 2). The BAL fluid levels of PGD 2 and CysLTs of OVA-challenged sPLA 2 -V 2/2 mice were decreased by 45% (P = 0.03) and 30% (P = 0.04) respectively compared to the OVA-treated wild-type mice ( Figure 2).

Effect of sPLA 2 -V Deficiency on Chronic Asthma Phenotype
Lung sPLA 2 -V expression was examined on d 76 by immunocytochemistry in wild-type mice and in sPLA 2 -V 2/2 mice as a control. sPLA 2 -V was undetected in saline-treated sPLA 2 -V +/+ controls and in sPLA 2 -V 2/2 mice after saline or OVA treatment ( Figure 3). sPLA 2 -V expression was observed in the airway columnar epithelial cells, airway smooth muscle cells, and mononuclear leukocytes infiltrating the lung interstitium of OVA-treated wild-type mice ( Figure 3). sPLA 2 -V was not detected in lungs from OVA-treated wild-type mice when immunocytochemistry was performed with pre-immune serum (not shown).
The effect of sPLA 2 -V deficiency on allergen-induced persistent infiltration of lung tissue by eosinophils and other inflammatory cells and airway goblet cell metaplasia, subepithelial fibrosis, and collagen and VEGF gene expression was examined in a chronic asthma model of lung remodeling (Figures 4-7). On d 76, sPLA 2 -V +/+ mice had a dense infiltrate in the lung interstitium of eosinophils and other inflammatory cells ( Figure 4) and increase in airway goblet cells ( Figure 5). By morphometric analysis, the total inflammatory cell and eosinophil infiltration was reduced by 61% (P = 0.01) and 67% (P = 0.001) respectively and the goblet cell metaplasia was diminished by 58% (P = 0.02) in OVA-treated sPLA 2 -V 2/2 mice compared to wild-type controls ( Figure 6). After long-term OVA challenge, the sPLA 2 -V +/+ mice had increased deposition of subepithelial collagen and increased lung collagen content compared to saline-treated controls ( Figure 7A,B). The subepithelial fibrosis and increased collagen content observed in OVA-treated wild-type mice were modestly reduced by 24% (P = 0.03) and 31% (P = 0.05) respectively in the allergen-challenged sPLA 2 -V 2/2 mice ( Figure 7B). By quantitative real-time PCR (qPCR), the OVA-treated sPLA 2 -V 2/2 mice had marked impairment in collagen (i.e., COL1a2 and COL3a1), and VEGF (i.e., VEGF-A, VEGF-A2, VEGF-B, and VEGF-C) gene expression in their lungs compared to OVA-treated wild-type controls ( Figure 7C).
To understand the nature of the Th2 cytokine defect in the sPLA 2 -V-deficient mice, CD4 + T cell and DC proliferation, and DC antigen processing and eicosanoid production was studied ( Figures 12 and 13). Although both sPLA 2 -V-deficient and wildtype splenic CD4 + T cells had a marked increase in proliferation under Th2 polarizing conditions in response to IL-2 and IL-4 after culture in anti-CD3-coated plates in vitro, the magnitude of this response was slightly decreased by 19% (P = 0.009) in the sPLA 2 -V-deficient mice ( Figure 12A). The mixed lymphocyte reaction (MLR) cell proliferative response was compared between sPLA 2 -V 2/2 and sPLA 2 -V +/+ splenic DCs. Under basal conditions, increasing numbers (0-50000 DCs per well containing 2610 5 allogeneic CD4 + T cells) of DCs from wild-type mice led to a marked increase in cell proliferation that was modestly reduced by 12% (P = 0.028) and 21% (P = 0.044) with 5000 and 50000 sPLA 2 -V 2/2 DCs/well respectively ( Figure 12B). The effect of sPLA 2 -V-deficiency on proliferation of BMDCs in culture with GM-CSF and IL-4 was studied. At baseline, no difference in proliferation was observed between the sPLA 2 -V 2/2 and sPLA 2 -V +/+ BMDCs  Figure 12C). In contrast, proliferation of the sPLA 2 -V-deficient BMDCs was reduced by 33% (P = 0.0064) compared to wild-type controls by d 9 in culture ( Figure 12C).
The effect of sPLA 2 -V deficiency on BMDC-derived eicosanoid production was determined. OVA (1 mg/ml) did not induce a significant increase in the 5-LO arachidonic acid products LTB 4 or CysLTs by either the sPLA 2 -V +/+ or sPLA 2 -V 2/2 BMDCs compared to saline-treated controls (data not shown). In contrast, OVA triggered a marked release of PGE 2 by sPLA 2 -V +/+ BMDCs that was significantly increased over the levels of saline-treated controls ( Figure 13C). This increased release of PGE 2 induced by OVA was reduced by 36% (P = 0.0015) in BDMCs of sPLA 2 -V 2/ 2 mice ( Figure 13C).

