acid-releasing

Oxidation and lipolytic remodeling of LDL are believed to stimulate LDL entrapment in the arterial wall, expanding the inflammatory response and promoting atherosclerosis. However, the cellular responses and molecular mechanisms underlying the atherogenic effects of lipolytically modified LDL are incompletely understood. Human THP-1 monocytes were prelabeled with [(3)H]arachidonic acid (AA) before incubation with LDL or LDL lipolytically modified by secretory PLA(2) (sPLA(2)) or bacterial sphingomyelinase (SMase). LDL elicited rapid and dose-dependent extracellular release of AA in monocytes. Interestingly, LDL modified by sPLA(2) or SMase displayed a marked increase in AA mobilization relative to native LDL, and this increase correlated with enhanced activity of cytosolic PLA(2) (cPLA(2)) assayed in vitro as well as increased monocyte tumor necrosis factor-alpha secretion. The AA liberation was attenuated by inhibitors toward cPLA(2) and sPLA(2), indicating that both PLA(2) enzymes participate in LDL-induced AA release. In conclusion, these results demonstrate that LDL lipolytically modified by sPLA(2) or SMase potentiates cellular AA release and cPLA(2) activation in human monocytes. From our results, we suggest novel atherogenic properties for LDL modified by sPLA(2) and SMase in AA release and signaling, which could contribute to the inflammatory gene expression observed in atherosclerosis.

Atherosclerosis, the underlying cause of myocardial infarction, stroke, and vascular occlusive disease, may be characterized as an inflammatory disease that is initiated by the entrapment of LDL in the arterial wall (1).In this process, the oxidation and lipolytic remodeling of LDL is recognized to be important for the subendothelial retention of LDL, causing LDL aggregation, foam cell formation, endothelial dysfunction, and macrophage chemotaxis (2).Secretory nonpancreatic phospholipase A 2 (sPLA 2 ) and sphingomyelinase (SMase) have been implicated in the lipolytic modification of LDL in vitro, thereby generating LDL that has increased binding strength to human aortic proteoglycans and stimulates foam cell formation (3)(4)(5)(6).Both sPLA 2 and SMase are found in the arterial intima (7,8), and several reports link these enzymes to atherogenesis: atherosclerotic lesions and levels of biologically active oxidized phospholipids are increased in sPLA 2 transgenic mice (9), and sPLA 2 has been identified as an independent risk factor for coronary artery disease (10).Also, mice that are knockouts for the acid SMase gene show resistance to atherosclerotic lesion development (11).Finally, the activities of sPLA 2 and secretory SMase are upregulated during acute systemic inflammation, which could specifically link these enzymes to the inflammatory events in atherosclerosis (12).
PLA 2 hydrolyzes the sn -2 fatty acid ester bond of glycerophospholipids (13), whereas SMase cleaves sphingo-Abbreviations: AA, arachidonic acid; BEL, bromoenol lactone; cPLA 2 , cytosolic phospholipase A 2 ; iPLA 2 , Ca 2 ϩ -independent phospholipase A 2 ; LDL nat , native LDL; MAFP, methyl arachidonyl fluorophosphonate; MDA, malondialdehyde equivalents; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OA, oleic acid; PLA 2 , phospholipase A 2 ; SMase, sphingomyelinase; sPLA 2 , secretory phospholipase A 2 ; TNF-␣ , tumor necrosis factor-␣ . 1 To whom correspondence should be addressed.e-mail: marit.w.anthonsen@medisin.ntnu.nomyelin that is present in cell membranes and lipoproteins (14).The phospholipid hydrolysis of LDL by PLA 2 and SMase generates FFA and lipid mediators (lysophospholipids and ceramide) that could possibly initiate diverse atherogenic processes in various cell types at sites of lipoprotein deposition in the arterial wall.Also, the physiochemical properties, including the charge and conformation of LDL, are changed upon phospholipid hydrolysis by sPLA 2 and SMase, which could account for the observed enhanced interaction between lipolytically modified LDL and cells in the arterial wall.Despite the findings that lipolytic modification of LDL enhances the retention of aortic proteoglycans and accelerates foam cell formation, the molecular mechanisms underlying the atherogenic properties of lipolytically modified LDL are incompletely understood.Therefore, the present study was undertaken to examine other cellular effects of lipolytically modified LDL.Stimulus-induced mobilization of arachidonic acid (AA), mediated by the group IV Ca 2 ϩdependent cytosolic PLA 2 (cPLA 2 ) and the 14 kDa Ca 2 ϩdependent sPLA 2 s, has been assigned an important role in lipid-mediated cellular signaling associated with inflammatory responses (15).In this study, we investigated whether LDL and lipolytically modified LDL may contribute to monocyte AA signaling.Our findings show that 1 ) LDL elicits rapid AA release mediated by PLA 2 enzymes in human THP-1 monocytes, and 2 ) LDL lipolytically modified by SMase or sPLA 2 exerts strongly enhanced AA-inducing capacity compared with native LDL (LDL nat ).These results suggest novel atherogenic properties for LDL, sPLA 2 -modified LDL, and SMase-modified LDL through the induction of monocyte AA release that may contribute to inflammatory signaling and gene expression promoting atherogenesis.

