Inhibitory effects of surfactant protein A on surfactant phospholipid hydrolysis by secreted phospholipases A2

Hydrolysis of surfactant phospholipids by secreted phospholipases A(2) (sPLA(2)) contributes to surfactant dysfunction in acute respiratory distress syndrome. The present study demonstrates that sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X efficiently hydrolyze surfactant phospholipids in vitro. In contrast, sPLA(2)-IIC, -IID, -IIE, and -IIF have no effect. Since purified surfactant protein A (SP-A) has been shown to inhibit sPLA(2)-IIA activity, we investigated the in vitro effect of SP-A on the other active sPLA(2) and the consequences of sPLA(2)-IIA inhibition by SP-A on surfactant phospholipid hydrolysis. SP-A inhibits sPLA(2)-X activity, but fails to interfere with that of sPLA(2)-V. Moreover, in vitro inhibition of sPLA(2)-IIA-induces surfactant phospholipid hydrolysis correlates with the concentration of SP-A in surfactant. Intratracheal administration of sPLA(2)-IIA to mice causes hydrolysis of surfactant phosphatidylglycerol. Interestingly, such hydrolysis is significantly higher for SP-A gene-targeted mice, showing the in vivo inhibitory effect of SP-A on sPLA(2)-IIA activity. Administration of sPLA(2)-IIA also induces respiratory distress, which is more pronounced in SP-A gene-targeted mice than in wild-type mice. We conclude that SP-A inhibits sPLA(2) activity, which may play a protective role by maintaining surfactant integrity during lung injury.     

Macrophage secretory phospholipase A2 group X enhances anti-inflammatory responses, promotes lipid accumulation, and contributes to aberrant lung pathology

Secreted phospholipase A2 group X (sPLA(2)-X) is one of the most potent enzymes of the phospholipase A(2) lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and low-density lipoproteins (LDL), has implicated sPLA(2)-X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA(2)-X enzyme activity in vitro and in vivo, by generating sPLA(2)-X-overexpressing macrophages and transgenic macrophage-specific sPLA(2)-X mice. Our results show that sPLA(2)-X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta12,14-prostaglandin J(2) (PGJ(2)). Moreover, we found that overexpression of active sPLA(2)-X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA(2)-X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA(2)-X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA(2)-X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA(2)-X in inflammatory lung disease.     

Differences between group X and group V secretory phospholipase A2 in lipolytic modification of lipoproteins

Secretory phospholipases A(2) (sPLA(2)s) are a diverse family of low molecular mass enzymes (13-18 kDa) that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA(2) (sPLA(2)-X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA(2) (sPLA(2)-V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA(2)-X in several respects. Although sPLA(2)-V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA(2)-X. In addition, the requirement of Ca(2+) for the lipolysis of LDL was about 10-fold higher for sPLA(2)-V than sPLA(2)-X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA(2)-V in the presence of sodium citrate, which contrasted with the potent response to sPLA(2)-X. Moreover, sPLA(2)-X, but not sPLA(2)-V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA(2)-X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (K(d) = 3.1 nM) in the presence of Ca(2+). Selective interaction of sPLA(2)-X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.     

Induction of distinct sets of secretory phospholipase A(2) in rodents during inflammation

Although the expression of the prototypic secretory phospholipase A(2) (sPLA(2)), group IIA (sPLA(2)-IIA), is known to be up-regulated during inflammation, it remains uncertain if other sPLA(2) enzymes display similar or distinct profiles of induction under pathological conditions. In this study, we investigated the expression of several sPLA(2)s in rodent inflammation models. In lipopolysaccharide (LPS)-treated mice, the expression of sPLA(2)-V, and to a lesser extent that of sPLA(2)-IID, -IIE, and -IIF, were increased, whereas that of sPLA(2)-X was rather constant, in distinct tissues. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, in which the expression of sPLA(2)-IID, -IIF and -V was increased, was significantly reduced by YM-26734, a competitive sPLA(2)-IIA inhibitor that turned out to inhibit sPLA(2)-IID, -IIE, -V and -X as well. In contrast, sPLA(2)-IIA was dominant in carageenin-induced pleurisy in rats, where the accumulation of exudate fluids and leukocytes was significantly ameliorated by YM-26734. These results indicate that distinct sPLA(2)s can participate in inflammatory diseases according to tissues, animal species, and types of inflammation.     