Discussion
We have previously shown that disruption of the gene for sPLA 2 -X in mice leads to a dramatic reduction in airway inflammation, remodeling, and hyperresponsiveness in a mouse model of airway asthma [12]. Given the large number of sPLA 2 s in the mammalian genome, it seems prudent to examine the involvement of other sPLA 2 s in an asthma model. Because sPLA 2 -V and sPLA 2 -X sPLA 2 are very active at hydrolyzing the membranes of mammalian cells, these two enzymes have been our first priority in genetic disruptions studies of sPLA 2 s in complex disease models. In prior work, Munoz et al reported a study of airway inflammation in OVA-administered sPLA 2 -V knockout mice [19]. Their observations are consistent with what we find in our independent study. In their and our ( Figure 3) studies, sPLA 2 -V expression is upregulated after OVA sensitization, and the protein is found in airway epithelium, mononuclear cells, and smooth muscle cells. Both studies report that loss of sPLA 2 -V leads to a reduction in inflammatory cell infiltration into the airways in response to OVA. (i.e., the influx of total inflammatory cells and eosinophils, was decreased by 45% and 57% respectively in the Munoz study [19] and 59% and 54% respectively in the present report ( Figure 1A). Similar to Munoz et al. [19], we also found that sPLA2-V-deficiency impaired allergen-induced airway hyperresponsiveness to methacholine ( Figure 1B).
The study by Munoz et al. [19], did not examine the effect of group V sPLA 2 -deficiency on allergen-induced airway remodeling (i.e., goblet cell metaplasia and subepithelial fibrosis) and provided little mechanistic insight (i.e., arachidonic acid metabolism and T cell/dendritic cell function was not examined) into the mechanism(s) by which group V sPLA 2 regulates allergic pulmonary inflammation. Our study thus adds the important dimension that a drop in airway inflammation in this model due to sPLA 2 -V deletion is likely due to a reduction in the primary immune response as reflected by changes in eicosanoid and Th2 cytokine generation.
Giannattasio et al. [20] used house dust mite Dermatophagoides farinae as antigen to induce pulmonary inflammation without systemic immunization in a mouse asthma model to explore the effect of sPLA 2 -V deficiency on the adaptive immune response. In this dust mite asthma model, they observed a greater reduction in eosinophil infiltration into the BAL fluid and goblet cell metaplasia (95% and 80% reductions respectively) than we did in the OVA model (54% and 58% reductions respectively) compared to wildtype controls. We also showed impairment in collagen and VEGF gene expression and modest reductions in lung collagen deposition in the OVA model (Figure 7), effects not examined in the Giannattasio study [20]. Whereas, no differences in eicosanoid production were observed between sPLA 2 -V 2/2 and sPLA 2 -V +/+ mice in the dust mite asthma model, a moderate reduction in eicosanoids (30% decrease in CysLTs and 45% decrease in PGD 2 ) was seen in the BAL fluid of OVA-treated sPLA 2 -V 2/2 mice compared to wild-type controls (Figure 2). In both the D. farinae and OVA (Figure 8) asthma models, antigen-specific IgE levels were reduced in the sPLA 2 -V-deficient mice compared to controls.   In the dust mite asthma model, Giannattasio et al. [20] observed significant reductions in total lung IL-5 and IL-13 transcripts in the sPLA 2 -V 2/2 knockouts and decreased Il-4, IL-5, and IL-13 Th2 cytokine levels from pulmonary lymph node cells after ex vivo restimulation with D. farinae [20]. In the acute asthma model, we found a 55% reduction in IL-4 transcripts in the OVAtreated sPLA 2 -X knockout mice but no decrease in IL-5 or IL-13 transcripts. In contrast, in the chronic asthma model ( Figure 9B), each of the Th2 cytokine transcripts was modestly reduced in the sPLA 2 -deficient mice (i.e., reductions of 22% for IL-4, 19% for IL-5, an 49% for IL-13). Similar to the Giannattasio study [20], restimulation of lung lymph node cells with OVA allergen ex vivo in the chronic OVA model led to marked reductions in IL-4, IL-5, and IL-13 production ( Figure 10A) with lesser reductions in these Th2 cytokines by OVA-restimulated spleen cells ( Figure 10B). We also observed moderate impairment of sPLA 2 -V-deficient splenic T cell production of IL-4, IL-5, and IL-13 cytokines after ex vivo allergen-specific, protein kinase C, and TCR activation ( Figure 11) and a small reduction in the ability of sPLA 2 -Vdeficient splenic CD4 + T cells to proliferate under Th2 polarizing conditions ( Figure 12A). Our data and that of Giannattasio et al. [20] suggest that the reduced airway inflammation in the OVAdriven sPLA 2 -V knockout mouse compared to the wild-type controls can be explained by a decrease in the primary immune response, leading to lower levels of OVA-specific IgE as well as Th2 cytokines. IL-4 induces class switching and release of IgE by B cells, and IL-5 plays an important role in the induction of the eosinophil influx into the lungs in allergen-driven models of asthma [21,22]. IL-13 is a key mucus secretagogue and profibrotic cytokine that causes fibroblast proliferation and collagen deposition in the airways [23]. IL-13 is also the primary Th2 cytokine in induction of airway hyperresponsiveness [24].
