Multiple Roles of Phospholipase A₂ during Lung Infection and Inflammation

Expression of sPLA₂ in the lung.Several studies have detected the presence of sPLA₂ in the lung (5, 88). In conditions such as asthma, ARDS, and bronchial pneumonia, elevated amounts of sPLA₂ are found in the bronchial/alveolar lavage fluid (BALF) and lung tissue of patients (5, 7, 18, 50, 72, 88, 108). In fact, a positive correlation exists between increased levels of sPLA₂ and a poor clinical outcome (5, 88). A major source of sPLA₂ is the alveolar macrophage (5). Group IIA sPLA₂ expression in the alveolar macrophage is induced by inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), as well as bacterial products, including lipopolysaccharide (LPS) (4, 5, 133). Increased expression of sPLA₂ colocalizes with areas of intense inflammation (5). In addition to macrophages, the epithelial cells along the respiratory tract may be an important source of sPLA₂s, as these cells express many sPLA₂ isoforms (44, 65, 66, 109). Human bronchial and nasal cell lines, as well as primary nasal mucosa, have displayed detectible mRNA expression of all sPLA₂ isoforms (65, 66). Increased expression of group IIA and IID is observed when epithelial cells are treated with gamma interferon (IFN-γ) (66). In a study investigating mRNA expression in the nasal mucosa, group X sPLA₂ displayed the highest expression level among the sPLA₂ isoforms (65). In studies examining human whole-lung tissue, groups V and X are generally highly expressed in the epithelium (72, 109). The results of one study comparing the protein expression levels of various sPLA₂ isoforms in normal lung tissue to the levels in lung tissue from patients having infectious bronchial pneumonia displayed discrete isoform localization in both control and diseased states (72). Under normal conditions, staining of group II isoforms is negligible, whereas group V displays a scattered expression pattern in the bronchial epithelium. Group X is strongly expressed in the alveolar epithelium and the interstitial tissue below the bronchial epithelium (72). In patients with pneumonia, group V is enhanced, with strong expression in the bronchial epithelium, lung fibroblasts, and macrophages. Group X remains highly expressed in the epithelium and the fibroblasts of patients with pneumonia. Group IIA, which is undetectable in control healthy lungs, is significantly expressed in the pulmonary arterial walls and the chondrocytes from pneumatic patients (72). The group IID isoform is increased in both macrophages and bronchial epithelial cells but decreased in alveolar epithelial cells. This study also reported that the protein expression levels for groups IIE and IIF are negligible in the lungs of both healthy controls and patients with pneumonia (72). There exists speculation that sPLA₂ contributes to lung inflammation occurring in the disease CF (75). The group IIA isoform is expressed at a significantly higher level in human lung epithelial cell lines possessing a defective CF transmembrane receptor (CFTR) than in genetically matched CFTR-normal epithelial cells (75). Many sPLA₂ isoforms have also been observed to be expressed in the mouse lung, and studies have revealed that intranasal or intratracheal instillation of antigen, LPS, or P. aeruginosa in mice, rats, and guinea pigs leads to severe inflammation, including the influx of immune cells into the airspace in a process that requires sPLA₂ (6, 25, 45, 67). Taken together, it is clear from data obtained from both humans and mice that various sPLA₂s are produced by multiple lung cell types in response to a diversity of stimuli. The role of each distinct isoform in a given lung cell type during inflammatory disease is not entirely clear; however, significant progress has been made over the past decade, and multiple distinctive functions have been ascribed to certain sPLA₂ isoforms.

Induction of cytokines and chemokines.Cytokines and chemokines are critical to the inflammatory and immune responses by serving as signals that orchestrate the actions of immune cells. Lung microvascular endothelial cells produce various CXC neutrophil chemokines, such as interleukin-8 (IL-8), Groα, and ENA-78, in response to LPS treatment (12). Group IB and IIA potentiate this pathway through the activation of NF-κB and the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) pathway (12). Group IB sPLA₂ is also capable of inducing the production of cytokines and chemokines by lung macrophages (38, 39). Upregulation of IL-8, MCP-1, and MIP-1β chemokine expression, as well as TNF-α, IL-6, and IL-10 cytokine expression, is observed when macrophages are treated with group IB sPLA₂. These events occur through the activation of multiple kinase signaling pathways, including NF-κB and ERK (38). In addition to group IB, group X can induce TNF-α and IL-6 expression in macrophages (39). Interestingly, stimulation of cytokines by group IB and X may not require sPLA₂ enzymatic activity. Instead, certain sPLA₂ isoforms bind to a specific receptor known as M-type receptor (or PLA₂R) and activate kinase signaling pathways through engagement of this mannose-type receptor (43).

