Type IIA Secreted Phospholipase A2 in Host Defense against Bacterial Infections.

The enzyme type IIA secreted phospholipase A2 (sPLA2-IIA) is crucial for mammalian innate host defense against bacterial pathogens. Most studies have investigated the role of sPLA2-IIA in systemic bacterial infections, identifying molecular pathways of bacterial resistance against sPLA2-IIA-mediated killing, and providing insight into sPLA2-IIA mechanisms of action. Sensitization of (antibiotic-resistant) bacteria to sPLA2-IIA action by blocking bacterial resistance or by applying sPLA2-IIA to treat bacterial infections might represent a therapeutic option in the future. Because sPLA2-IIA is highly expressed at mucosal barriers, we also discuss how sPLA2-IIA is likely to be an important driver of microbiome composition; we anticipate that future research in this area may bring new insights into the role of sPLA2-IIA in health and disease.

Regulatory Functions of Phospholipase A2.

Phospholipase A2 (PLA2) plays crucial roles in diverse cellular responses, including phospholipid digestion and metabolism, host defense and signal transduction. PLA2 provides precursors for generation of eicosanoids, such as prostaglandins (PGs) and leukotrienes (LTs), when the cleaved fatty acid is arachidonic acid, platelet-activating factor (PAF) when the sn-1 position of the phosphatidylcholine contains an alkyl ether linkage and some bioactive lysophospholipids, such as lysophosphatidic acid (lysoPA). As overproduction of these lipid mediators causes inflammation and tissue disorders, it is extremely important to understand the mechanisms regulating the expression and functions of PLA2. Recent advances in molecular and cellular biology have enabled us to understand the molecular nature, possible function, and regulation of a variety of PLA2 isozymes. Mammalian tissues and cells generally contain more than one enzyme, each of which is regulated independently and exerts distinct functions. Here we classify mammalian PLA2s into three large groups, namely, secretory (sPLA2), cytosolic (cPLA2), and Ca2+-independent PLA2s, on the basis of their enzymatic properties and structures and focus on the general undestanding of the possible regulatory functions of each PLA2 isozyme. In particular, the roles of type II sPLA2 and cPLA2 in lipid mediator generation are discussed.

Group V secretory phospholipase A2 reveals its role in house dust mite-induced allergic pulmonary inflammation by regulation of dendritic cell function.

We have previously shown that group V secretory phospholipase A(2) (sPLA(2)) regulates phagocytosis of zymosan and Candida albicans by a mechanism that depends on fusion of phagosomes with late endosomes in macrophages. In this study, we report that group V sPLA(2) (Pla2g5)-null mice exposed to an extract of house dust mite Dermatophagoides farinae had markedly reduced pulmonary inflammation and goblet cell metaplasia compared with wild-type (WT) mice. Pla2g5-null mice had also impaired Th2-type adaptive immune responses to D. farinae compared with WT mice. Pla2g5-null bone marrow-derived dendritic cells (BMDCs) activated by D. farinae had delayed intracellular processing of allergen and impaired allergen-dependent maturation, a pattern recapitulated by the native lung DCs of D. farinae-challenged mice. Adoptively transferred D. farinae-loaded Pla2g5-null BMDCs were less able than D. farinae-loaded WT BMDCs to induce pulmonary inflammation and Th2 polarization in WT mice. However, Pla2g5-null recipients transferred with WT or Pla2g5-null D. farinae-loaded BMDCs exhibited significantly reduced local inflammatory responses to D. farinae, even though the transfer of WT BMDCs still induced an intact Th2 cytokine response in regional lymph nodes. Thus, the expression of group V sPLA(2) in APCs regulates Ag processing and maturation of DCs and contributes to pulmonary inflammation and immune response against D. farinae. Furthermore, an additional yet to be identified resident cell type is essential for the development of pulmonary inflammation, likely a cell in which group V sPLA(2) is upregulated by D. farinae, and whose function is also regulated by group V sPLA(2).

T-cell and antibody responses to phospholipase A2 from different species show distinct cross-reactivity patterns.