At this point, the role of sPLA 2 -V in augmenting the primary immune response is becoming better understood. Prior studies have demonstrated that peritoneal macrophages from sPLA 2 -V 2/2 mice phagocytose zymosan particles significantly less well than wild-type mice [25], clearance of immune complexes is reduced in sPLA 2 -V 2/2 mice [26], and that the presence of sPLA 2 -V in dendritic cells is key for cell maturation and antigen processing in dust miteinduced lung inflammation [20] suggesting that perhaps sPLA 2 -V deficiency may affect processing of allergen by dendritic cells. CysLTs have important actions on DC maturation and function. CysLTs augment the antigen-presenting capacity of dendritic cells in the lung [27]. LTC 4 , but not LTD 4 or LTB 4 , matures DCs in a superior fashion than PGE 2 to stimulate DC-driven CD4 + T cell responses and antigen-specific T cell induction [28]. CysLTs also increase the capacity of BMDCs to induce Th2 immune responses in the lungs after adoptive transfer in mice [29]. CysLTs promote the migration of dendritic cells to lymph nodes [30]. Pretreatment of asthmatics with a CysLT 1 receptor antagonist reduces the allergeninduced decrease in circulating CD33 + DCs indicating a role for CysLTs in the trafficking of myeloid DCs in vivo [31]. LTB 4 through its BLT 1 [32] and BLT 2 [33] receptors also promotes DC migration. CysLT-mediated activation and chemotaxis of monocyte-derived immature dendritic cells is inhibited by the immunoregulatory cytokine IL-10 suggesting a link between IL-10 regulatory responses and the 5-LO pathway [34]. Human DCs differentially express the CysLT receptors CysLT 1 and CysLT 2 depending upon maturation signals such as the Toll-like receptor (TRL) 4 agonist lipopolysaccharide (LPS) [35]. The TRL2 agonist zymosan down-regulates CysLT 1 receptor expression on human monocyte-derived DCs diminishing their responsiveness to LTD 4 [36]. In the D. farinae mouse asthma model, CysLT 2 receptor negatively regulates CysLT 1 receptor activation of BMDCs and also Figure 7. Impaired chronic allergen-induced airway remodeling and collagen deposition in sPLA 2 -V 2/2 mice. Lung tissue was obtained on d 76 from sPLA 2 -V +/+ and sPLA 2 -V 2/2 mice treated with either saline or OVA. A. Sections underwent Masson's trichrome staining. Scale bar, 50 mm. Micrographs are representative from 4-5 mice per group. B. Airway subepithelial fibrosis (0-4+ scale) was determined by morphometry, and lung collagen deposition (mg/lung) was determined by Sircol TM assay (n = 4-5, each group). C. Collagen (COL1a2 and COL3a1) and VEGF (VEGF-A, VEGF-A2, VEGF-B, and VEGF-C) gene expression in OVA-treated sPLA 2 -V +/+ (black bars) and sPLA 2 -V 2/2 (blue bars) mice was determined by qPCR; nd = not detected in samples from the sPLA 2 -V 2/2 mice. *P,0.05 OVA-treated sPLA 2 -V +/+ versus sPLA 2 -V 2/2 mice (n = 4-5, each group). doi:10.1371/journal.pone.0056172.g007 the expression of the CysLT 1 receptor on the surface of these DCs suggesting that a competitive balance between these two CysLT receptors may regulate allergic lung inflammation [37].
In this report, we also found that BMDCs from sPLA 2 -Vdeficient mice have decreased proliferation after stimulation. The sPLA 2 -V 2/2 DCs exhibit defects in their uptake of OVA and ability to activate CD4 + cell proliferation and present OVA to OVA-transgenic T cells. These defects in DC function are associated with impairment in PGE 2 production by these immunoregulatory cells. PGE 2 has key effects on cytokine production by antigen-presenting cells such as DCs and T cells affecting DC and T helper cell differentiation and effector actions [38][39][40]. PGE 2 promotes DC maturation, activation, and migration [41]. Recent studies have shown that PGE 2 signaling through its EP1 and EP3 receptors is needed for optimal survival of DC progenitors and DC development in vivo via regulation of the receptor tyrosine kinase Flt3 on the DC progenitor cells [42]. Thus, impairment in eicosanoid generation (both 5-LO and COX arachidonate metabolites) in sPLA 2 -V 2/2 mice may lead to a diminution in both Th2 and DC responses in the airways. With the development of selective sPLA 2 inhibitors [12], blockade of group V sPLA 2 may provide a novel therapeutic opportunity in the treatment of asthma and other allergic disorders.