A recent study investigating single-nucleotide polymorphisms (SNPs) has revealed an association between an SNP in the gene for sPLA₂ group IID and increased weight loss contributing to a worse prognosis in COPD. This SNP in group IID results in a single amino acid change which may modify the activity of the group IID enzyme and, as speculated by the authors, alter the inflammatory process by enhancing sPLA₂-mediated cytokine production (116). Induced expression of chemokines and cytokines likely contributes to the process of immune effector cell recruitment. Consistent with this notion, studies have demonstrated a dramatic influx of neutrophils into the lung upon intratracheal administration of sPLA₂ (38). Diseases where sPLA₂ levels are elevated, such as ARDS, asthma, and pneumonia, are marked by excessive neutrophil influx.

Certain PLA₂ groups also impact the adaptive immune system through the ability to activate signaling pathways that result in the generation of cytokines (94, 97). The maturation of dendritic cells (DCs) from monocytes has been demonstrated to occur as a result of treatment with group III sPLA₂s (94, 97). Group III sPLA₂ can activate transcription factors, such as NF-κB, AP-1, and NFAT, in DCs which likely generate a diversity of bioactive molecules involved in organizing the effector immune response. DCs matured by sPLA₂ group III display increased migratory capacity and, when mixed with T cells, cause T-cell release of IFN-γ. T-cell production of IFN-γ is an important step in the generation of an inflammatory Th1-type adaptive immune response. Interestingly, this ability of sPLA₂ to induce maturity of DCs, which can modulate T-cell activity, is dependent on sPLA₂ enzymatic activity rather than involving binding to a receptor (94). Clearly, sPLA₂ stimulation of cytokines and chemokines in various cell types involves multiple distinct mechanisms and these processes have dramatic effects on immune and inflammatory processes.

Generation of eicosanoids.The enzymatic function of PLA₂ is to cleave phospholipids into free fatty acids and lysophospholipids (Fig. 1). In the case where arachidonic acid (AA) is the free fatty acid in the sn-2 position of the phospholipid target, AA release from phospholipids can subsequently be converted into a variety of functionally diverse lipid mediators collectively known as eicosanoids (Fig. 2) (106). Eicosanoids are a class of bioactive lipids derived from AA, which includes prostaglandins (PGs), leukotrienes (LTs), hepoxilins, and lipoxins (LXs), as well as several others that are synthesized by the actions of lipoxygenases and cycloxygenases on the common substrate, AA (Fig. 2) (110). Eicosanoids, such as the leukotriene LTB₄ and the hepoxilin HXA₃, serve as neutrophil chemoattractants, playing a key role in the inflammatory process. On the other hand, PGs, including PGE₂, and LXs, such as LXA₄, can protect against excessive inflammation in the lung, as well as participate in lung tissue repair (110, 122). The generation of AA by PLA₂ is considered to be the rate-limiting step in the synthesis of eicosanoids, and thus, PLA₂ plays a critical role in the generation of all eicosanoids. Unlike other PLA₂ families, sPLA₂s do not selectively target phospholipids that possess AA at the sn-2 position, which has led many to speculate on a less-prominent role for sPLA₂s in eicosanoid synthesis. Nevertheless, there are numerous cases where sPLA₂ isoforms are fully capable of independently generating AA for conversion to eicosanoids, in addition to possessing the ability to cooperate with other PLA₂s to mediate this important function (32, 99).

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FIG. 2.

Represents a simplified schematic of some eicosanoid synthesis pathways occurring within a mammalian cell. PLA₂ hydrolyzes membrane phospholipids, releasing AA. AA can then be converted to eicosanoids, such as LTs, LXs, and hepoxilins, through the actions of lipoxygenases (LOs), or PGs, through the actions of cyclooxygenases (COX). Enzymes are depicted within boxes.