New phospholipase A(2) isozymes with a potential role in atherosclerosis

PURPOSE OF REVIEW: Inflammation is an integral feature of atherosclerosis, in which inflammatory processes contribute to the initiation, progression and rupture of lipid-rich atherosclerotic plaques. Recent studies have suggested the involvement of the proinflammatory secretory phospholipase A2 (sPLA2)-IIA in the development of atherosclerosis. This enzyme has been proposed to hydrolyze phosphatidylcholine (PC) in lipoproteins to liberate lyso-PC and free fatty acids in the arterial wall, thereby facilitating the accumulation of bioactive lipids and modified lipoproteins in atherosclerotic foci. However, the recent discovery of several novel sPLA2 isozymes has raised the question of which types of sPLA2 truly contribute to the atherosclerotic process. RECENT FINDINGS: Amongst the 10 mammalian sPLA2 isozymes, sPLA2-X, -V, -IIF and -III exhibit much more potent PC-hydrolyzing activity than do the others, and can release free fatty acids and lysophospholipids from the PC-rich outer leaflet of the cellular plasma membrane. In particular, sPLA2-X and sPLA2-V hydrolyze PC in lipoproteins far more efficiently than does sPLA2-IIA. Moreover, sPLA2-X promotes foam cell formation in vitro and is expressed in the atherosclerotic arterial walls of apolipoprotein E deficient mice in vivo. SUMMARY: PC-hydrolyzing sPLA2 isozymes, particularly sPLA2-V and sPLA2-X, are attractive candidates for proatherosclerotic factors that may act in place of sPLA2-IIA. However, their expression in human atherosclerotic lesions requires confirmation by specific methods that can distinguish between the different sPLA2 isozymes.     

Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.     

Group V and X secretory phospholipase A(2)s-induced modification of high-density lipoprotein linked to the reduction of its antiatherogenic functions

The quantitative or qualitative decline of high-density lipoprotein (HDL) is linked to the pathogenesis of atherosclerosis because of its antiatherogenic functions, including the mediation of reverse cholesterol transport from the peripheral cells to the liver. We have recently shown that group X secretory phospholipase A(2) (sPLA(2)-X) is involved in the pathogenesis of atherosclerosis via potent lipolysis of low-density lipoprotein (LDL) leading to macrophage foam cell formation. We demonstrate here that sPLA(2)-X as well as group V secretory PLA(2) (sPLA(2)-V), another group of sPLA(2) that can potently hydrolyze phosphatidylcholine (PC), also possess potent hydrolytic potency for PC in HDL linked to the production of a large amount of unsaturated fatty acids and lysophosphatidylcholine (lysoPC). In contrast, the classical types of group IB and IIA secretory PLA(2)s evoked little, if any, lypolytic modification of HDL. Treatment with sPLA(2)-X or -V also caused an increase in the negative charge of HDL with no oxidation and little modification of apolipoprotein AI (apoAI). Modification with sPLA(2)-X or -V resulted in significant decrease in the capacity of HDL to cause cellular cholesterol efflux from lipid-loaded macrophages. Immunohistochemical analysis revealed significant expression of sPLA(2)-X in foam cell lesions in the arterial intima of Watanabe heritable hyperlipidemic (WHHL) rabbit. These findings suggest that lipolytic modification of HDL by sPLA(2)-X or -V causes drastic change of HDL in terms of the production of a large amount of unsaturated fatty acids and lysoPC linked to the reduction of its antiatherogenic functions. These sPLA(2)-mediated modifications of plasma lipoproteins might be relevant to the pathogenesis of atherosclerosis.     