Lipoprotein purification
Human LDL (d 1.019-1.050g/ml) was isolated from the plasma of normolipidemic donors by sequential ultracentrifugation (16) in the presence of EDTA (1.08 mM) and thereafter extensively dialyzed at 4 Њ C against RPMI 1640 with EDTA (10 mM) and subjected to a short (1-2 h) dialysis in RPMI 1640.LDL lipid composition was determined to check the purity of each preparation, and protein concentration was determined by a pyrogallol red technique (Elitech Diagnostics, Sees, France) (17), which gave results similar to those of the bicinchoninic acid kit.Determination of the content of thiobarbituric acid-reactive substances, expressed as malondialdehyde equivalents (MDA), provided an estimation of the degree of lipid peroxidation (18).Typically LDL nat preparations contained Ͻ 0.9 Ϯ 0.1 nmol MDA/ mg LDL protein.Also, conjugated dienes were determined by monitoring the changes in absorbance at 234 nm (19).

Treatment of LDL with sPLA 2 and SMase
LDL (100 g/ml) was incubated with purified human recombinant group IIa sPLA 2 or B. cereus SMase in RPMI 1640 medium with 5 mM MgCl 2 and 2 mM CaCl 2 at 37 Њ C for the periods of time indicated in the figures.Lipolysis was stopped by the addition of EDTA to a final concentration of 10 mM EDTA.SMaseand sPLA 2 -treated LDL are denoted SMase/LDL and sPLA 2 / LDL, respectively.The oxidative status of LDL (measured as thiobarbituric acid-reactive substances or conjugated dienes) was not changed after incubation with sPLA 2 or SMase or after incubation of LDL species with THP-1 cells for up to 2 h.

Measurement of AA and OA release
THP-1 cells were labeled with [ 3 H]AA (0.4 Ci/ml) and [ 14 C]OA (0.07 Ci/ml) as previously described (20).Extracellular release of total [ 3 H]AA and [ 14 C]OA radioactivity was assessed by liquid scintillation counting of supernatants.The results are given as released [ 3 H]AA or [ 14

Statistical analysis
Data are expressed as means Ϯ SD of triplicate determinations within separate experiments.Similar results were obtained in three to five experiments.Differences between means were determined by Student's t -test for paired samples, and those at P Ͻ 0.05 were considered significant.In the figures, asterisks indicate that values are statistically different from the given reference values.

LDL induces AA release in human monocytes
Stimulus-induced mobilization of AA has been assigned important roles in cellular signaling that regulates inflammatory activation (15,24).To investigate whether LDL also stimulates the extracellular release of AA and OA in monocytes, THP-1 cells were prelabeled with [ 3 H]AA and [ 14 C]OA before the addition of LDL.LDL rapidly and reproducibly increased cellular AA and OA liberation by 1.4-to 2.5-fold (Fig. 1), showing dose-dependent effects without exerting cytotoxic effects at the time and concentrations applied (as assessed by the MTT assay; data not shown).