Group IB and group II sPLA₂s are capable of inducing AA release in both bronchial epithelial cells and mast cells, with group IB displaying greater efficiency than group II (6). When group IIF, V, and X sPLA₂s are transfected into bronchial lung epithelial cells and lung fibroblasts, significant AA release and subsequent PGE₂ production occurs. This is not observed for groups IIA, IID, and IIE. The catalytic activity of the sPLA₂s is required for the increased AA to occur in these transfection studies, suggesting direct enzymatic involvement of these sPLA₂s in this process (72). Group V and X are much more efficient than group IIA at hydrolyzing phosphatidylcholine (PC), which potentially accounts for the greater effectiveness of group V and X at generating AA from mammalian cell membrane targets. High levels of eicosanoids have been reported in the BALF of CF patients (75). Baseline and LPS-induced sPLA₂ group IIA expression, as well as production of PGE₂, are enhanced in CF epithelial cells. Increased AA and subsequent PGE₂ release precedes sPLA₂ group IIA expression, suggesting that the enzymatic source of increased AA and PGE₂ in CF epithelial cells is likely the result of cPLA₂, rather than sPLA₂, activity (75). In some instances it has been reported that sPLA₂s induce AA release indirectly by binding to its receptor and stimulating kinase pathways that lead to the phosphorylation and activation of cPLA₂, which is known to be directly responsible for the generation of AA (32, 99).

Neutrophils treated with group V, but not group IIA phospholipases, release AA and demonstrate increased LTB₄ synthesis (57). The mechanism mediating increased LTB₄ synthesis does not simply involve the hydrolysis of neutrophil extracellular-membrane phospholipids by group V sPLA₂. Instead, the extracellular-membrane hydrolysis products resulting from group V sPLA₂ activity, lysophospholipids, and AA serve to stimulate cPLA₂ activity in the perinuclear region of neutrophils. The actions of cPLA₂ generate a discrete pool of AA derived from perinuclear phospholipids that can then be converted to LTB₄ by 5-lipoxygenase, also located in the perinuclear region. Furthermore, LTB₄ produced by the neutrophils is also released, causing a second wave of cPLA₂ activation by an autocrine positive feedback mechanism. LTB₄ interacts with LTB₄ receptors on the neutrophil surface and further activates cPLA₂ through calcium influx and activation of the ERK pathway, which leads to further LTB₄ production (57). For this complex mechanistic process to occur in vivo, the initial group V sPLA₂ activation of neutrophils requires an alternative, nonneutrophil cell source of group V sPLA₂, which is speculated to be provided by other inflammatory cells, such as mast cells or macrophages (57). Indeed, transcellular sPLA₂ activity for the purposes of eicosanoid generation has been reported in other studies (77, 79, 128). Bronchial epithelial cells produce group V sPLA₂ in response to endothelin-1, which is then capable of directly hydrolyzing eosinophil membranes to produce AA for subsequent conversion to cysteinyl LTs, such as LTC₄. LTC₄s, as well as other eicosanoids, are frequently found to be enhanced in the BALF of asthmatic patients (64, 77, 128). Of particular note, AA release leading to LTC₄ synthesis in eosinophils was shown to occur independently of cPLA₂, demonstrating the ability of sPLA₂ group V to directly generate AA for eicosanoid synthesis (77, 79, 128). A mouse model where the sPLA₂ group V gene has been knocked out results in significantly reduced eicosanoid generation in ex vivo-stimulated macrophages, further emphasizing the role of group V in eicosanoid generation (102). A more-recent study exploring the role of group V sPLA₂ in a mouse model of airway hyperresponsiveness has further established the importance of group V sPLA₂ to the lung inflammatory process in vivo (78).

Group X sPLA₂ appears to possess the ability to act directly on the extracellular-membrane surface for mediating AA release and PGE₂ production (99). A recent study exploring the role of group X sPLA₂ in a mouse model of asthma establishes a critical role for group X in immune cell recruitment, cytokine production, and airway remodeling (45). sPLA₂ group X-knockout mice produce significantly less PGs and LTs upon challenge with antigen, and this appears to underlie the much-attenuated asthmatic response to allergen of group X-knockout mice compared with the response of wild-type mice (45).

Evidence for the ability of sPLA₂ group III to facilitate the production of PGE₂ has recently been reported (81). Group III exhibits a lack of sequence homology with other members of the sPLA₂ group, except in the catalytic site and the calcium binding loop where significant sequence homology exists between group III and the other sPLA₂ isoforms (81). The role of group III sPLA₂s in contributing to the synthesis of eicosanoids in the setting of lung inflammation is largely unexplored.

Eicosanoids are major contributors to the lung inflammatory process, and as described above, many of the sPLA₂ isoforms are capable of generating their common substrate, AA. Significant evidence establishes a role of sPLA₂ in eicosanoid synthesis, either by direct enzymatic action on membrane phospholipids or through receptor signaling leading to activation of the cPLA₂ group.

Effects on lung surfactant.Surfactant is a complex combination of lipids and proteins that is secreted by airway epithelial cells into the airspace for the purpose of reducing surface tension, thereby preventing alveolar collapse during the breathing process (101). Lung inflammatory conditions, such as ARDS and asthma, are marked by surfactant dysfunction (101). Surfactant provides a barrier to pathogens as well, and thus, deficiencies in surfactant may predispose individuals to infectious pneumonia (101). As discussed above, various sPLA₂ isoforms are produced in the lung, mainly by macrophages and epithelial cells, and they appear to have direct affects on lung surfactant.