Purified group X secretory phospholipase A(2) induced prominent release of arachidonic acid from human myeloid leukemia cells

Group X secretory phospholipase A(2) (sPLA(2)-X) possesses several structural features characteristic of both group IB and IIA sPLA(2)s (sPLA(2)-IB and -IIA) and is postulated to be involved in inflammatory responses owing to its restricted expression in the spleen and thymus. Here, we report the purification of human recombinant COOH-terminal His-tagged sPLA(2)-X, the preparation of its antibody, and the purification of native sPLA(2)-X. The affinity-purified sPLA(2)-X protein migrated as various molecular species of 13-18 kDa on SDS-polyacrylamide gels, and N-glycosidase F treatment caused shifts to the 13- and 14-kDa bands. NH(2)-terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA(2)-X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH(2) termini of the mature protein. Separation with reverse-phase high performance liquid chromatography revealed that N-linked carbohydrates are not required for the enzymatic activity and pro-sPLA(2)-X has a relatively weak potency compared with the mature protein. The mature sPLA(2)-X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA(2) groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA(2)-IB and -IIA with concomitant production of prostaglandin E(2). A prominent release of arachidonic acid was also observed in sPLA(2)-X-treated human U937 and HL60 cells. Immunohistochemical analysis of human lung preparations revealed its expression in alveolar epithelial cells. These results indicate that human sPLA(2)-X is a unique N-glycosylated sPLA(2) that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA(2)-IB and -IIA.     

Alveolar lipoproteinosis in an acid sphingomyelinase-deficient mouse model of Niemann-Pick disease

Types A and B Niemann-Pick disease (NPD) are lipid storage disorders caused by the deficient activity of acid sphingomyelinase (ASM). In humans, NPD is associated with the dysfunction of numerous organs including the lung. Gene targeting of the ASM gene in transgenic mice produced an animal model with features typical of NPD, including pulmonary inflammation. To assess mechanisms by which ASM perturbed lung function, we studied lung morphology, surfactant content, and metabolism in ASM-deficient mice in vivo. Pulmonary inflammation, with increased cellular infiltrates and the accumulation of alveolar material, was associated with alterations in surfactant content. Saturated phosphatidylcholine (SatPC) content was increased twofold, and sphingomyelin content was increased 5.5-fold in lungs of the ASM knockout (ASMKO) mice. Additional sphingomyelin enhanced the sensitivity of surfactant inhibition by plasma proteins. Clearance of SatPC from the lungs of ASMKO mice was decreased. Catabolism of SatPC by alveolar macrophages from the ASMKO mouse was significantly decreased, likely accounting for decreased pulmonary SatPC in vivo. In summary, ASM is required for normal surfactant catabolism by alveolar macrophages in vivo. Alterations in surfactant composition, including increased sphingomyelin content, contributed to the abnormal surfactant function observed in the ASM-deficient mouse.     

Eosinophil cysteinyl leukotriene synthesis mediated by exogenous secreted phospholipase A2 group X