LDL-elicited AA release is mediated by PLA 2 activity
The activation of PLA 2 enzymes provides a direct pathway for AA release (13).To examine whether PLA 2 enzymes mediate LDL-induced AA mobilization, we first investigated whether the observed AA release correlated with an increased activity of the AA-selective cPLA 2 enzyme.The activity of cPLA 2 was determined in vitro using sonicated vesicles of arachidonoylphosphatidylcholine as a substrate.Indeed, LDL enhanced the activity of cPLA 2 (Fig. 2), which was blocked by the cPLA 2 /Ca 2ϩ -independent PLA 2 (iPLA 2 ) inhibitor MAFP (25) (data not shown).These results indicate that cPLA 2 , at least in part, mediates LDL-induced AA liberation.To further examine the role of PLA 2 enzymes in LDL-elicited AA release in THP-1 cells, we next investigated the influence of specific inhibitors targeted against distinct PLA 2 enzymes on LDL-induced fatty acid release.Pretreatment of THP-1 cells with the dual cPLA 2 /iPLA 2 inhibitor MAFP (25, 26) potently reduced LDL induced AA release, resulting in complete inhibition at 10 M (Fig. 3).Likewise, the active site-directed, sPLA 2 -specific inhibitor SB203347, displaying 40-fold selectivity for sPLA 2 over cPLA 2 (27), dose-dependently attenuated LDL-stimulated AA liberation, displaying ‫%08ف‬ inhibition at 10 M (Fig. 3).In contrast, the iPLA 2 inhibitor BEL or vehicles had no effect on AA release, and inhibitors were not cytotoxic as determined by MTT assay (data not shown).Therefore, these results suggest that both cPLA 2 and sPLA 2 contribute to the LDL-elicited AA release observed in THP-1 monocytes.

Lipolytic modification of LDL with sPLA 2 or SMase potentiates monocyte AA release
To examine whether the modification of LDL with sPLA 2 or SMase affects the AA-inducing capacity of LDL, LDL nat was pretreated with either recombinant human group IIa sPLA 2 or B. cereus SMase before incubation with THP-1 monocytes for different time periods.Indeed, induction of AA cellular release was markedly enhanced after treatment of LDL with sPLA 2 or SMase, repeatedly resulting in up to a 4.5-fold increase in AA release (relative to untreated cells; Fig. 4), i.e., a 2-to 3-fold enhancement relative to LDL nat .Importantly, the enhanced AA release observed for sPLA 2or SMase-treated LDL compared with LDL nat correlated with the increased induction of cPLA 2 activity by the lipolytically modified LDL (Fig. 2), indicating the possible involvement of cPLA 2 in LDL-stimulated AA release.
To investigate the importance of preincubation time of sPLA 2 or SMase with LDL for the AA-releasing capacity of LDL, LDL was preincubated with either sPLA 2 or SMase for various time periods before incubation with the THP-1 monocytes.We observed that the cellular AA release increased with increasing preincubation times of LDL with sPLA 2 or SMase, resulting in maximal release for LDL that had been pretreated with sPLA 2 for 15-18 h (Fig. 5A).In contrast, the maximal effect of SMase on LDL modification was obtained after 40 min of preincubation with LDL (Fig. 5B).Thus, under our experimental conditions, the SMase-mediated modification of LDL that is responsible for the enhanced AA release occurs far more rapidly than the corresponding sPLA 2 -induced LDL modification.Also, increasing concentrations of sPLA 2 (Fig. 6A) or SMase (Fig. 6B) during preincubation with LDL dose-dependently enhanced AA liberation.
To address the question of whether sPLA 2 activity is of significance for the AA release stimulated by sPLA 2 -modi-fied LDL, we used an anti-group IIa activity-neutralizing antibody or the sPLA 2 inhibitor SB203347 to block enzyme activity.Addition of an anti-group IIa sPLA 2 activityneutralizing antibody or SB203347 during sPLA 2 /LDL preincubation completely inhibited the effect of sPLA 2 on cellular AA liberation (Fig. 7A).Thus, sPLA 2 catalytic activity is of crucial significance for the LDL modification that causes AA potentiation.Likewise, the presence of 10 mM EDTA to inhibit SMase during the SMase/LDL preincubation completely abrogated the enhancing effect of SMase-treated LDL on AA liberation (Fig. 7B).Hence, the catalytic activity of SMase is responsible for LDL modification, generating LDL with increased capacity for AA release in human monocytes.
To partially address the binding mechanisms by which sPLA 2 -/SMase-modified LDL promotes cellular AA release, we used different strategies aimed at modulating cellular LDL binding or LDL degradation.Cell surface proteoglycans have been implicated in the binding and subsequent internalization and degradation of atherogenic lipoproteins (28,29).To test if heparan sulfate-or chondroitin sulfate-containing proteoglycans are involved in AA release stimulated by sPLA 2 -or SMase-modified  LDL, we investigated whether the preincubation of cells with heparinase or chondroitinase ABC affected AA liberation.Heparinase and chondroitinase ABC lyase had no effects on AA release (Table 1).To examine if the CD36 scavenger receptor is involved in LDL-induced AA liberation, THP-1 cells were preincubated with an anti-CD36 blocking antibody before incubation with SMase-treated LDL.In the presence of the anti-CD36 antibody, AA liberation was reduced to 83% of that of SMase/LDL-stimulated cells (Table 1).Thus, we suggest that the CD36 scavenger receptor in part mediates AA release in response to SMase-modified LDL.In contrast, pretreatment of the cells with chloroquine or cytochalasin D to inhibit lysosomal activity and actin-dependent endocytosis did not significantly reduce LDL-induced AA release.

