Enhancement of leukocyte response to

Enhancement of leukocyte response to lipopolysaccharide by secretory group IIA phospholipase A2 Tian-Quan Cai, Nathalie Thieblemont, Birming Wong, Rolf Thieringer, Brian P. Kennedy,* and Samuel D. Wright Department of Lipid Biochemistry, Merck Research Laboratories, Rahway, New Jersey; and *Department of Biochemistry & Molecular Biology, Merck Frosst, Center for Therapeutic Research, Pointe-Claire-Dorval, Quebec, Canada

Abstract: Secretory nonpancreatic group IIA phospholipase A2 (sPLA2), a lipolytic enzyme found in plasma, is thought to play an important role in inflammation. In patients with sepsis, a strong positive correlation is observed between the plasma level of sPLA2 and poor clinical outcome in sepsis. We have thus asked whether sPLA2 could play a role in enabling responses of cells to bacterial lipopolysaccharide (LPS), a key contributor to sepsis. In the presence of sPLA2, cellular responses to LPS were significantly increased. This was demonstrated in assays of LPS-stimulated interleukin-6 (IL-6) production in whole blood and binding of freshly isolated human polymorphonuclear neutrophils (PMN) to fibrinogen-coated surfaces. We further found that sPLA2 enhanced binding of labeled LPS to PMN, and that the sPLA2-mediated cell responses to LPS were all blocked by monoclonal antibodies directed against membrane CD14. Two properties of sPLA2 may contribute to its activity to mediate responses to LPS. sPLA2 appears to bind LPS because pre-exposure of sPLA2 to LPS led to a dose-dependent increase in its ability to hydrolyze phospholid substrate, and incubation of sPLA2 with BODIPY-LPS micelles resulted in enhanced fluorescence, presumably from the disaggregation of the LPS aggregates. Additional studies demonstrated that the esterolytic function of sPLA2 is also needed both for the disaggregation of LPS and CD14dependent cell stimulation. The precise mechanisms by which LPS-binding and esterolytic activity contribute to sPLA2 activity are not clear but our data strongly suggest that these activities result in interaction of LPS with CD14 and subsequent cell activation. J. Leukoc. Biol. 65: 750–756; 1999.

bacteria. LPS is an amphipathic molecule that forms aggregates in aqueous buffer and diffuses very slowly from these aggregates. As a consequence, spontaneous association of LPS with cells occurs very slowly, and a high concentration of LPS is generally needed to initiate a cell response. Two serum proteins, LPS-binding protein (LBP) and CD14, have been demonstrated to have a role in enhancing cellular responses to LPS [1–3]. CD14 is a 55-kDa glycoprotein found both as a soluble protein in plasma (soluble CD14) and as a glycosylphosphatidylinositol-linked protein at the membrane (membrane CD14). LBP can transfer LPS to CD14 [4], and the resulting LPS-CD14 complexes initiate functional responses of cells [5]. LBP can also transfer LPS to lipoprotein particles, resulting in a functional neutralization of LPS [6]. In addition to CD14 and LBP, several other factors have also been demonstrated to affect the biological activity of LPS. For example bactericidal/permeability-increasing protein (BPI) [7], phospholipid transfer protein (PLTP) [8], and tissue factor pathway inhibitor (TFPI) [9] have been shown to play a role in the neutralization of LPS. Secretory nonpancreatic group IIA phospholipase A2 (sPLA2) is a 14-kDa protein found in normal plasma [for review see ref. 10]. sPLA2 is a lipolytic enzyme that catalyzes the hydrolysis of the acyl ester bond at the sn-2position of phospholipids. sPLA2 is thought to be an important inflammatory agent because it is induced by inflammatory cytokines such as interleukin (IL)-1b, IL-6, and tumor necrosis factor a (TNF-a), and its activation can lead to the release of arachidonic acid and subsequent production of various proinflammatory mediators such as prostaglandins, leukotrienes, and platelet-activating factor [10–12]. sPLA2 rises in the acute phase [10]. Moreover, the regulatory sequences of the gene encoding sPLA2 contain a putative IL-6-response element, homologous to that found in several other genes encoding

Key Words: CD14 · polymorphonuclear neutrophils · sPLA2

Abbreviations: PMN, polymorphonuclear leukocytes; LPS, lipopolysaccharide; sPLA2, secretory nonpancreatic group IIA phospholipase A2; LBP, LPS-binding protein; BODIPY, boron dipyrromethene difluoride; PBS, Dulbecco’s phosphate-buffered saline; DFP, diisopropyl fluorophosphate; IL-6, interleukin-6; BPI, bactericidal permeability-increasing protein; PLTP, phospholipid transfer protein; TNF-a, tumor necrosis factor a; CFSF, 5-(and 6)carboxyfluorescein diacetate succinimidyl ester. Correspondence: Tian-Quan Cai, Department of Lipid Biochemistry, Merck Research Laboratories, Rahway, NJ 07065. E-mail: [email protected] Received November 13, 1998; revised February 17, 1999; accepted February 18, 1999.