The lipid component of surfactant predominantly consists of PC, but also contains a small percentage of phosphatidylglycerol, phosphatidylinositol, and phosphatidylethanolamine (PE) (51). Various sPLA₂ isoforms have different specificities for hydrolyzing particular phospholipid substrates with particular head groups. Hydrolysis of surfactant phospholipids results in a buildup of free fatty acids and lysophospholipids (Fig. 1). Both a reduction in surfactant phospholipid content and the accumulation of released lysophospholipids can be detrimental to surfactant function. Groups IB and V favor hydrolysis of PC, whereas group IIA is far more effective at hydrolyzing phosphatidylglycerol. Thus, it appears that different sPLA₂ isoforms act on distinctive regions of the lipid portion of surfactant (50, 52). Mice that are transgenic for group V expire immediately after birth due to an inability to breathe resulting from a marked reduction in lung surfactant (87). Thus, group V, at least in mice, is quite effective at hydrolyzing surfactant.

In addition to serving as a target of sPLA₂ activity, surfactant appears to exert a certain degree of control over sPLA₂ (133, 134). Both lipid constituents and protein components of surfactant, such as the collectin protein SP-A, can cause a major reduction in the expression of sPLA₂ group IIA in alveolar macrophages (134). In fact, SP-A has been shown to specifically bind to group IIA sPLA₂ and to reduce the ability of sPLA₂ group IIA to hydrolyze phosphatidylglycerol (5, 18). Decreased SP-A levels in BALF are observed in ARDS patients, where sPLA₂ activity is more abundant (18). Thus, during inflammatory lung disease processes, a counterbalance between sPLA₂ and surfactant appears to be deranged, ultimately manifesting in surfactant degradation. The degradation of surfactant and subsequent loss of phospholipids and collectins, such as SP-A, relieves their repressive effects on sPLA₂, further exacerbating surfactant function and perpetuating the lung inflammatory process.

Bactericidal activity.In addition to the numerous contributions toward the enhancement of the inflammatory response attributable to sPLA₂s, several isoforms exert an innate defensive role by virtue of their inherent bactericidal activity (15). Gram-positive organisms, such Listeria monocytogenes, Staphylococcus aureus, and Bacillus anthracis, appear to be directly susceptible to sPLA₂ activity both in vitro and in vivo owing to the ability of sPLA₂ to hydrolyze bacterial-membrane phospholipids (36, 41, 42, 61, 96). Interestingly, lethal factor produced by B. anthracis is capable of down-regulating sPLA₂ group IIA expression by alveolar macrophages, a potential virulence mechanism utilized by anthrax to protect itself from destruction by host defenses (36). Gram-negative bacteria, such as Escherichia coli, are also susceptible to sPLA₂ bactericidal activity; however, additional factors, such as bactericidal permeability-increasing protein or components of complement, are required for effective sPLA₂-mediated bacterial destruction (60, 62). Interestingly, not all gram-negative bacteria require cofactors, as the gram-negative lung pathogen P. aeruginosa is potently susceptible to direct sPLA₂-mediated killing. P. aeruginosa killing by sPLA₂ occurs even under the high salt or acidic conditions that are found in the lungs of individuals with CF. This is in contrast to other bactericidal factors that are less effective under the conditions present in the CF lung (24). Bacterial membranes exhibit a different phospholipid composition than mammalian cell membranes, with a greater presence of PE and phosphatidylglycerol and a lesser presence of PC (42). This attribute of bacterial membranes likely accounts for the observation that group IIA exerts the greatest bactericidal activity among the sPLA₂ isoforms against gram-positive bacteria (59). Groups V and X possess bactericidal activity to gram-positive pathogens, albeit to a lesser extent than group IIA, and group IB has minimal bactericidal activity (59). Almost all sPLA₂ groups require cofactors to kill gram-negative bacteria, such as E. coli. Group XII represents an exception, however, and is able to directly kill E. coli without need of additional factors (59). The bactericidal activity of sPLA₂ isoforms represents a defense mechanism against infectious lung diseases through the ability of sPLA₂ to eliminate bacterial pathogens prior to initiating or exacerbating the inflammation process.