Effect of sPLA 2 -or SMase-modified LDL on TNF-␣ secretion
LDL promotes immune reactions by modulating the production of various cytokines and the expression of vascular adhesion molecules in the arterial wall (1).Therefore, we examined whether the enhanced AA mobilization observed for sPLA 2 -or SMase-modified LDL cor-related with the increased production of the proinflammatory mediator TNF-␣.Indeed, LDL premodified with sPLA 2 or SMase augmented TNF-␣ secretion from THP-1 cells, resulting in a 2-to 3-fold increase compared with LDL nat (Fig. 8).

DISCUSSION
Subendothelial retention of LDL with subsequent oxidation and lipolytic remodeling of LDL are considered to be critical events during early atherosclerosis development.Human sPLA 2 and SMase have recently been implicated in the lipolytic modification of LDL in vitro, and roles for these enzymes in the LDL modification, aggregation, and atherogenic lipid accumulation in vivo have been suggested (4)(5)(6)(7)(8)(9)29).However, the molecular mechanisms underlying the atherogenic effects of lipolytically  A: LDL nat (100 g/ml) was preincubated at 37ЊC for 12 h with 1 g/ml human recombinant group IIa sPLA 2 in the presence or absence of the sPLA 2 inhibitor SB203347 (SB; 10 M) or the anti-sPLA 2 activity-neutralizing antibody BA11 (mAB BA11; 30 g/ml) before addition to THP-1 monocytes and treatment for 30 min.B: LDL nat (100 g/ml) was preincubated at 37ЊC for 1 h with 500 mU/ml B. cereus SMase in the presence or absence of EDTA (10 mM) before addition to THP-1 monocytes and treatment for 30 min.Extracellular release of [ 3 H]AA was quantified as described in Methods.Data are expressed as means Ϯ SD of triplicate determinations within separate experiments.modified LDL are poorly understood.Therefore, we addressed the question of whether the LDL modified by human group IIa sPLA 2 or bacterial SMase may contribute to the AA mobilization and proinflammatory activation of human monocytes.
Interestingly, we found that modification of LDL by human group IIa sPLA 2 or bacterial SMase markedly enhances the AA-inducing potential of LDL, which is accompanied by increased TNF-␣ secretion compared with LDL nat .Importantly, no oxidative changes in LDL (measured as thiobarbituric acid-reactive substances and conjugated dienes) were detected after dialysis and treatment with SMase or sPLA 2 for 1 or 12 h, respectively.Likewise, incubation of LDL, SMase-treated LDL, or sPLA 2 -treated LDL with THP-1 cells for 2 h did not alter the oxidative status of LDL.Collectively, these findings suggest that AA release in response to LDL or enzymatically modified LDL is not attributable to oxidative changes in LDL, as have been reported previously to occur (30), and indicate that LDL modifications produced by sPLA 2 and SMase hydrolysis bring about AA liberation.Hence, although it has been found that prior sPLA 2 treatment may render LDL more susceptible to oxidation (31), LDL oxidation appears not to mediate AA release in our system.Our results show that lipolytically modified LDL rapidly induces AA mobilization, entailing maximal release after 2 h of LDL exposure.Cholesterol loading of the cells does not occur during this incubation period.Also, because pretreatment of the cells with chloroquine (to inhibit lysosomal activity) had no significant effect on the AA mobilization induced by lipolytically modified LDL (data not shown), these results indicate that AA release occurs independently of LDL internalization and degradation.Hence, more rapid LDL signaling mechanisms appear to participate in the observed cellular AA liberation.In this regard, it is noteworthy that oxidized LDL or ligand binding to the macrophage scavenger receptor A rapidly (within minutes) stimulates kinase activities (32)(33)(34).Thus, such early LDL signaling events may mediate the cPLA 2 activation and AA release observed in our study.Pretreatment of the cells with chloroquine or cytochalasin D to inhibit lysosomal activity and actin-dependent endocytosis did not significantly reduce LDL-induced AA release, indicating that AA release occurs independently of LDL internalization and degradation.
The LDL molecular properties that are changed by sPLA 2 or SMase treatment and that cause the enhanced AA mobilization for lipolytically treated LDL relative to LDL nat are unknown, but they may involve altered physiochemical properties [e.g., aggregation, as is known to occur under experimental conditions similar to those used in our experiments (4,6)] or structural changes in apolipoprotein B [as reviewed in ref. (35)].One alternative mechanism for the induction of AA release by enzymatically modified LDL could be through LDL-derived lipid mediators generated by sPLA 2 or SMase, such as lysophosphatidylcholine and ceramide.However, we found that lysophosphatidylcholine and ceramide induce AA release in THP-1 cells more rapidly than LDL (maximal within 10-20 min, in contrast to 2 h for LDL treatment).Hence, the AA-releasing capacity of enzymatically modified LDL is probably not mediated by lysophosphatidylcholine and ceramide.