INTRODUCTION Introduction of bacteria into mammalian tissues elicits a strong and immediate innate immune response. A key factor responsible for mediating such a response is thought to be bacterial lipopolysaccharide (LPS), a membrane lipid of gram-negative 750

Journal of Leukocyte Biology

Volume 65, June 1999

http://www.jleukbio.org

acute-phase proteins [11]. Recent studies from patients with sepsis revealed a strong correlation between the plasma levels of sPLA2 and sepsis [13, 14]. Plasma levels of sPLA2 were significantly higher in patients who died of sepsis than in those who survived the illness. Nevertheless, the biological significance for sPLA2 in septic shock remains unclear. Here we report a role for sPLA2 in enabling responses of leukocytes to LPS. We demonstrate that sPLA2 enhances cellular responses to LPS by a potential direct interaction with LPS.

MATERIALS AND METHODS

weight for BODIPY-LPS was estimated to be approximately 3000. To study the physical interaction of sPLA2 with BODIPY-LPS, BODIPY-LPS (100 ng/mL), sPLA2, and various other reagents diluted in PBS were added to a 96-well plate (Costar). Time-dependent changes in fluorescence were measured using a multi-well plate reader, Cytofluor II-4000 series (PerSeptive Biosystems). Fluorescence emission at 530 nm was digitally recorded over time, with excitation at 485 nm.

Measurement of sPLA2 activity sPLA2 activity was measured using an enzyme assay kit provided by Cayman Chemical Co. (Ann Arbor, MI). This assay uses the 1,2-dithio analog of diheptanoyl phosphatidylcholine as a substrate for sPLA2. To study the effect of LPS on sPLA2 activity, increasing concentrations of Re-LPS were incubated with sPLA2 for 20 min at 37°C. Enzyme activity of sPLA2 was then measured.

Reagents LPS from Salmonella minnesota strain R595 (Re-LPS or Ra-LPS) was purchased from List Biological Laboratories, Inc. (Campbell, CA). Monoclonal antibody MY4 (anti-CD14) was from Coulter Immunology (Hialeah, FL). MEM18 (anti-CD14) was from Caltag (San Francisco, CA). 3C10 (anti-CD14) [15], W6/32 (anti-class I histocompatibility Ag) [16], and OKT3 (anti-CD3) [16] were purified from ascites fluid by chromatography on protein G-Sepharose. Human neutrophil lysozyme was purchased from Calbiochem-Novabiochem International (San Diego, CA). DFP, diisopropyl fluorophosphate, was purchased from Sigma Chemical Co. (St. Louis, MO). Purified recombinant sPLA2 was prepared as described previously [17]. By Limulus amebocyte lysate assay this preparation had less than 0.1 ng LPS/mg sPLA2.

Expression and purification of recombinant soluble human CD14 and recombinant human LBP The cDNA encoding the complete open reading frame for human LBP [3, 7] was obtained by polymerase chain reaction from human liver cDNA (Clontech, Palo Alto, CA) and cloned into the multiple cloning site of the expression vector pRmHa3 [18]. The resulting construct was cotransfected with pUChsneo [19] into Schneider-2 cells [20] using the calcium phosphate transfection method with a commercially available kit as outlined by the manufacturer (Life Technologies, Gaithersburg, MD). Stable transfectants were selected with 0.5 mg/mL G418 sulfate (Life Technologies) in Schneider insect medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies). After selection, the cells were adapted to growth on serum-free Ex-cell 401 medium (JRH Biosciences, Lenexa, KS) supplemented with 2 mM L-glutamine, 10 units/mL penicillin, 10 µg/mL streptomycin, and 0.5 mg/mL G418 sulfate. Expression of LBP was induced by growing the cells in serum-free medium in the presence of 1 mM CuSO4. LBP expression was confirmed by Western blotting using a polyclonal antibody against human LBP. Protein from the conditioned medium containing recombinant LBP was subjected to ammonium sulfate precipitation (70% w/v). The pellet was dissolved in 10 mM HEPES, pH 7.3, 2 mM EDTA, 10% (v/v) glycerol (Buffer A) and dialyzed against Buffer A. The dialyzed sample was loaded onto a Mono Q column to which a Mono S column was attached (both columns from Pharmacia Biotech, Piscataway, NJ). Both columns were pre-equilibrated in Buffer A. Under these conditions, most of the contaminating proteins will bind to the Mono Q column, whereas LBP appears in the follow-through of the Mono Q and subsequently binds to the Mono S column. After washing with Buffer A, the Mono Q column was disconnected and a gradient of 0–1 M NaCl in Buffer A was applied to the Mono S. Fractions containing LBP were pooled. All purification steps were carried out at 4°C. This purification protocol resulted in LBP to apparent homogeneity as judged by sodium dodecyl sulfate (SDS)polyacrylamide gels stained with Coomassie blue (not shown). The purification of recombinant human CD14 from conditioned medium of Schneider-2 insect cells transfected with cDNA encoding human CD14 was as described previously [21].