BACKGROUND: Secreted phospholipases A2 (sPLA2) represent antigens to which humans may be rarely or frequently exposed. Thus, the investigation of humoral and cellular immune responses to sPLA2s from different species can provide a suitable model in the study of antibody and T-cell cross-reactivity. METHODS: Specific IgE, IgG1, IgG4, and IgA antibodies were analyzed by ELISA against sPLA2s from pancreas of Bos taurus (BT), Apis mellifera (AM) bee venom, Daboia russellii (DR) and Naja mossambica (NM) snake venoms, and human group III (hGIII) sPLA2 using sera of nonallergic beekeepers, AM-allergic patients, and healthy controls. T-cell cross-reactivity was investigated in PBMC, and T-cell clones (TCC) are generated against AM sPLA2. RESULTS: Hyperimmune and allergic individuals showed high levels of sPLA2-specific IgG4 and significant IgG4 cross-reactivity between BT, DR, and NM sPLA2s. Furthermore, IgE, IgA, and IgG1 cross-reactivities against BT, DR, and NM sPLA2s were also detectable in the range of 22.2-44.8%. Allergic patients showed significant T-cell proliferative response to NM sPLA2 together with increased IFN-γ and IL-13 production even though they had never been exposed to cobra venom. Although nonallergic healthy controls show no cross-reactivity at T-cell level, they did have low levels of IgG4 and IgA against BT, DR, and NM sPLA2s. Human TCC spanning three major T-cell epitopes of AM sPLA2 showed minor proliferative response to NM and hGIII sPLA2s. CONCLUSIONS: This study shows that T cells and antibodies may show cross-reactivity between different species without being naturally exposed to sPLA2s.

Roles of secreted phospholipase A2 group IIA in inflammation and host defense.

Among all members of the secreted phospholipase A2 (sPLA2) family, group IIA sPLA2 (sPLA2-IIA) is possibly the most studied enzyme. Since its discovery, many names have been associated with sPLA2-IIA, such as "non-pancreatic", "synovial", "platelet-type", "inflammatory", and "bactericidal" sPLA2. Whereas the different designations indicate comprehensive functions or sources proposed for this enzyme, the identification of the precise roles of sPLA2-IIA has remained a challenge. This can be attributed to: the expression of the enzyme by various cells of different lineages, its limited activity towards the membranes of immune cells despite its expression following common inflammatory stimuli, its ability to interact with certain proteins independently of its catalytic activity, and its absence from multiple commonly used mouse models. Nevertheless, elevated levels of the enzyme during inflammatory processes and associated consistent release of arachidonic acid from the membrane of extracellular vesicles suggest that sPLA2-IIA may contribute to inflammation by using endogenous substrates in the extracellular milieu. Moreover, the remarkable potency of sPLA2-IIA towards bacterial membranes and its induced expression during the course of infections point to a role for this enzyme in the defense of the host against invading pathogens. In this review, we present current knowledge related to mammalian sPLA2-IIA and its roles in sterile inflammation and host defense.

A non-venomous sPLA2 of a lepidopteran insect: Its physiological functions in development and immunity.

Eicosanoids are oxygenated C20 polyunsaturated fatty acids that mediate various physiological processes in insects. Eicosanoid biosynthesis begins with a C20 precursor, arachidonic acid (5,8,11,14-eicosatetraenoic acid: AA). AA is usually released from phospholipids at sn-2 position by catalytic activity of phospholipase A2 (PLA2). Although various PLA2s classified into 16 gene families (= Groups) are known in various biological systems, few PLA2s are known in insects. Only two PLA2s involved in intracellular calcium independent PLA2 (iPLA2) group have been identified in lepidopteran insects with well known eicosanoid physiology. This study reports the first secretory PLA2 (sPLA2) in lepidopteran insects. A partial open reading frame (ORF) of PLA2 was obtained by interrogating Spodoptera exigua transcriptome. Subsequent 3'-RACE resulted in a full ORF (Se-sPLA2A) encoding 194 amino acid sequence containing signal peptide, calcium-binding domain, and catalytic site. Phylogenetic analysis indicated that Se-sPLA2A was clustered with other Group III sPLA2s. Se-sPLA2A was expressed in most larval instars except late last instar. Its expression was inducible by immune challenge and juvenile hormone analog injection. RNA interference of Se-sPLA2A significantly suppressed cellular immunity and impaired larval development. These results suggest that non-venomous sPLA2 plays a crucial role in immune and developmental processes in S. exigua, a lepidopteran insect.