Ethics Statement
All animal use procedures were approved by the IACUC of the University of Washington (Animal Welfare Assurance No. A346401).

Pulmonary Function Testing
On d 23 (40 min after the last aerosol challenge with OVA or saline in the acute model), invasive pulmonary mechanics were measured in mice in response to methacholine as described [12]. Mice received aerosolized solutions of methacholine (0, 3.125, 6.25, 12.5, 25, and 50 mg/ml in normal saline) with R L determined from measures of pressure and flow and expressed

BAL Fluid and Blood Collection
After completion of plethysmography on d 23, the left lung was tied off at the mainstem bronchus, and the right lung lavaged three times with 0.5 ml of normal saline. After centrifugation at 2506g for 5 min at 4uC, total BAL fluid cells were counted with eosinophils stained with 0.05% eosin [46]. BAL fluid eicosanoid analyses were performed only in mice that did not get the invasive R L measurements; the supernatant was processed for eicosanoid assays as described below. Lung tissue was collected for qPCR assay of Th2 cytokines. Plasma samples were obtained on d 23 and assayed for OVA-specific IgE.

Eicosanoid Analyses
For CysLTs and PGE 2 analyses, BAL fluid supernatant was processed on solid-phase extraction cartridges followed by detection using enzyme immunoassay (EIA) kits from Cayman Chemical Company (Ann Arbor, MI) as described [12]. PGD 2 was analyzed using a 0.25 ml aliquot of the BAL fluid supernatant using the PGD 2 -MOX EIA kit (Cayman Chemical Company) [12].