Labeling of LPS with BODIPY and measurement of fluorescence Re-LPS was labeled with fluorophore BODIPY FL C3 as previously described [22], using a kit from Molecular Probes, Inc. (Eugene, OR). The molecular

Binding of LPS to the surfaces of PMN Freshly isolated PMN (5 3 106 cells/mL) diluted in HAP (Dulbecco’s PBS with 0.05% human serum albumin, 3 mM D-glucose) were incubated with BODIPYLPS (100 ng/mL) in the presence of varying concentrations of sPLA2 or LBP for 30 min at 37°C. Alternatively, cells were incubated in the presence of various antibodies (10 µg/mL). After washing, the cell surface-associated fluorescence was analyzed on a FACScan flow cytometer (Becton-Dickinson Immunocytometry System, San Jose, CA).

Stimulation of PMN by LPS The biological activity of LPS was analyzed using an assay measuring the adhesion of PMN to fibrinogen-coated surfaces as previously described [23]. Briefly, freshly isolated PMN (5 3 106 cells/mL) diluted in HAP were fluorescently labeled with 5-(and 6) carboxyfluorescein diacetate, succinimidyl ester (CFSE). Equal volumes of LPS preincubated with sPLA2 for 20 min at 37°C were mixed with fluorescein-labeled PMN and incubated for 10 min at 37°C. PMN were washed with HAP and added to a 72-well Terasaki plate precoated with fibrinogen. After a 15-min incubation at 37°C, adhesion of PMN to the plate was quantitated. Alternatively, 5 µL of agonists diluted in HAP was added to the plate already containing the cells. After 25 min at 37°C, adhesion of PMN to the plate was quantitated. The fluorescence in each well was measured with the use of a Cytofluor 2300 fluorescence plate reader (Millipore Corp.) before and after washing. Binding is expressed as the percentage of cells remaining in the well after the washing step. Donor to donor variation in maximal responses prohibited averaging results of separate experiments but the pattern of response was highly reproducible.

Stimulation of IL-6 production in whole blood Heparinized blood was obtained by venipuncture from healthy human volunteers. Monoclonal antibodies and mixtures containing LPS and sPLA2 diluted in RPMI-1640 (BioWhittaker, Walkersville, MD) to the concentrations indicated were added in 200 µL heparinized whole blood and incubated in 5% CO2 at 37°C for 5 h. Samples were centrifuged for 6 min at 500 g. Plasmas were collected and stored at 4°C. IL-6 levels were measured using a commercially available human IL-6 ELISA kit (Central Laboratories of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), modified for use with smaller volumes of reagents and for detection of IL-6 with a fluorescent alkaline phosphatase substrate as described [24].

RESULTS sPLA2 mediates stimulation of PMN by LPS To test whether increases in sPLA2 concentration have an effect on cellular responses to LPS, we measured the effect of sPLA2 on LPS-induced integrin-mediated adhesion of PMN to fibrinogen-coated surfaces (Fig. 1). LPS alone was incapable of stimulating PMN but addition of sPLA2 enabled a strong, dose-dependent adhesion response to as little as 1 ng/mL of Cai et al.

Interaction of LPS with sPLA2 751

maximal with 100 ng/mL. This high sensitivity is comparable to that seen with LBP measured in parallel (Fig. 1B). LBPmediated responses to LPS require CD14 [5], and further studies showed that antibodies against CD14 (MY4) also effectively blocked the adhesion of PMN mediated by sPLA2 and LPS, whereas control antibody (W6/32) had no effect (Fig. 1C).

sPLA2 mediates IL-6 production in whole blood by LPS To determine whether sPLA2 also affects responses of monocytes, LPS-mediated IL-6 production in whole blood was measured in the presence or absence of sPLA2 (Fig. 2) As previously reported [5], incubation of LPS alone led to an increase in IL-6 production. Although addition of sPLA2 alone did not significantly change IL-6 production, addition of sPLA2 with LPS resulted in a further significant increase of IL-6 production. In nine separate studies, addition of sPLA2 raised IL-6 production 1.62 6 0.29-fold. The sPLA2-mediated IL-6 production was effectively blocked by antibodies against CD14 (MY4 and 3C10), whereas a control antibody (W6/32) did not show any effect.