cPLA₂s.cPLA₂ consists of four known isoforms in humans, designated group IVA (cPLA₂-α), IVB (cPLA₂-β), IVC (cPLA₂-γ), and the newly identified IVD (cPLA₂-δ) (33, 34, 106). The most-extensively investigated cPLA₂ isoform is the group IVA isoform (cPLA₂-α), which is ubiquitously expressed in human tissue, including the lung, with prominent expression in the lung epithelium, endothelium, fibroblasts, macrophages, and neutrophils (33, 106). The group IVA cPLA₂-α resides within the cytosol of these various cell types but shifts to the nuclear membrane upon stimulation, where it then gains access to phospholipids (33, 106). Unlike sPLA₂s, cPLA₂s have a substrate preference for phospholipids with AA in the sn-2 position, rendering cPLA₂ more selective for the purposes of eicosanoid generation (Fig. 1) (33, 106). In addition to translocation to nuclear membranes, cPLA₂ is also activated by phosphorylation, whereby the addition of phosphate to certain amino acid residues serves to enhance the specific enzymatic activity (33, 106). The catalytic activity of cPLA₂ is not dependent on calcium, in contrast to sPLA₂s, which require millimolar calcium for enzyme action. However, the translocation of cPLA₂ from the cytosol to the membrane does require calcium, making calcium-mobilizing agents potent activators of cPLA₂ (33, 106).

Stimulants of cPLA₂ in lung epithelial cells include cytokines, such as TNF-α, IFN-γ, and IL-1α, as well as oxidative stress (93, 130, 131, 132, 136). Oxidative stress is also a source of cPLA₂ stimulation in lung fibroblasts (68). The results of several studies suggest that protein signaling kinases, such as ERK1/2, protein kinase C, and p38, play a prominent role in cPLA₂ activation by mediating cPLA₂ phosphorylation (93, 129). These protein kinase signaling pathways are frequently activated by cytokines, such as TNF-α, in many cell types. Cytokines are also capable of stimulating cPLA₂ by inducing mRNA and protein expression, as observed in both lung epithelial cells and lung fibroblasts (23, 131, 132).

cPLA₂ has been proposed to play a significant role in airway inflammatory diseases, such as ARDS, as cPLA₂ is significantly elevated in lung tissues in response to multiple pathological stimuli (83). The primary function of cPLA₂ in lung tissues is considered to be the generation of AA from membrane phospholipids for the synthesis of eicosanoids (106). Since multiple eicosanoids have divergent functions during lung inflammation, cPLA₂ serves as a key enzyme in mediating many aspects of the lung inflammatory process. AA metabolites produced by cPLA₂ also appear to be involved in mediating the transcription of genes involved in lung inflammation, such as IL-8, through interaction with the ligand-associated nuclear transcription factor peroxisome proliferator-activated receptor (91, 92). A role for cPLA₂ in promoting apoptosis has also been reported (58).

A number of studies have supported the notion of cPLA₂ as a key enzyme in the synthesis of eicosanoids during lung inflammation (83, 84, 98, 121). cPLA₂-α-knockout mice have been generated and exploited in the context of airway disease models (83, 84, 98, 121). An evaluation of the role of cPLA₂-α during the pathological process of ARDS has been conducted whereby both wild-type and cPLA₂-α-knockout mice are subjected to intratracheal installation of LPS, zymosan, or HCl. Treatment of wild-type mice with the various insults results in a significant enhancement of the quantity of eicosanoids, including trioxilins and Cys-LTs, in the BALF (83). In parallel to the increase in the quantity of eicosanoids, severe lung edema, neutrophil infiltration, and exacerbation of gas exchange occur in wild-type mice subjected to treatment. The pathological findings are markedly less prominent in the cPLA₂-α-knockout mice, with low levels of detectable eicosanoids present in the BALF after insult. Such results suggest that cPLA₂ plays a critical role in facilitating this robust inflammatory process in the airway, likely through the generation of eicosanoid mediators (83). In a model of acute allergy, antigen challenge of the mouse airway, which normally causes a severe anaphylactic response, results in reduced anaphylaxis in cPLA₂-α-knockout mice compared with the anaphylaxis in wild-type mice (121). Macrophages from cPLA₂-knockout mice are completely unable to produce PGE₂, Cys-LTs, and PAF in response to LPS or calcium ionophors. Thus, data from studies employing cPLA₂-α-knockout mice support a mechanistic model in which cPLA₂ facilitates the production of eicosanoids in the lung (121).