Distinguishing septic from normal donors by detection of sPLA2-IIA from human plasma using a microsieve-based immunoassay.

Bloodstream infections that progress to septic shock are responsible for hundreds of thousands of deaths each year, and are associated with significant healthcare costs. Recent studies have shown that a member of the secreted phospholipase protein family, termed sPLA2-IIA, may play a role during the innate immune response to bacterial infections, and is elevated in the plasma of septic patients. In this report, the feasibility of a simple microsieve-based sPLA2-IIA detection immunoassay was explored. Microsieves containing 5µm pores were covalently coupled with a sPLA2-IIA-specific monoclonal antibody at 0.1, 1.0, and 10µg/mL and then assayed with plasma-based positive and negative controls to determine the optimal coating concentration. Recombinant sPLA2-IIA was then serially diluted to a final concentration of 200, 100, 50, 25, 12.5, and 6.25ng/mL and tested alongside a non-spiked sample to estimate the detection limit of the prototype assay. Recombinant sPLA2-IIA was also spiked into serum, EDTA-plasma, and Lithium-Heparin plasma, in an effort to evaluate assay performance when analyzing these sample matrices. The preliminary limit of detection studies suggests that the microsieve assay is able to distinguish approximately 6-12ng/mL of sPLA2-IIA from a non-spiked sample. When compared to an immunoassay diluent, the microsieve assay also yielded acceptable percent recoveries for each of the three sample matrices spiked with clinically significant levels of sPLA2-IIA. The sPLA2-IIA microsieve assay prototype also clearly distinguished five samples from septic patients from five normal donor samples, and the results were in good agreement with a comparator ELISA test system (R2=0.9347).

Emerging roles of secreted phospholipase A2 enzymes: the 3rd edition.

Within the phospholipase A2 (PLA2) superfamily, secreted PLA2 (sPLA2) enzymes comprise the largest family that contains 11 to 12 mammalian isoforms with a conserved His-Asp catalytic dyad. Individual sPLA2s exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting distinct biological roles. Individual sPLA2s are involved in diverse biological events through lipid mediator-dependent or -independent processes and act redundantly or non-redundantly in a given microenvironment. In the past few years, new biological aspects of sPLA2s have been clarified using their transgenic and knockout mouse lines in combination with mass spectrometric lipidomics to unveil their target substrates and products in vivo. In the 3rd edition of this review series, we highlight the newest understanding of the in vivo functions of sPLA2s in pathophysiological conditions in the context of immunity and metabolism. We will also describe the latest knowledge on PLA2R1, the best known sPLA2 receptor, which may serve either as a clearance or signaling receptor for sPLA2 or may even act independently of sPLA2 function.

Roles of secreted phospholipases A2 in the mammalian immune system.

Secreted phospholipase A2 (sPLA2) molecules constitute a family of proteins that are involved functionally in many biological processes. In particular, they participate in diverse pathophysiological settings as enzymes that release free fatty acids and lysophospholipids from phospholipids in biological membranes, or as ligands for various cellular receptors. In this review the confirmed or expected functions of sPLA2s in the mammalian immune system are surveyed. Some of the twelve mammalian sPLA2 molecules constitute part of the so-called innate immune system by virtue of their antibacterial, antiviral and antifungal activities. They are also involved in acute inflammation, a protective reaction of the body to infection or injury. The acute inflammation sometimes escapes regulation, becomes chronic and can evolve into a severe pathology. One or more types of sPLA2 are involved in asthma, rheumatoid arthritis, sepsis, atherosclerosis, myocardial infarction, Crohn's disease, ulcerative colitis and cancer. sPLA2s are thus important therapeutic targets as well as biotherapeutic molecules. Improving the selectivity of inhibitors of sPLA2s to be able to target a particular sPLA2 could therefore be one of the most important tasks for future research.