sPLA2 enables binding of BODIPY-LPS to PMN To ask whether sPLA2 would affect LPS-mediated cell activation by changing cellular uptake of LPS, we measured the role of sPLA2 on binding of BODIPY-LPS to PMN (Fig. 3). Consistent with previous observations [25], incubation of BODIPY-LPS alone with PMN in the absence of serum proteins such as LBP and soluble CD14 led to only a modest binding to PMN surfaces. When sPLA2 was added with BODIPY-LPS, the binding of BODIPY-LPS to PMN was significantly increased. Further studies showed that, as with the PMN adhesion to fibrinogen-coated surfaces (Fig. 1C), antibodies against CD14 also effectively blocked the binding of BODIPY-LPS to PMN induced by sPLA2. Together with the cell adhesion experiment, these observations suggest that sPLA2 may enhance cell

Fig. 1. sPLA2 enables PMN response to LPS. Fluorescently labeled PMN (2.5 3 106 cells/mL) were incubated with (A) increasing concentrations of Re-LPS in the presence of buffer (open triangles), sPLA2 (2.5 µg/mL), or LBP (0.5 µg/mL) or (B) Re-LPS (100 ng/mL) in the presence of buffer (open triangles) and increasing concentrations of sPLA2, LBP, or (C) Re-LPS (100 ng/mL) and indicated monoclonal antibodies (10 µg/mL) in the presence or absence of sPLA2 (2.5 µg/mL) for 10 min at 37°C. After wash, cells were added to plates coated with fibrinogen and incubated for 15 min at 37°C. PMN adhesion was measured as described in Materials and Methods. Results are means of triplicate determinations of a representative experiment repeated four times.

LPS. The magnitude of the sPLA2-mediated response to LPS was identical to that mediated by LBP (Fig. 1A). Additional studies with varied doses of sPLA2 revealed that as little as 10 ng/mL caused a measurable response and responses were 752



Fig. 2. sPLA2 mediates IL-6 production by LPS in whole blood. Whole peripheral blood was incubated for 5 h with 10 ng/mL of LPS and indicated monoclonal antibodies (50 µg/mL) in the presence or absence of sPLA2 (2.5 µg/mL). The supernatants were assayed for IL-6 production by ELISA as described in Materials and Methods. Results are means of triplicate determinations of a representative experiment repeated five times.


Fig. 3. sPLA2 enables binding of BODIPY-LPS to PMN. PMN (5.0 3 106 cells/mL) were incubated with BODIPY-LPS (100 ng/mL) and indicated monoclonal antibodies (10 µg/mL) in the presence or absence of sPLA2 (2.5 µg/mL) for 30 min at 37°C. Cells were washed and cell-associated BODIPYLPS was analyzed on a FACScan flow cytometer. Results were expressed as mean fluorescence intensity of a representative experiment repeated five times.

responses to LPS by facilitating LPS interaction with membrane CD14.

LPS increases enzymatic activity of sPLA2 To ask whether sPLA2 would facilitate cellular uptake of LPS by interacting with LPS directly, we first studied the effect of LPS on the enzyme activity of sPLA2 (Fig. 4). Pre-exposure of sPLA2 to LPS led to a dose-dependent increase in its ability to hydrolyze phospholipid substrate, suggesting a physical interaction between LPS and sPLA2 (or between LPS/PC mixed lipid system and sPLA2). This is consistent with the observation that ceramide, a molecule sharing structural similarity to LPS [26], enhances sPLA2 activity [27]. It should be noted that our analysis cannot rule out an effect of LPS on the substrate (PC). This, however, appears unlikely because LPS interacts with PC very slowly in the absence of lipid transfer proteins [28].

sPLA2 causes dequenching of BODIPY-LPS To further examine the interaction of sPLA2 with LPS, we measured the effect of sPLA2 on fluorescence of LPS labeled with the fluorophore BODIPY. In aqueous buffer, BODIPY-LPS