Lung Tissue, Paratracheal Lymph Node, and Spleen Collection
On d 76 (24 h after the last i.n. dose of OVA or saline, chronic model), the lungs were collected for histopathology, collagen, and qPCR analyses. Paratracheal lymph nodes and spleens were collected for cytokine analyses and isolation of spleen cells, CD4 + T cells, and DCs as described below. Plasma samples were also obtained on d 76 and assayed for OVA-specific IgE.

Lung Histopathology
The upper and lower lobes of the left lung were collected and 5 mm sections prepared [46]. Ten airways (0.4-0.7 mm in diameter and surrounded by smooth muscle cells) per mouse were randomly selected for morphometric analysis by individuals blinded to the protocol design [46]. The sections were stained with hematoxylin and eosin (H&E) to determine total inflammatory cell infiltration [46] on a semi-quantitative scale (0-4+), and eosinophil number per unit lung tissue area (2,200 mm 2 ) [47,48]. Alcian blue staining was used to identify airway goblet cells.
Immunocytochemistry sPLA 2 -V expression in mouse lung was determined by immunocytochemistry using light microscopy [49]. The sections were incubated with the primary antibody, polyclonal rabbit anti-sPLA 2 -V-specific antisera [50] at a 1:50 dilution for 25 min at room temperature followed by rinsing in PBS and incubation with the secondary antibody, goat anti-rabbit antibody conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA) at a 1:20 dilution for 25 min. As controls, PBS or normal rabbit IgG (Vector Laboratories) were used in place of the primary antibody. To detect peroxidase, the sections were incubated with 0.5% 39, 39-diaminobenzidine tetrachloride (Sigma-Aldrich Corporation) in PBS and 0.15% hydrogen peroxide for 15 min at room temperature; nuclei were counterstained with 1% methyl green in distilled water for 3 min.

Collagen Assay
Collagen content of the right lung was determined by the Sircol TM collagen assay (Biocolor Ltd., Newtownabbey, Northern Ireland, UK).

OVA-specific IgE Assay
For assay of OVA-specific IgE, Nunc 96-well flat bottomed plates (Nalge Nunc International, Rochester, NY) were coated with OVA (50 mg/ml) in 1X PBS overnight at room temperature, washed 3 times with 1X PBS containing 0.05% Tween-20, and blocked with 1X PBS containing 3% BSA for 60 min at room temperature. 50 ml plasma samples (1:1 in 1X PBS) or varying concentrations of internal standard (Clone C38-2 anti-mouse IgE, BD Biosciences, San Diego, CA) were added per well and incubated for 90 min at 37uC, and washed/blotted dry. After addition of 100 ml (1:100 in 1X PBS) biotin-conjugated rat antimouse IgE monoclonal antibody (Clone R35-72; BD Biosciences) to each well, the plates were incubated overnight at 4uC; then 100 ml Streptavidin-HRP-conjugated secondary antibody (1:1000 in 1X PBS, BD Biosciences) was added per well and plates incubated at 37uC for 90 min. 100 ml of 2,29-azinobis (3-ethylbenzthiazoline-sulfonic acid (i.e., one tablet dissolved in 100 ml of 0.05 M phosphate-citrate buffer, pH 5.0 and 25 ml 30% H 2 O 2 ; Sigma-Aldrich Corporation) substrate solution was added per well and after incubation for 30 min at room temperature, the plates were read at OD 405 nm with a standard curve constructed by linear regression analysis of the absorbances in comparison to serial dilutions of known concentrations of mouse IgE. qPCR Total RNA was isolated from the right lung using an RNeasy mini kit (QIAGEN Inc., Valencia, CA), and mRNA levels for IL-4, IL-5, IL-13, COL1a2, COL3a1, vascular endothelial growth factor (VEGF)-A, VEGF-A2, VEGF-B, VEGF-C, and GAPDH determined by qPCR using a model 7900HT Fast Real-Time PCR System [Applied Biosystems (ABI), Foster City, CA] as described [12]. PCR DNA sizes were ,100 bp and confirmed by gel electrophoresis.