As predicted from the results of the knockout studies, inhibitors of cPLA₂ can partially reduce the neutrophil infiltration observed in LPS-induced lung injury in normal mice and thus may represent potential therapeutics for pathological lung inflammatory processes (84). However, despite the seemingly detrimental role cPLA₂-α plays in certain pathological lung situations, such as ARDS and acute allergy, it likely exhibits a protective role in other situations. Tracheal instillation of live E. coli, rather than LPS alone, in the cPLA₂-α-knockout mice, has revealed a severe defect in the ability of cPLA₂-deficient neutrophils to eradicate the bacteria, suggesting the importance of cPLA₂ in the clearance of bacterial infection (98). The inability of cPLA₂-α-knockout mice to eliminate infection appears to be directly due to the inability of cPLA₂-α-deficient neutrophils to generate sufficient AA for the production of eicosanoids. Although the administration of cPLA₂ inhibitors may aid in relieving overly active and potentially detrimental aspects of inflammation, in the context of an active infection, such treatment may cause more harm by allowing an infection to thrive (98).

A role for cPLA₂ in multiple animal models of asthma has also been reported (20, 21, 82). Both mast cells and eosinophils appear to require cPLA₂ in order to exert their effector functions in the lung (20, 21, 82). Both cell types are important sources of eicosanoids, and eicosanoids, such as Cys-LTs, are critical modulators of asthmatic disease.

The heritable condition CF, which results in bouts of severe lung inflammation associated with chronic bacterial infection over the life span of afflicted individuals, appears to involve cPLA₂ dysregulation in the lung. CF epithelial cells have been reported to produce more eicosanoids and have a higher amount of cPLA₂ activity than normal epithelial cells (14). Heightened cPLA₂ activity leading to overproduction of eicosanoids may exacerbate the inflammatory process, leading to severe lung injury as is observed in CF (14). Our group and others have demonstrated that the bacterial pathogen most relevant to CF, P. aeruginosa, can induce activation of cPLA₂ in lung epithelial cells (53, 58), perhaps further enhancing cPLA₂-mediated eicosanoid generation. Clearly, cPLA₂ is central to numerous lung diseases that are marked by a severe inflammatory process.

Although the majority of research regarding the involvement of cPLA₂ in lung inflammation has focused on cPLA₂-α, group IVC cPLA₂-γ has recently been implicated in airway inflammation in certain cases as well (70). Alveolar macrophages pretreated with the hormone leptin produce PGE₂ and LTB₄ when challenged with zymosan or calcium ionophors through a mechanism that is completely independent of cPLA₂-α but is associated with an increased expression of cPLA₂-γ (70). Leptin is believed to be increased during lung inflammatory conditions, including pneumonia and asthma (70). Thus, cPLA₂-γ should also be considered as a potential contributor to eicosanoid generation during lung disease.

Clearly the PLA₂ group known as cPLA₂ plays a significant role in many lung inflammatory diseases, including pneumonia, asthma, ARDS, and CF. Its main function as a key enzyme in eicosanoid synthesis make it an attractive target for therapeutics, but it should always be considered that other PLA₂ groups are also capable of generating eicosanoids. A better understanding of the role played by individual PLA₂ isoforms in the generation of distinct eicosanoids by certain tissues and cell types during particular stages of disease processes will be crucial information for designing effective therapeutic intervention.

iPLA₂s.iPLA₂s have many characteristics in common with cPLA₂s, including the facts that both groups are of comparable size, both groups possess a similar active-site structure involving a key serine residue, both groups are expressed in the cytosol, and both groups act on phospholipids derived from internal membranes (8, 106). One crucial difference between the two groups is that the activation of iPLA₂ does not require calcium (8, 106). Also, unlike cPLA₂s, iPLA₂ isoforms do not show a substrate preference for phospholipids with AA at the sn-2 position (8, 106). The two known isoforms that represent the iPLA₂ group are VIA and VIB, or iPLA₂-β and iPLA₂-γ, respectively. Group VIA has five identified mRNA splice variants, with two of the splice variants lacking enzymatic activity and possibly serving an inhibitory role (8). Group IVA has been more widely investigated, and much of the information gathered regarding the iPLA₂ group is based on studies with group IVA. In addition to enhanced gene expression, alternative splicing, and/or enhanced protein expression of iPLA₂ upon stimulation, posttranslational mechanisms of iPLA₂ activation have been described (8). For example, the activity of iPLA₂ has been demonstrated to be modulated by interaction with calmodulin, the presence of ATP, phosphorylation, oligomerization via ankyrin repeats, proteolytic processing, and substrate availability (8). The mechanism of activation of iPLA₂ depends greatly on the particular stimulus, as well as the cell type involved. Alveolar macrophages and airway epithelial cells are capable of expressing iPLA₂, and activity in both cell types appears to be inducible (8, 65, 106, 124).