exists in aggregates and fluorescence is low because of quenching. Disaggregation of the BODIPY-LPS with detergent or by LBP-mediated transfer of LPS monomer to CD14 results in a sharp rise in fluorescence [22]. Consistent with these published results, we found that incubation of CD14 (5 µg/mL) and LBP (0.5 µg/mL) with BODIPY-LPS resulted in a timedependent increase in fluorescence (Fig. 5 , curve 6). We observed that sPLA2 (2.5 µg/mL) also caused a fluorescence increase (Fig. 5, curve 4). The amount of fluorescence increase induced by sPLA2 was lower than that of CD14 and LBP but was significantly higher than that of background (Fig. 5, curve 1, BODIPY-LPS alone). Dequenching of BODIPY-LPS is not a general property of proteins because, as previously reported, human serum albumin did not cause an increase in fluorescence [ref. 8, and data not shown]. sPLA2 is an extremely basic protein [29]. To test whether the effect of sPLA2 on BODIPYLPS was due to a simple electrostatic interaction, we used lysozyme as a control protein. Lysozyme has a similar molecular weight and charge as sPLA2 (14.7 kDa and 8 positive charges for lysozyme, 13.9 kDa and 15 charges for sPLA2), and lysozyme is known to bind to LPS aggregates but is unable to disaggregate them [35]. As shown in Figure 5 (curve 2), the same concentration of lysozyme did not cause any changes in fluorescence of BODIPY-LPS. We conclude that sPLA2 binds to LPS and that the binding alters the aggregation state of LPS. LBP catalyzes the association of LPS with CD14 [22]. In the absence of LBP, the kinetics for the association of LPS with CD14 are very slow, but addition of LBP dramatically enhances the reaction rate [22]. Adding LBP to the mixture of sPLA2 and BODIPY-LPS micelles neither changed the reaction rate nor changed the maximal level of fluorescence induced by sPLA2 (Fig. 6 , curve 4). This observation suggests that LBP does not catalyze binding of LPS to sPLA2. In contrast, on addition of CD14 with sPLA2 and BODIPY-LPS, a small but consistent fluorescence increase was observed (Fig. 6, curve 6). Because incubation of BODIPY-LPS with CD14 alone for 14 min did not increase the fluorescence level (Fig. 6, curve 2), these results suggest a small portion of LPS may be transferred from the sPLA2-LPS complexes to CD14. The maximal fluorescence reached at plateau was dependent on the concentration of sPLA2 (Fig. 7 ). Addition of 0.5 µg/mL sPLA2 induced fluorescence that was almost maximal under these conditions. This molar concentration of BODIPY-LPS (30 nM) was similar to the molar concentration of sPLA2 (35 nM), suggesting a stoichiometric interaction.

Role of esterolytic activity in the interaction of sPLA2 with LPS

Fig. 4. LPS increases sPLA2 activity. sPLA2 (0.20 µg/mL) was incubated with increasing concentrations of Re-LPS for 20 min at 37°C. Samples were then subjected to sPLA2 enzyme activity assay as described in Materials and Methods.

To ask whether the enzymatic activity of sPLA2 plays a role in mediating its interaction with LPS, sPLA2 was treated with or without DFP, a non-reversible enzyme inhibitor of sPLA2. After dialysis to remove the free DFP, DFP-treated sPLA2 was incubated with LPS and its effect on LPS-induced adhesion of PMN to fibrinogen-coated surfaces was measured (Fig. 8 ). Similar to the result shown in Figure 1, addition of untreated sPLA2 enabled a strong adhesion response to LPS. In contrast, sPLA2 treated with DFP failed to enable an adhesion in response to LPS. In a similar experiment, we found that IL-6 Cai et al.


Fig. 5. Dequenching of BODIPY-LPS by sPLA2. BODIPY-LPS (100 ng/mL) was mixed with PBS (curve 1), with lysozyme (2.5 µg/mL, curve 2), with sPLA2 (2.5 µg/mL), and EDTA (2.5 mM, curve 3), with sPLA2 (2.5 µg/mL, curve 4), with LBP (0.5 µg/mL), CD14 (5 µg/mL), and EDTA (2.5 mM, curve 5), or with LBP (0.5 µg/mL) and CD14 (5 µg/mL, curve 6). The fluorescence emission at 530 nm was measured over time as described in Materials and Methods.

production in whole blood mediated by sPLA2 was also blocked by DFP treatment (data not shown). Additional studies with DFP and EDTA (which also blocks sPLA2 activity) showed that catalytic activity also appears necessary for the effect of sPLA2 on the LPS aggregation state. EDTA had no effect on the fluorescence increase of BODIPY-LPS mediated by CD14 and LBP (Fig. 5, curve 5). In contrast, the increase of fluorescence induced by sPLA2 was completely abolished by EDTA (Fig. 5, curve 3). Similar results were obtained with DFP (data not shown).

DISCUSSION Our results suggest that a direct interaction of sPLA2 with LPS occurs. sPLA2 enhances the fluorescence of BODIPY-LPS aggregates (Fig. 5), suggesting that it binds and disaggregates LPS aggregates. LPS also enhances the catalytic activity of sPLA2 (Fig. 4), also suggesting a binding interaction. sPLA2 is a very positively charged molecule with 23 arginines and lysines but only eight glutamic and aspartic acid residues [29]. These basic residues might interact with the strongly anionic LPS molecule. Several of the sPLA2 basic residues encircle a hydrophobic channel that appears as a deep pocket. Such a structure is believed to account for this enzyme’s striking

Fig. 6. Effect of LBP and CD14 on sPLA2-mediated dequenching of BODIPY-LPS. BODIPY-LPS (100 ng/mL) was mixed with PBS (curve 1), with CD14 (5 µg/mL, curve 2), with LBP (0.5 µg/mL, curve 3), with LBP (0.5 µg/mL), and sPLA2 (2.5 µg/mL, curve 4), with sPLA2 (2.5 µg/mL, curve 5), or with CD14 (5 µg/mL) and sPLA2 (2.5 µg/mL, curve 6). The fluorescence emission at 530 nm was measured over time as described.