Spleen Cell and CD4 + T Cell Isolation
Spleens were placed in RPMI-1640 with 25 mmol/L HEPES (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Invitrogen), cut into small pieces with scissors, and strained through a 70 mm BD Falcon TM cell strainer (BD Biosciences, San Jose, CA) to create single-cell suspensions. Red cells were lysed using BD PharmLyse TM lysing buffer (BD Biosciences). CD4 + T cells were purified from splenic lymphocytes by magnetic depletion of B cells, macrophages, DCs, NK cells, granulocytes, erythroid precursors, and CD8 + T cells using MACSH CD4 + T Cell Isolation Kit (Miltenyi Biotec Inc., Auburn, CA).

Spleen DC Purification
Spleens were placed in 3 ml RPMI with 1 mg/ml DNase (Worthington Biochemical Corporation, Lakewood, NJ) and 50 mg/ml collagenase (Worthington Biochemical Corporation) in 6-well plates. 200 ml of this RPMI was injected in each spleen that was then cut into small pieces and strained through a 70 mm cell strainer (BD Biosciences). Red cells were lysed using BD PharmLyse TM lysing buffer (BD Biosciences). After washing with AutoMACS TM Rinsing solution (Miltenyi Biotec Inc.), DCs were purified with CD11cMicroBeadsH (Miltenyi Biotec Inc.) [51]. MLR 2610 5 allogeneic CD4 + cells from C57Bl6 mice were cultured with irradiated (3000 rad) splenic DCs (0-50000 cells/well) from wild-type or sPLA 2 -V 2/2 mice in complete RPMI-1640 at 37uC in 5% CO 2 for 72 h. The MTT assay was used to determine cell proliferation [52].

BMDC Culture
Murine DCs were generated from bone marrow of sPLA 2 -V 2/2 mice and wild-type mice. Briefly, bone marrow was harvested by flushing femurs and tibias with PBS containing 1% FBS. Cells were resuspended at 2610 6 cells/ml in GIBCOH RPMI-1640 (Invitrogen Corporation) supplemented with 5 ng/ml of recombinant mouse GM-CSF (R&D Systems, Inc., Minneapolis, MN) and 10 ng/ml of recombinant mouse IL-4 (BD Biosciences). On d 3 and 5 of culture, half of the medium was replaced with fresh medium containing GM-CSF and IL-4. On d 6, loosely adherent cells were harvested and DCs purified with CD11c MicroBeadsH according to the manufacturer's instructions (Miltenyi Biotec Inc.) [52].

BMDC Cell Proliferation
BMDC cells from d 0, 3, and 6 in culture were cultured in triplicate at 2610 5 cells per well in 96-well plates for 3 d in complete GIBCOH RPMI-1640 (Invitrogen Corporation) supplemented with 5 ng/ml of recombinant mouse GM-CSF (R&D Systems, Inc.) and 10 ng/ml of recombinant mouse IL-4 (BD Biosciences) at 37uC in 5% CO 2 and assayed for cell proliferation using the MTT cell proliferation kit [51].

BMDC Cell OVA-Alexa Fluor 488 Phagocytosis
OVA-Alexa Fluor 488 (Invitrogen Corporation) was added to 1610 6 BMDCs at a final concentration of 0.1 mg/ml. Endocytosis of the tracer was halted at 2 h by rapid cooling of the cells on ice and BMDC cells washed with ice-cold HBSS. The fluorescence intensity of the cells was analyzed by flow cytometry (FACSCanto TM Flow Cytometry System, BD Biosciences). Incubation of cells with endocytic tracer on ice was used as background control. The mean fluorescence intensity (MFI) represented the amount of incorporated tracer by APC-CD11c + cells (eBioscience) [53].

Statistical Analysis
The data are reported as the mean 6 SE of the mean. Differences were analyzed for significance (p,0.05) by analysis of variance (ANOVA) using the least significant difference method.