During cell growth, iPLA₂ serves to facilitate membrane remodeling by deacylation and reacylation of membrane phospholipids (8, 106, 124). In studies examining alveolar epithelial cells, the bioactive lipid sphingosine 1-phosphate (Sph-1-P) has been associated with regulation of cell proliferation (124). Treatment of alveolar epithelial cells with Sph-1-P results in the release of AA, which has also been shown to contribute to cell proliferation (124). Interestingly, specific inhibitors of iPLA₂ prevent AA release in response to Sph-1-P but did not prevent AA release in response to alternative stimuli, such as calcium-mobilizing agents (124). This observation allows one to speculate that the specific PLA₂ used to generate AA may depend on the stimulus and the intended consequence of the AA (i.e., inducing cell proliferation versus being metabolized for eicosanoid generation).

Despite the notion that iPLA₂ mainly serves a housekeeping role in cells, more-recent studies have pointed to the involvement of iPLA₂ in eicosanoid generation during inflammation (8, 35, 106). Using an in vivo rat model of acute pleurisy, the inflammatory process was investigated in discrete stages: influx of inflammatory cells, persistence of inflammatory cells, and resolution (35). It was observed that both the eicosanoids present, as well as the PLA₂ enzymes operating, were altered from one stage of the inflammatory process to the next. In this model, iPLA₂ appeared to act exclusively at the onset of inflammation, generating eicosanoids, such as LTB₄, to recruit inflammatory cells. At later stages in the inflammatory process, sPLA₂ and cPLA₂ were the predominant generators of eicosanoids, synthesizing the distinct anti-inflammatory eicosanoids LXA₄ and PGD₂, respectively. Proresolving eicosanoids, such as LXA₄ and PGD₂, aid in the repair of tissue damage resulting from inflammatory cell influx (35). The results of this study suggest not only that iPLA₂ is capable of participating in eicosanoid generation but also that certain cell types involved in inflammation possess specific and distinct mechanisms for activating the appropriate PLA₂ group or isoform at the appropriate stage of the inflammatory process. A better understanding of these mechanisms will greatly enhance therapies geared toward preventing the generation of potentially detrimental eicosanoids while preserving the ability to produce potentially beneficial eicosanoids during active disease and resolving processes.

Another consequence of iPLA₂ activity that has been documented is the promotion of apoptosis in certain cell types (8, 9, 106). Membrane damage resulting from iPLA₂ activity results in cellular accumulation of free fatty acids and lysophospholipids. These products are capable of acting as signals during apoptotic processes (8, 106). In addition, the apoptotic effector enzyme caspase-3 has been shown to cleave iPLA₂, causing enhanced activity, further implicating a connection between apoptosis and iPLA₂ (8, 106). The consequences of apoptotic processes mediated by iPLA₂ in lung inflammatory disease have not been widely explored; however, several lung cell types, including alveolar macrophages, alveolar epithelial cells, and the nasal mucosa, appear to express iPLA₂, and these cells routinely experience apoptosis under normal and inflammatory conditions (8, 65, 106, 124).

Pseudomonas aeruginosa.The lung pathogen P. aeruginosa produces a toxin known as ExoU that is extremely cytotoxic to a diverse range of eukaryotic host cells (103). ExoU is injected directly into host cells in the lung by P. aeruginosa via a bacterial type III secretion system (103). Once inside the cell, ExoU interacts with a cofactor, Cu²⁺-Zn²⁺-superoxide dismutase 1, and translocates to the cell membrane, where it encounters phospholipid substrates (105). ExoU has broad substrate specificity, also possessing the ability to hydrolyze lysophospholipids and operate in a calcium-independent manner (117). Like cPLA₂ and iPLA₂, ExoU contains a patatin domain with an active-site serine (95, 104). ExoU is incapable of acting on the extracellular surface because interaction with the intracellular enzyme superoxide dismutase 1 is required for PLA₂ activity (105). In the lung, ExoU is thought to target epithelial cells, endothelial cells, and macrophages (100, 117).

ExoU is not present in all pathogenic clinical isolates of P. aeruginosa; however, infection with ExoU-positive strains of P. aeruginosa is strongly associated with a significantly more-severe pneumonia with a much higher rate of complications and a greater degree of mortality (103). Animal models of acute pneumonia have clearly demonstrated that ExoU plays a significant role in virulence by directly mediating acute lung injury and bacterial dissemination, resulting in the development of septic shock (90, 103, 112). The pathology associated with ExoU can be dramatically reduced by infection with a P. aeruginosa strain expressing a catalytically inactive (S142A or D344A) ExoU mutant (90, 103, 112). In vitro cell culture models have revealed that the PLA₂ activity of ExoU is responsible for rapid, necrotic host cell lysis through disruption of lipid metabolism, phospholipid membrane cleavage, and loss of cell membrane integrity (90, 103, 112). This phenomenon is believed to account for the invasive nature of ExoU-positive P. aeruginosa clinical isolates in both animal models and human disease (90, 103, 112).