754



preference for negatively charged substrates [29]. It is possible that such a deep pocket encircled by positively charged molecules may also enable an interaction with LPS. sPLA2 promotes binding of LPS to cells and strongly increases CD14-dependent cell stimulation. Although it appears likely that the binding of sPLA2 to LPS underlies this phenomenon, the mechanism by which sPLA2 facilitates these events is not clear at this time. LPS can be catalytically transferred from micelles or aggregates to membrane or soluble CD14 by the action of LBP [4]. The resulting complexes of LPS and CD14 can deliver monomeric LPS to cells and induce cell activation with no further requirement for LBP [5]. Although the action of sPLA2 may resemble that of LBP in some aspects, it certainly differs in others. The PMN adhesion in response to LPS was nearly equal in the presence of sPLA2 or LBP (Fig. 1), but the efficiency with which sPLA2 transferred BODIPY-LPS to soluble CD14 (Fig. 6) was far less than that seen with LBP [22]. There could be a number of reasons for such differences. For example, LPS may be more effectively transferred from sPLA2-LPS complex to membrane CD14 than that to soluble CD14. sPLA2 is known to bind to cell surfaces [30], and it is possible that sPLA2 may also need to associate with a membrane in order to transfer LPS efficiently. sPLA2 could also have a dual effect on cells, both hydrolyzing membrane phospholipids and transferring LPS. In this regard, we found that pre-exposure of sPLA2 to LPS resulted in an increase of enzyme activity of sPLA2 (Fig. 4), and that the ability of sPLA2

Fig. 7. Fluorescence at plateau of BODIPY-LPS depends on the concentration of sPLA2. BODIPY-LPS (100 ng/mL) and increasing concentrations of sPLA2 were mixed and incubated for 20 min at 37°C. The fluorescence emission at 530 nm was measured.


clear that mechanisms in addition to LBP exist in mediating responses to LPS. It is also clear that animals express a growing ‘‘family’’ of sPLA2 and a contribution from these enzymes, for example a recently described acidic sPLA2 [34], can also be contemplated.

ACKNOWLEDGMENTS We thank Drs. Carl Sparrow, Matt Anderson, Paul Meechan, and Jeanine Regenthal for technical advice at various stages.

Fig. 8. Effect of DFP on sPLA2-mediated PMN response to LPS. sPLA2 (2.5 µg/mL) was incubated with or without DFP (5 mM) for 20 min at 37°C. Free DFP was removed by dialyzing against PBS. sPLA2 samples were first incubated with Re-LPS (25 ng/mL) for 20 min at 37°C and then incubated with fluorescently labeled PMN (2.5 3 106 cells/mL) for 20 min at 37°C with plates coated with fibrinogen. PMN adhesion was measured as described in Materials and Methods. Results are means of triplicate determinations of a representative experiment repeated three times.

to dequench aggregates of BODIPY-LPS and to enhance cell responses to LPS were blocked by enzyme inhibitors of sPLA2 (Figs. 5 and 8). It is thus possible that the enzyme activity may contribute importantly to the action of sPLA2 and LPS. Lipid hydrolysis is known to dramatically enhance lipid transfer from HDL to liver cells [31], and a similar phenomenon may be involved in the transfer of LPS to cells mediated by sPLA2. Esterolytic activity of sPLA2 clearly influences sPLA2mediated activities of LPS on cells (Fig. 8). LPS bears fatty acids esterified to the 3-OH of other fatty acids, but it is unlikely that sPLA2 acts by cleaving these fatty acids. Cleavage of these fatty acids has been shown not only to inactivate LPS but also to convert LPS into an antagonist [32]; our data showed the opposite (Figs. 1 and 2). sPLA2 might enhance fluorescence by cleaving the BODIPY fluorophore from the LPS. However, we found that incubation of sPLA2 with BODIPY-LPS did not release free BODIPY as determined by thin-layer chromatography (data not shown). Finally, if LPS were a substrate for sPLA2 one would expect that (1) adding LPS would decrease cleavage of phospholipids by competition, but the opposite results were observed, and (2) one sPLA2 molecule would interact with multiple molecules of LPS, but optimal fluorescence dequenching was not observed until one sPLA2 was added per LPS monomer (Fig. 7). We thus prefer the view that sPLA2 does not cleave LPS but instead might cleave phospholipid at the cell surface. The mechanism by which this cleavage could enhance LPS action, however, is not clear at this time. sPLA2 has been described as a component of the acute phase response since 1991 [11] but a clear function for this enzyme remains unclear. Our results suggest that sPLA2 may act to enhance cellular responses to LPS. We cannot, however, determine at this time whether this action of sPLA2 plays a significant physiological role. On one hand, LBP is more abundant than sPLA2 and can efficiently transfer LPS to CD14, thus making sPLA2 potentially irrelevant. On the other hand, LBP knock-out animals respond identically to LBP-sufficient animals when challenged with LPS intravenously [33]. It is thus