We observed that the preincubation time necessary to obtain LDL with enhanced AA-releasing capacity was extended for human sPLA 2 treatment compared with that of bacterial SMase.These results are in accordance with previous reports showing that LDL hydrolysis by sPLA 2 is slower than that by B. cereus SMase, which is also reflected by slower LDL aggregation in response to the sPLA 2 treatment (29,36).Also, SMase induces both aggregation and fusion, whereas sPLA 2 induces only aggregation in the absence of proteoglycans.Hence, the preincubation times of either lipolytic enzyme with LDL necessary to induce AA release appear to parallel the different LDL hydrolytic rates and aggregation and/or fusion of LDL particles induced by sPLA 2 or SMase, which suggests that LDL aggregation and/or fusion is important for the enhanced AA release.In an attempt to explore this possibility, we separated LDL treated with agarose-coupled bee venom sPLA 2 into native-sized and aggregated LDL particles [by gel filtration chromatography (6)] before incubation of these distinct LDL species with THP-1 cells.However, we found that sPLA 2 -treated native-sized particles stimulated AA release to a similar extent as did sPLA 2 -induced aggregated LDL; therefore, further investigations are needed to resolve this issue.Interestingly, sPLA 2 or SMase treatment of LDL is known to affect LDL interaction with cells (5, 7), but the exact mechanisms involved in cellular binding of sPLA 2or SMase-modified LDL are unknown.Cellular binding of LDL may generally be mediated by different LDL receptors or by distinct cell surface proteoglycans, such as heparin sulfate-or chondroitin sulfate-containing proteoglycans (28,29,37).We found that preincubation of THP-1 cells with heparinase or chondroitinase ABC did not significantly affect AA release induced by LDL.In contrast, the presence of an anti-CD36 receptor-blocking antibody partially reduced AA liberation induced by lipolytically modified LDL, which suggests that the CD36 receptor contributes to AA release.However, the exact mechanisms by which lipolytically remodeled LDL contributes to AA release should be addressed in detail in future studies.
Our findings show that the cPLA 2 /iPLA 2 inhibitor MAFP and the sPLA 2 inhibitor SB203347, but not the iPLA 2 -specific inhibitor BEL, attenuated the LDL-elicited AA release (by 80-100% at 5-10 M).Also, we found that cPLA 2 activity in monocytes was promoted by LDL lipolytically modified with sPLA 2 or SMase.Moreover, we simultaneously measured AA and OA release in THP-1 monocytes and found that OA release, regulated by sPLA 2 s (because iPLA 2 is probably not involved in the observed fatty acid release, as discussed previously), which show no fatty acid preference, largely paralleled AA release upon exposure to sPLA 2 -or SMase-treated LDL (data not shown).By the use of macrophages from cPLA 2 (Ϫ/Ϫ) mice and the iPLA 2 -specific inhibitor BEL, it has been found that cPLA 2 , but not iPLA 2 , mediates AA release in response to oxidized LDL (30).Hence, based on these observations, we propose that both sPLA 2 s and the AA-selective cPLA 2 , but probably not iPLA 2 , participate in LDLstimulated AA release in THP-1 monocytes.The necessity of both sPLA 2 and cPLA 2 in AA liberation could be attributable to a sequential interplay between the enzymes leading to maximal AA release, as reported in response to inflammatory stimuli in other cells, leading to the activation of the immunomodulatory transcription factor nuclear factor-B (22,24).Several closely related sPLA 2 isoforms are expressed in THP-1 monocytes (38), but which of these that participate in LDL-elicited AA release is uncertain.
In conclusion, we demonstrate that lipolytic modification of LDL with sPLA 2 or SMase markedly potentiates LDL-induced monocyte AA release and cPLA 2 activation, accompanied by increased TNF-␣ secretion.Importantly, as both sPLA 2 and SMase are present in human atherosclerotic tissue, bind to arterial proteoglycans, and are im-plicated in inflammation (6,7,9,10,39), our results should be of physiological relevance.AA mobilization plays a crucial role in inflammatory signaling.Thus, sPLA 2 -or SMase-modified LDL may contribute to atherosclerosis through the induction of AA and AA-derived lipid mediators that could exert autocrine and/or paracrine control of cellular processes, such as inflammation, chemotaxis, apoptosis, proliferation, and differentiation, at sites of lipoprotein retention in the arterial wall.Therefore, our results suggest novel atherogenic effects of lipolytically modified LDL.
C]OA in the supernatant relative to [ 3 H]AA or [ 14 C]OA incorporated into the cells (dissolved in 1 M NaOH) and are normalized to show fold induction of treated cells relative to untreated cells.