Phospholipase A2 in experimental allergic bronchitis: a lesson from mouse and rat models.

BACKGROUND: Phospholipases A2 (PLA2) hydrolyzes phospholipids, initiating the production of inflammatory lipid mediators. We have previously shown that in rats, sPLA2 and cPLA2 play opposing roles in the pathophysiology of ovalbumin (OVA)-induced experimental allergic bronchitis (OVA-EAB), an asthma model: Upon disease induction sPLA2 expression and production of the broncho-constricting CysLTs are elevated, whereas cPLA2 expression and the broncho-dilating PGE2 production are suppressed. These were reversed upon disease amelioration by treatment with an sPLA2 inhibitor. However, studies in mice reported the involvement of both sPLA2 and cPLA2 in EAB induction. OBJECTIVES: To examine the relevance of mouse and rat models to understanding asthma pathophysiology. METHODS: OVA-EAB was induced in mice using the same methodology applied in rats. Disease and biochemical markers in mice were compared with those in rats. RESULTS: As in rats, EAB in mice was associated with increased mRNA of sPLA2, specifically sPLA2gX, in the lungs, and production of the broncho-constricting eicosanoids CysLTs, PGD2 and TBX2 in bronchoalveolar lavage (BAL). In contrast, EAB in mice was associated also with elevated cPLA2 mRNA and PGE2 production. Yet, treatment with an sPLA2 inhibitor ameliorated the EAB concomitantly with reverting the expression of both cPLA2 and sPLA2, and eicosanoid production. CONCLUSIONS: In both mice and rats sPLA2 is pivotal in OVA-induced EAB. Yet, amelioration of asthma markers in mouse models, and human tissues, was observed also upon cPLA2 inhibition. It is plausible that airway conditions, involving multiple cell types and organs, require the combined action of more than one, essential, PLA2s.

The standard mouse assay of anti-venom quality does not measure antibodies neutralising the haemorrhagic activity of Vipera ammodytes venom.

The venom of Vipera ammodytes ammodytes (Vaa), like the venoms of other Viperinae snakes, is largely haemorrhagic and necrotising, and only to a lesser extent neurotoxic to humans. The components most extensively studied so far, and most probably involved in generating the observed pathologies, are haemorrhagins (H), members of the metalloproteinase group of enzymes, and neurotoxic ammodytoxins (Atxs), that belong to the secretory phospholipases A2. Rabbit antisera were prepared containing functional antibodies specific for each class of pathology-inducing venom constituents and for both classes together. The involvement of these antibodies in neutralising the toxicity of whole Vaa venom was assessed using the ED50 assay in mice. This assay is the only regulatorily approved assay for estimating anti-venom potency and as such has the task to quantify the active compound neutralising venom-induced pathology of the anti-venom. Fully functional anti-Atx antibodies were shown to be responsible for neutralising the portion of venom toxicity, while anti-H antibodies were not protective in this assay. Thus, the mouse ED50 assay, intended to measure the active principle of the anti-venom, does not measure antibodies specific for Vaa venom haemorrhagins, and consequently does not fulfil its primary task from the regulatory point of view.

Route and type of nutrition and surgical stress influence secretory phospholipase A2 secretion of the murine small intestine.

BACKGROUND: The function of secretory phospholipase A2 (sPLA2) is site dependent. In tissue, sPLA2 regulates eicosanoid production; in circulation, sPLA2 primes neutrophils; and in the intestinal lumen, sPLA2 provides innate bactericidal immunity as a defensin-related protein. Since parenteral nutrition (PN) primes leukocytes while suppressing intraluminal mucosal immunity, the authors hypothesized that (1) PN would diminish luminal sPLA2 activity but increase activity in intestinal tissue and serum and (2) stress would accentuate these changes. METHODS: Mice received chow, a complex enteral diet (CED), intragastric PN (IG-PN), or PN in experiment 1 and chow, chow+stress, PN, or PN+stress in experiment 2. RESULTS: In experiment 1, luminal sPLA2 activity was greatest in chow and decreased in CED, IG-PN, and PN, with PN lower than CED and IG-PN. Compared to that after chow, serum sPLA2 activity dropped after CED, IG-PN, and PN. Serum sPLA2 was higher in portal than systemic serum. In experiment 2, PN lowered luminal sPLA2 activity vs chow. Stress lowered luminal sPLA2 activity in chow, without change in PN. Following stress, luminal immunoglobulin A increased in chow but not PN. Serum sPLA2 activity increased in PN. CONCLUSIONS: PN attenuates sPLA2 activity in intestinal fluid, consistent with suppressed innate mucosal defense. Stress suppresses luminal fluid sPLA2 activity in chow but not the immunoglobulin A response; PN impairs both. Stress significantly elevates serum sPLA2 in PN-fed mice, consistent with known increased neutrophil priming with PN. PN reduces innate bactericidal immunity of the gut but upregulates serum proinflammatory products poststress.