REFERENCES 1. Wright, S. D., Tobias, P. S., Ulevitch, R. J., Ramos, R. A. (1989) Lipopolysacharide (LPS) binding protein opsonizes LPS-bearing particles for recognition by a novel receptor on macrophages. J. Exp. Med. 170, 1231–1241. 2. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J., Mathison, J. C. (1990) CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249, 1431–1433. 3. Schumann, R. R., Leong, S. R., Flaggs, G. W., Gray, P. W., Wright, S. D., Mathison, J. C., Tobias, P. S., Ulevitch, R. J. (1990) Structure and function of lipopolysaccharide binding protein. Science 249, 1429–1431. 4. Hailman, E., Lichenstein, H. S., Wurfel, M. M., Miller, D. S., Johnson, D. A., Kelley, M., Busse, L. A., Zukowski, M. M., Wright, S. D. (1994) Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J. Exp. Med. 179, 269–277. 5. Hailman, E., Vesselon, T., Kelley, M., Busse, L. A., Hu, M. C. T., Lichenstein, H. S., Detmers, P. A., Wright, S. D. (1996) Stimulation of macrophages and neutrophils by complexes of lipopolysaccharide and soluble CD14. J. Immunol. 156, 4384–4390. 6. Wurfel, M. M., Kunitake, S. T., Lichenstein, H., Kane, J. P., Wright, S. D. (1994) Lipopolysaccharide (LPS)-binding protein is carried on lipoprotein and acts as a cofactor in the neutralization of LPS. J. Exp. Med. 180, 1025–1035. 7. Wilde, C.G., Seilhamer, J. J., McGrogan, M., Ashton, N., Snable, J. L., Lane, J. C., Leong, S. R., Thornton, M. B., Miller, K. L., Scott, R. W., Marra, M. N. (1994) Bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein: LPS binding properties and effects on LPS-mediated cell activation. J. Biol. Chem. 269, 17411–17416. 8. Hailman, E., Albers, J. J., Wolfbauer, G., Tu, A., Wright, S. D. (1996) Neutralization and transfer of lipopolysaccharide by phospholipid transfer protein. J. Biol. Chem. 271, 12172–12178. 9. Park, C. T., Creasey, A. A., Wright, S. D. (1997) Tissue factor pathway inhibitor blocks cellular effects of endotoxin by binding to endotoxin and interfering with transfer to CD14. Blood 89, 4268–4274. 10. Vadas, P., Browning, J., Edelson, J., Pruzanski, W. (1993) Extracellular phospholipase A2 expression and inflammation: the relationship with associated disease states. J. Lipid Mediat. 8, 1. 11. Crowl, R. M., Stoller, T. J., Conroy, R. R., Stoner, C. R. (1991) Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response. J. Biol. Chem. 266, 2647–2651. 12. Kudo, I., Murakami, M., Hara, S., Inoue, K. (1993) Mammalian nonpancreatic phospholipase A2. Biochim. Biophys. Acta 1170, 217–231. 13. Endo, S., Inada, K., Nakae, H., Takakuwa, T., Yamada, Y., Suzuki, T., Taniguchi, S., Yoshida, M., Ogawa, M., Teraoka, H. (1995) Plasma levels of type II phospholipase A2 and cytokines in patients with sepsis. Res. Commun. Mol. Pathol. Pharmacol. 90, 413–421. 14. Guidet, B., Poit, O., Masliah, J., Barakett, V., Maury, E., Bereziat, G., Offenstadt, G. (1996) Secretory non-pancreatic phospholipase A2 in severe sepsis: relation to endotoxin, cytokines and thromboxane B2. Infection 24, 103–108. 15. Van Voorhis, W. C., Steinman, R. M., Hair, L. S., Luban, J., Witmer, M. D., Koide, S., Cohn, Z. A. (1983) Specific antimononuclear phagocyte monoclonal antibodies: amplification to the purification of dendritic cells and the tissue localization of macrophages. J. Exp. Med. 158, 126–145. 16. Barnstable, C. J., Bodmer, W. F., Brown, G., Galfre, G., Milstein, C., Williams, A. F., Ziegler, A. (1979) Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens: new tools for genetic analysis. Cell 14, 9.

Cai et al.