Secreted phospholipase A(2) group X (sPLA(2)-X) has recently been identified in the airways of patients with asthma and may participate in cysteinyl leukotriene (CysLT; C(4), D(4), and E(4)) synthesis. We examined CysLT synthesis and arachidonic acid (AA) and lysophospholipid release by eosinophils mediated by recombinant human sPLA(2)-X. We found that recombinant sPLA(2)-X caused marked AA release and a rapid onset of CysLT synthesis in human eosinophils that was blocked by a selective sPLA(2)-X inhibitor. Exogenous sPLA(2)-X released lysophospholipid species that arise from phospholipids enriched in AA in eosinophils, including phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine as well as plasmenyl phosphatidylcholine and phosphatidylethanolamine. CysLT synthesis mediated by sPLA(2)-X but not AA release could be suppressed by inhibition of cPLA(2)α. Exogenous sPLA(2)-X initiated Ser(505) phosphorylation of cPLA(2)α, an intracellular Ca(2+) flux, and translocation of cPLA(2)α and 5-lipoxygenase in eosinophils. Synthesis of CysLTs in response to sPLA(2)-X or lysophosphatidylcholine was inhibited by p38 or JNK inhibitors but not by a MEK 1/2 inhibitor. A further increase in CysLT synthesis was induced by the addition of sPLA(2)-X to eosinophils under conditions of N-formyl-methionyl-leucyl-phenylalanine-mediated cPLA(2)α activation. These results indicate that sPLA(2)-X participates in AA and lysophospholipid release, resulting in CysLT synthesis in eosinophils through a mechanism involving p38 and JNK MAPK, cPLA(2)α, and 5-lipoxygenase activation and resulting in the amplification of CysLT synthesis during cPLA(2)α activation. Transactivation of eosinophils by sPLA(2)-X may be an important mechanism leading to CysLT formation in the airways of patients with asthma.     

Physiological roles of group X-secreted phospholipase A2 in reproduction, gastrointestinal phospholipid digestion, and neuronal function

Although the secreted phospholipase A(2) (sPLA(2)) family has been generally thought to participate in pathologic events such as inflammation and atherosclerosis, relatively high and constitutive expression of group X sPLA(2) (sPLA(2)-X) in restricted sites such as reproductive organs, the gastrointestinal tract, and peripheral neurons raises a question as to the roles played by this enzyme in the physiology of reproduction, digestion, and the nervous system. Herein we used mice with gene disruption or transgenic overexpression of sPLA(2)-X to clarify the homeostatic functions of this enzyme at these locations. Our results suggest that sPLA(2)-X regulates 1) the fertility of spermatozoa, not oocytes, beyond the step of flagellar motility, 2) gastrointestinal phospholipid digestion, perturbation of which is eventually linked to delayed onset of a lean phenotype with reduced adiposity, decreased plasma leptin, and improved muscle insulin tolerance, and 3) neuritogenesis of dorsal root ganglia and the duration of peripheral pain nociception. Thus, besides its inflammatory action proposed previously, sPLA(2)-X participates in physiologic processes including male fertility, gastrointestinal phospholipid digestion linked to adiposity, and neuronal outgrowth and sensing.     

Group X secretory PLA2 in neutrophils plays a pathogenic role in abdominal aortic aneurysms in mice

Group X secretory PLA(2) (sPLA(2)-X) is expressed in neutrophils and plays a role in the pathogenesis of neutrophil-mediated tissue inflammation and injury. This study tested the hypothesis that sPLA(2)-X in neutrophils may contribute to the pathogenesis of abdominal aortic aneurysms (AAA) using sPLA(2)-X(-/-) mice. AAA was created by application of CaCl(2) to external surface of aorta. As a result, the aortas of sPLA(2)-X(-/-) mice had smaller diameters (percent increase from baseline; 24.8 ± 3.5% vs. 49.9 ± 9.1%, respectively; P < 0.01), a reduced grade of elastin degradation, and lower activities of elastase and gelatinase (26% and 19% lower, respectively) after CaCl(2) treatment compared with sPLA(2)-X(+/+) mice. In sPLA(2)-X(+/+) mice, immunofluorescence microscopic images showed that the immunoreactivity of sPLA(2)-X was detected only in neutrophils within aortic walls 3 days, 1, 2, and 6 wk after CaCl(2) treatment, whereas the immunoreactivity was not detected in macrophages or mast cells in aortic walls. sPLA(2)-X immunoreactivity also was colocalized in cells expressing matrix metalloproteinase (MMP)-9. Neutrophils isolated from sPLA(2)-X(-/-) mice had lower activities of elastase, gelatinase, and MMP-9 in response to stimuli compared with sPLA(2)-X(+/+) mice. The attenuated release of elastase and gelatinase from sPLA(2)-X(-/-) neutrophils was reversed by exogenous addition of mouse sPLA(2)-X protein. The adoptive transfer of sPLA(2)-X(+/+) neutrophils days 0 and 3 after CaCl(2) treatment reversed aortic diameters and elastin degradation grades in the lethally irradiated sPLA(2)-X(+/+) mice reconstituted with sPLA(2)-X(-/-) bone marrow to an extent similar to that seen in sPLA(2)-X(+/+) mice. In conclusion, sPLA(2)-X in neutrophils plays a pathogenic role in AAA in a mice model.     