Fig. 1 .
Fig. 1.LDL stimulates [ 3 H]arachidonic acid (AA) and [ 14 C]oleic acid (OA) mobilization in THP-1 cells.[ 3 H]AA-and [ 14 C]OA-labeled THP-1 cells were incubated with 100 g/ml native LDL (LDL nat ) for the indicated time periods (A) or with increasing concentrations of LDL nat for 30 min (B) before determination of [ 3 H]AA and [ 14 C]OA in the supernatants as described in Methods.Data are expressed as means Ϯ SD of triplicate determinations within separate experiments.

Fig. 2 .
Fig. 2. LDL nat and LDL modified by secretory phospholipase A 2 (sPLA 2 ) or sphingomyelinase (SMase) stimulates cytosolic phospholipase A 2 (cPLA 2 ) activation.cPLA 2 activity was determined in lysates from THP-1 monocytes treated with 100 g/ml LDL nat or LDL that had been modified with sPLA 2 or B. cereus SMase.For LDL modification, LDL was pretreated with sPLA 2 (12 h) or SMase (1 h) at 37ЊC as described in Methods before addition to THP-1 monocytes.Data are expressed as means Ϯ SD of triplicate determinations within separate experiments.Asterisks indicate that values are statistically different from untreated (*) or LDL nat -treated (**) cells.