17. Tremblay, N. M., Kennedy, B. P., Street, I. P., Kaupp, W. J., Laliberte, F., Weech, P. K. (1993) Human group II phospholipase A2 expressed in trichoplusia-ni larvae—Isolation and kinetic properties of the enzyme. Protein Expression Purification 4, 490–498. 18. Bunch, T.A., Grinblat, Y., Goldstein, L. S. B. (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res. 15, 1043. 19. Steller, H., Pirrotta, V. (1985) A transposable P vector that confers selectable G418 resistance to Drosophila larvae. EMBO J. 4, 167. 20. Schneider, I. (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27, 353–365. 21. Detmers, P. A., Zhou, D., Polizzi, E., Thieringer, R., Hanlon, W. A., Vaidya, S., Bansal, V. S. (1998) Role of stress-activated mitogen-activated protein kinase (p38) in beta 2-integrin-dependent neutrophil adhesion and the adhesion-dependent oxidative burst. J. Immunol. 161, 1921–1929. 22. Yu, B., Wright, S. D. (1996) Catalytic properties of lipopolysacchride (LPS) binding protein: Transfer of LPS to soluble CD14. J. Biol. Chem. 271, 4100–4105. 23. van Kessel, K. P. M., Park, C. T., Wright, S. D. (1994) A fluorescence microassay for the quantitation of integrin-mediated adhesion of neutrophil. J. Immunol. Meth. 172, 25. 24. Kusunoki, T., Hailman, E., Juan, T. S. C., Lichenstein, H. S., Wright, S. D. (1995) Molecules from Staphylococcus aureus that bind CD14 and stimulate innate immune responses. J. Exp. Med. 182, 1673–1682. 25. Wurfel, M. M., Hailman, E., Wright, S. D. (1995) Soluble CD14 acts as a shuttle in the neutralization of lipopolysaccharide (LPS) by LPS-binding protein and reconstituted high density lipoprotein. J. Exp. Med. 181, 1743–1754. 26. Joseph, C. K., Wright, S. D., Bornmann, W. G., Randolph, J. T., Kumar, E. R., Bittman, R., Liu, J., Kolesnick, R. N. (1994) Bacterial lipopolysaccharide has structural similarity to ceramide and stimulates ceramideactivated protein kinase in myeloid cells J. Biol. Chem. 269, 17606– 17610.

756



27. Huang, H-W., Goldberg, E. M., Zidovetzki, R. (1996) Ceramide induces structural defects into phosphatidylcholine bilayers and activates phopholipase A2. Biochem. Biophys. Res. Commun. 220, 834–838. 28. Wurfel, M. M., Wright, S. D. (1997) Lipopolysaccharide-binding protein and soluble CD14 transfer lipopolysaccharide to phospholipid bilayers: Preferential interaction with particular classes of lipid. J. Immunol. 158, 3925–3934. 29. Wery, J. P., Schevitz, R. W., Clawson, D. K., Bobbitt, J. L., Dow, E. R., Gamboa, G., Goodson, T., Jr, Hermann, R. B., Kramer, R. M., McClure, D. B., et al. (1991) Structure of recombinant human rheumatoid arthritic synovial fluid phospholipase A2 at 2.2 A resolution. Nature 352, 79–82. 30. Lambeau, G., Ancian, P., Barhanin, J., Lazdunski, M. (1994) Cloning and expression of a membrane receptor for secretory phospholiase A2. J. Biol. Chem. 269, 1575–1578. 31. Bamberger, M., Glick, J. M., Rothblat, G. H. (1983) Hepatic lipase stimulates the uptake of high density lipoprotein cholesterol by hepatoma cells. J. Lipid Res. 24, 869–876. 32. Kovach, N. L., Yee, E., Munford, R. S., Raetz, C. R., Harlan, J. M. (1990) Lipid IVA inhibits synthesis and release of tumor necrosis factor induced by lipopolysaccharide in human whole blood ex vivo. J. Exp. Med. 172, 77–84. 33. Wurfel, M. M., Monks, B. G., Ingalls, R. R., Dedrick, R. L., Delude, R., Zhou, D., Lamping, N., Schumann, R. R., Thieringer, R., Fenton, M. J., Wright, S. D., Golenbock, D. (1997) Targeted deletion of the lipopolysaccharide (LPS)-binding protein gene leads to profound suppression of LPS responsese ex vivo, whereas in vivo responses remain intact. J. Exp. Med. 186, 2051–2056. 34. Cupillard, L., Koumanov, K., Mattei, M.-G., Lazdunski, M., Lambeau, G. (1997) Cloning, chromosomal mapping and expression of a novel human secretory phospholipase A2. J. Biol. Chem. 272, 15745–15752. 35. Ohno, N., Morrison, D. C. (1989) Lipopolysaccharide interaction with lysozyme: binding of lipopolysacchride to lysozyme and inhibition of lysozyme enzymatic activity. J. Biol. Chem. 264, 4434–4441.