Potent modification of low density lipoprotein by group X secretory phospholipase A2 is linked to macrophage foam cell formation

The deposition of cholesterol ester within foam cells of the artery wall is fundamental to the pathogenesis of atherosclerosis. Modifications of low density lipoprotein (LDL), such as oxidation, are prerequisite events for the formation of foam cells. We demonstrate here that group X secretory phospholipase A2 (sPLA2-X) may be involved in this process. sPLA2-X was found to induce potent hydrolysis of phosphatidylcholine in LDL leading to the production of large amounts of unsaturated fatty acids and lysophosphatidylcholine (lyso-PC), which contrasted with little, if any, lipolytic modification of LDL by the classic types of group IB and IIA secretory PLA2s. Treatment with sPLA2-X caused an increase in the negative charge of LDL with little modification of apolipoprotein B (apoB) in contrast to the excessive aggregation and fragmentation of apoB in oxidized LDL. The sPLA2-X-modified LDL was efficiently incorporated into macrophages to induce the accumulation of cellular cholesterol ester and the formation of non-membrane-bound lipid droplets in the cytoplasm, whereas the extensive accumulation of multilayered structures was found in the cytoplasm in oxidized LDL-treated macrophages. Immunohistochemical analysis revealed marked expression of sPLA2-X in foam cell lesions in the arterial intima of high fat-fed apolipoprotein E-deficient mice. These findings suggest that modification of LDL by sPLA2-X in the arterial vessels is one of the mechanisms responsible for the generation of atherogenic lipoprotein particles as well as the production of various lipid mediators, including unsaturated fatty acids and lyso-PC.     

Clearance of group X secretory phospholipase A(2) via mouse phospholipase A(2) receptor.

Given the potent hydrolyzing activity toward phosphatidylcholine, group X secretory phospholipase A(2) (sPLA(2)-X) elicits a marked release of arachidonic acid linked to the potent production of lipid mediators in various cell types. We have recently shown that sPLA(2)-X can also act as a ligand for mouse phospholipase A(2) receptor (PLA(2)R). Here, we found that sPLA(2)-X was internalized and degraded via binding to PLA(2)R associated with the diminished prostaglandin E(2) (PGE(2)) formation in PLA(2)R-expressing Chinese hamster ovary (CHO) cells compared to CHO cells. Indirect immunocytochemical analysis revealed that internalized sPLA(2)-X was co-localized with PLA(2)R in the punctate structures in PLA(2)R-expressing CHO cells. Moreover, in mouse osteoblastic MC3T3-E(1) cells that endogenously express the PLA(2)R, the internalized sPLA(2)-X was localized in lysosomes. These findings demonstrate that PLA(2)R acts as a clearance receptor for sPLA(2)-X to suppress its strong enzymatic activity.     