Although the destruction of host cells through membrane hydrolysis is considered to be the main consequence of the PLA₂ activity of ExoU, several other PLA₂-dependent processes have been attributed to ExoU (22, 74, 100, 103). The alteration of host cell signaling pathways by ExoU has been observed to occur in respiratory epithelial cell lines with ExoU-specific activation of c-fos and c-jun, leading to the initiation of AP-1-mediated gene expression (22, 74). These alterations in cell signaling pathways can lead to ExoU-specific induction of cytokines, such as IL-6 and IL-8 (22, 74). The consequence of these processes may be to instigate an inflammatory response upon intoxication with ExoU, which likely contributes to the disease process. The results of a recent report have demonstrated that ExoU is also capable of mediating eicosanoid production in mammalian cells (100). PGE₂ and PGI₂ release from P. aeruginosa-infected endothelial cells is ablated when the active-site serine of ExoU is mutated, and less PGE₂ is recovered in the BALF of mice infected with ExoU-deficient P. aeruginosa (100). Whether it is through cytokine induction, eicosanoid generation, or direct host cell lysis, ExoU is a bacteria-derived PLA₂ that can clearly have an impact on human lung disease.

Legionella pneumophilia.Legionnaires’ disease manifests as severe pneumonia frequently accompanied by ARDS (31). Legionella pneumophilia, the bacterial pathogen responsible for Legionnaires’ disease, is inhaled and infects and destroys lung epithelial cells and macrophages. PLA₂ activity originating from L. pneumophilia is believed to be important in the destruction of airway cells, as well as a contributor to the disruption of lung surfactant (31). Several enzymes have been characterized as being responsible for the lypolytic activity observed in L. pneumophilia, including PlaA, PlaB, and PlaC (11, 29, 30). All three enzymes possess lysophospholipase (LPLA) activity, but only PlaB and PlaC possess PLA₁ and PLA₂ activity (Fig. 1) (11, 29, 30). The PLA activity of PlaB is bacterial cell associated, whereas PlaC-derived PLA activity is secreted into the external environment (11, 30). Although PLA₂s from L. pneumophilia have been shown to be capable of hydrolyzing surfactant, the individual roles of PlaB and PlaC in this process or in other aspects of disease pathogenesis have not been definitively characterized. PlaB does exhibit hemolytic activity, and this may contribute to the disease process (30). Of note, a recently identified protein of L. pneumophilia called VipD displays significant homology to the P. aeruginosa toxin ExoU. Both VipD and ExoU share the patatin domain with a conserved active-site serine. Whether VipD functions as a PLA₂ awaits further study (123).

Other lung bacterial pathogens.Several bacterial pathogens in addition to P. aeruginosa and L. pneumophilia that are noted to cause lung inflammation have been observed to possess functional PLA₂ enzymes (113). Gram-negative bacterial species implicated in cases of bacterial pneumonia, including Escherichia coli and Klebsiella pneumoniae, produce an approximately 31-kDa enzyme referred to as OMPLA and encoded by the pldA gene (113). This enzyme has broad specificity, is calcium dependent, and is expressed on the outer membrane of the bacterial cell surface of lung, as well as enteric, pathogens (113). OMPLA is believed to contribute to the cytolytic properties of bacterial pathogens; however, a role for OMPLA in the pathogenesis of lung disease has yet to be elucidated. Functional PLA₂ enzymes have also been described for gram-positive pathogens with relevance to respiratory disease (113). Various species of group A Streptococcus (Streptococcus pyogenes) bacteria secrete a small (19 kDa) enzyme with significant sequence similarity to sPLA₂s that include pancreatic bovine sPLA₂ and sPLA₂s found in snake venom (113). This enzyme, termed SlaA, plays a key role in upper respiratory tract colonization during group A Streptococcus infection of monkeys (113). As more bacterial pathogens are sequenced, it is likely that several novel bacterial PLA₂ enzymes will be discovered. Future challenges include determining the extent to which these enzymes play a role in the disease process, the molecular mechanisms underlying their involvement in lung disease, and whether targeting such factors represents a useful strategy for treatment.