Fig. 3 .
Fig. 3. Effect of the sPLA 2 inhibitor SB203347 and the cPLA 2 / iPLA inhibitor methyl arachidonyl fluorophosphate (MAFP) on LDL-stimulated [ 3 H]AA release.[ 3 H]AA-labeled THP-1 cells were preincubated with the indicated concentrations (conc.) of MAFP or SB203347 for 1 h before the addition of LDL nat (100 g/ml), further incubation for 30 min, and determination of [ 3 H]AA released to the supernatants as described in Methods.Data are expressed as means Ϯ SD of triplicate determinations within separate experiments.

Fig. 4 .
Fig. 4. Pretreatment of LDL nat with sPLA 2 or SMase potentiates LDL-elicited [ 3 H]AA mobilization in THP-1 monocytes.LDL nat (100 g/ml) was incubated at 37ЊC for 12 h with 1 g/ml human recombinant group IIa sPLA 2 , for 1 h with 350 mU/ml B. cereus SMase, or without enzymes.Samples of nontreated LDL (LDL nat ) and treated LDL (LDL nat /sPLA 2 and LDL nat /SMase) were subsequently incubated with THP-1 monocytes for the indicated time periods before determination of extracellular [ 3 H]AA release as described in Methods.Data are expressed as means Ϯ SD of triplicate determinations within separate experiments.Asterisks indicate that the values for LDL nat /sPLA 2 -and LDL nat /SMase-treated cells are statistically different from those for LDL nat -treated cells.

Fig. 5 .
Fig. 5. Preincubation time of LDL with sPLA 2 or SMase markedly affects [ 3 H]AA liberation.LDL nat (100 g/ml) was preincubated at 37ЊC for the indicated time periods with either 1 g/ml human recombinant group IIa sPLA 2 (A) or 500 mU/ml B. cereus SMase (B) before addition to THP-1 monocytes, treatment for 30 min, and determination of extracellular [ 3 H]AA release as described in Methods.Data are expressed as means Ϯ SD of triplicate determinations within separate experiments.

Fig. 6 .
Fig. 6.Effect of sPLA 2 and SMase concentration during LDL preincubation on [ 3 H]AA mobilization.LDL nat (100 g/ml) was incubated with the indicated concentrations of either human recombinant group IIa sPLA 2 (A) or B. cereus SMase (B) at 37ЊC for 12 or 1 h, respectively, before addition to THP-1 monocytes and treatment for 30 min.Extracellular release of [ 3 H]AA was quantified as described in Methods.Data are expressed as means Ϯ SD of triplicate determinations within separate experiments.

Fig. 7 .
Fig.7.Presence of the sPLA2 inhibitor SB203347 or EDTA during LDL preincubation with sPLA 2 or SMase blocks LDL-induced [ 3 H]AA release.A: LDL nat (100 g/ml) was preincubated at 37ЊC for 12 h with 1 g/ml human recombinant group IIa sPLA 2 in the presence or absence of the sPLA 2 inhibitor SB203347 (SB; 10 M) or the anti-sPLA 2 activity-neutralizing antibody BA11 (mAB BA11; 30 g/ml) before addition to THP-1 monocytes and treatment for 30 min.B: LDL nat (100 g/ml) was preincubated at 37ЊC for 1 h with 500 mU/ml B. cereus SMase in the presence or absence of EDTA (10 mM) before addition to THP-1 monocytes and treatment for 30 min.Extracellular release of [ 3 H]AA was quantified as described in Methods.Data are expressed as means Ϯ SD of triplicate determinations within separate experiments.

Fig. 8 .
Fig. 8. Effect of sPLA 2 -and SMase-modified LDL on tumor necrosis factor-␣ (TNF-␣) secretion.THP-1 cells were incubated for 12 h with 100 g/ml LDL nat , LDL treated with recombinant human group IIa sPLA 2 , or LDL modified with B. cereus SMase.Determination of TNF-␣ in the extracellular medium was performed as described in Methods.Untr, untreated.Data are expressed as means Ϯ SD of triplicate determinations within separate experiments.Asterisks indicate that values are statistically different from those of LDL nat -treated cells.