Reversibility of lung inflammation caused by SP-B deficiency

Whereas decreased concentrations of surfactant protein (SP)-B are associated with lung injury and respiratory distress, potential causal relationships between SP-B deficiency and lung inflammation remain unclear. A transgenic mouse in which human SP-B expression was placed under conditional control of doxycycline via the CCSP promoter was utilized to determine the role of SP-B in the initiation of pulmonary inflammation. Adult mice, made SP-B deficient by removal of doxycycline, developed severe respiratory failure within 4 days. Deficiency of SP-B was associated with increased minimal surface tension of the surfactant and perturbed lung mechanics. Four days of SP-B deficiency did not alter SP-C content or surfactant phospholipid content or composition. SP-B deficiency was associated with lung inflammation and increased soluble L-selectin, STAT-3, and phosphorylated STAT-3 in alveolar macrophages and alveolar epithelial cells. Alveolar IL-6, IL-1beta, and macrophage inflammatory protein-2 concentrations were increased after removal of doxycycline, indicating pulmonary inflammation. Restoration of SP-B expression following administration of doxycycline rapidly reversed SP-B-dependent abnormalities in lung mechanics and inflammation. SP-B deficiency is sufficient to cause lung dysfunction and inflammation in adult mice. SP-B reversed inflammation and maintained lung function in vivo, indicating its potential utility for the prevention and treatment of pulmonary injury and surfactant deficiency.     

Differential behavior of sPLA2-V and sPLA2-X in human neutrophils

Neutrophils and differentiated PLB-985 cells contain various types of PLA(2)s including the 85 kDa cytosolic PLA(2) (cPLA(2)), Ca(2+)-independent PLA(2) (iPLA(2)) and secreted PLA(2)s (sPLA(2)s). The present study focuses on the behavior of sPLA(2)s in neutrophils and PLB cells and their relationship to cPLA(2)alpha. The results of the present research show that the two types of sPLA(2) present in neutrophils, sPLA(2)-V and sPLA(2)-X, which are located in the azurophil granules, are differentially affected by physiological stimuli. While sPLA(2)-V is secreted to the extacellular milieu, sPLA(2)-X is detected on the plasma membranes after stimulation. Stimulation of neutrophils with formyl-Met-Leu-Phe (fMLP), opsonized zymosan (OZ) or A23187 resulted in a different kinetics of sPLA(2) secretion as detected by its activity in the neutrophil supernatants. Neutrophil priming by inflammatory cytokines or LPS enhanced sPLA(2) activity detected in the supernatant after stimulation by fMLP. This increased activity was due to increased secretion of sPLA(2)-V to the supernatant and not to release of sPLA(2)-X. sPLA(2) in granulocyte-like PLB cells exhibit identical characteristics to neutrophil sPLA(2), with similar activity and optimal pH of 7.5. Granulocyte-like cPLA(2)alpha-deficient PLB cells serve as a good model to study whether sPLA(2) activity is regulated by cPLA(2)alpha. Secretion and activity of sPLA(2) were found to be similar in granulocyte-like PLB cells expressing or lacking cPLA(2)alpha, indicating that they are not under cPLA(2)alpha regulation.     

Surfactant phospholipid catabolic rate is pool size dependent in mice

We increased surfactant pool size by surfactant treatment in mice to test if the catabolism of the major component of surfactant, saturated phosphatidylcholine (Sat PC), was rate limited. By intratracheal instillation, we gave mice trace doses, doses of 45 or 110 micromol/kg, or three doses of 110 micromol/kg of Sat PC in surfactant that contained radiolabeled dipalmitoylphosphatidylcholine (DPPC) and a radiolabeled phospholipase A-resistant ether analog of DPPC. Two strains of mice with 2-fold differences in alveolar and total Sat PC pool sizes were used; the mice with the higher pool sizes had a 2.3-fold higher steady-state catabolic rate. Acute increases in alveolar surfactant given by intratracheal instillation increased catabolic rates approximately 2-fold over the steady-state rates in both strains. There was minimal loss of the ether analog of DPPC from the lungs, and the alveolar macrophages did not accumulate more than 10% of the ether analog. In these two strains of mice, the catabolism of Sat PC was not rate limited because catabolic rate increased when alveolar pool sizes were increased.