A novel anti-inflammatory role for secretory phospholipase A2 in immune complex-mediated arthritis.

Phospholipase A2 (PLA2) catalyses the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA2 enzymes (sPLA2), numbering nine members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA2 in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene-deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA2 isoform plays a novel anti-inflammatory role that opposes the pro-inflammatory activity of group IIA sPLA2. Mechanistically, group V sPLA2 counter-regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti-inflammatory function for a PLA2 and identify group V sPLA2 as a potential biotherapeutic for treatment of immune-complex-mediated inflammation.

The role of antibodies specific for toxic sPLA2s and haemorrhagins in neutralizing potential of antisera raised against Vipera ammodytes ammodytes venom.

The contribution of antibodies directed against the two main toxic groups of proteins in the Vipera ammodytes ammodytes venom, haemorrhagic metalloproteinases (H) and neurotoxic sPLA2s (Atxs), to the overall protective efficacy of the whole venom antisera was investigated. Using ELISA assays we established a high correlation between the protective efficacy of the whole venom antisera in mice and their anti-Atxs antibody content. As the haemorrhage is the prevailing toxic effect of the venom in human, the lack of correlation also with anti-H IgG content exposed that the mouse model might not be optimal to evaluate the neutralizing potential of the venom-specific antisera for human therapy. We further revealed that Atxs and structurally very similar but non-toxic AtnI2 from the venom are not immuno cross-reactive.

Jak2 dampens the induction by IL-1beta of prostaglandin endoperoxide H synthase 2 expression in human orbital fibroblasts: evidence for divergent influence on the prostaglandin E2 biosynthetic pathway.

Prostaglandin endoperoxide H synthase 2 (PGHS-2) catalyzes the rate-limiting steps in the synthesis of PGE(2). It is substantially but transiently induced in human orbital fibroblasts treated with IL-1beta. In this study, we report that the induction of PGHS-2 by IL-1beta is dramatically enhanced and prolonged when Jak2 signaling is abrogated, either with the specific inhibitor AG490 or by transiently transfecting fibroblasts with a dominant negative mutant Jak2. Attenuating Jak2 increases PGHS-2 steady-state mRNA levels, a consequence of increased gene transcription and mRNA survival in IL-1beta-treated cultures. Surprisingly, interrupting Jak2 function also blocked the expected increase in PGE(2) synthesis usually provoked by IL-1beta. This resulted from the rapid loss of IL-1beta-dependent arachidonate release and by attenuation of group IIA secreted PLA(2) (sPLA(2)) gene induction. Supplying Jak2-compromised cultures with exogenous arachidonate failed to increase PGE(2) production in response to IL-1beta until cells were mechanically disrupted. However, transiently transfecting them with wild-type sPLA(2) fully restored prostanoid production to anticipated levels. sPLA(2) expression following transfection resulted in increased IL-1beta-dependent PGHS-2 and microsomal PGE(2) synthase levels. Thus, sPLA(2) plays important roles in PGE(2) synthesis in addition to its release of arachidonate. Our findings suggest that Jak2 ordinarily dampens and limits the duration of the PGHS-2 induction by IL-1beta. Moreover, it is required for IL-1beta-dependent signaling to sPLA(2), the expression and activity of which are necessary for up-regulating PGE(2) synthesis in orbital fibroblasts.