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PHAGOCYTES
From the Department of Pediatrics, Division of
Hematology/Oncology, and the Department of Internal Medicine, Division
of Nephrology, University of Michigan, Ann Arbor.
Exogenous C2-ceramide has been shown to inhibit
polymorphonuclear leukocyte (PMN) phagocytosis through inhibition of
phospholipase D (PLD) and downstream events, including activation of
extracellular signal-regulated kinases 1 and 2, leading to the
hyphothesis that the sphingomyelinase pathway is involved in
termination of phagocytosis. Here it is postulated that increased
PLD activity generating phosphatidic acid and diacylglycerol (DAG) is
essential for superoxide release and degranulation and that ceramide,
previously shown to be generated during PMN activation, inhibits PLD
activation, thereby leading to inhibition of PMN function. When PMNs
were primed with granulocyte colony-stimulating factor (G-CSF) and then
activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C2-ceramide (10 µM) completely inhibited release
of superoxide, lactoferrin, and gelatinase; the DAG analog
sn-1,2-didecanoylglycerol (DiC10) (10 µM) restored
oxidase activation and degranulation in the ceramide-treated cells.
Similarly, C2-ceramide inhibited oxidase activity and
degranulation of PMNs treated with cytochalasin B followed by FMLP, and
DiC10 restored function. In contrast, C2-ceramide did not
inhibit phosphorylation of p47phox or p38 mitogen-activated protein
kinase, or translocation of p47phox, PLD-containing organelles,
adenosine diphosphate-ribosylation factor 1, RhoA, protein kinase C
(PKC)- Polymorphonuclear leukocytes (PMNs) ingest
microbes, then destroy them by release of reactive oxygen intermediates
and granule enzymes to the phagosome. These processes must be closely
regulated temporally and spatially to effectively destroy microbes
without damage to surrounding tissue, suggesting a signaling linkage in which release of cytotoxins occurs after phagocytosis. Several signaling and effector processes are similar or shared among the PMN
functions of phagocytosis, oxidant production, and degranulation, including activation of mitogen-activated protein (MAP)
kinases,1-3 some protein kinase C (PKC)
isoforms,4,5 phospholipase D (PLD),6-8 and
cytoskeletal reorganization.9-11 Specifically, phagocytosis is dependent upon activation of PLD, extracellular signal-regulated kinase (ERK)-1, ERK2, and myosin light chain kinase,
and involves translocation of PKC- Activation of the nicotinamide adenine dinucleotide phosphate (reduced
form) (NADPH) oxidase is dependent on translocation of the
p47phox-p67phox-p40phox cytosolic complex and small GTPase Rac2 to the plasma or phagosomal membrane where they associate with
cytochrome b558 and Rap1A.18,19
Phosphorylation of p47phox, p67phox, and p40phox occurs, although only
p47phox phosphorylation may be required for oxidase activation. P47phox
is phosphorylated on several residues, mediated by PKA, PKC, a
phosphatidic acid-activated kinase, p21-activated kinases, and/or MAP
kinases.2,18,20,21 The MAP kinase p38 may be involved in
phosphorylation of p47phox, as evidenced by in vitro
phosphorylation2 and inhibition of oxidase by the p38
inhibitor SB203580.22 There is also evidence that ERKs 1 and 2 participate in phosphorylation of p47phox.2,3
We have previously shown that endogenous ceramide levels increase
with activation in adherent PMNs stimulated with FMLP and in PMNs
undergoing phagocytosis of antibody-opsonized particles. Ceramide is
the product of neutral sphingomyelinase, and plasma membrane-associated neutral sphingomyelinase activity increases during
FMLP stimulation of PMNs.23 Once maximal levels of
endogenous ceramide are achieved, PMN functional responses are
terminated.7,23,24 Since neutral sphingomyelinase activity
also increases concurrently with ceramide accumulation during
phagocytosis,7 we concluded that the neutral
sphingomyelinase pathway may be involved in terminating both
phagocytosis and oxidant production. Exogenous C2-ceramide inhibits oxidant production in both suspended and adherent
PMNs24-27 and inhibits the increase in PLD activity
occurring during oxidant production in adherent PMNs24 and
phagocytosis of antibody-coated particles by PMNs in
suspension.7 C2-ceramide also inhibits PLD
activity in cell-free systems28; in HL-60 cells, ceramide inhibits by preventing activation of PLD by the small GTPases adenosine
diphosphate-ribosylation factor (ARF)-1 and RhoA.29 Following inhibition of PLD activity, both phosphatidic acid (PA) and
diacylglycerol (DAG) fail to accumulate. The lack of DAG theoretically could lead to decreased activation of PKC isoforms We hypothesized that the sphingomyelinase pathway is involved in
terminating oxidant production, degranulation, and phagocytosis by
leading to ceramide inhibition of PLD activity. To examine this
possibility, we used suspended PMNs to measure oxidant release and
degranulation of secondary and tertiary granules in the presence of
exogenous C2-ceramide. PLD involvement was tested in 2 ways: by using a DAG analog to attempt restoration of oxidant and
granule release following ceramide treatment and by directly measuring C2-ceramide inhibition of PLD activity under conditions
that result in oxidant production and degranulation. In an effort
to determine the mechanism of PLD inhibition by ceramide,
translocation of ARF1, RhoA, PKC- Materials
Cells
Degranulation Incubation with C2-ceramide or dihydro-C2-ceramide was performed before stimulation as follows: PMNs were suspended at 2 × 106/mL (final) in PBS containing 1 mM Ca++, 1 mM Mg++, and 5 mM glucose; then lipids were added and cells incubated for 30 minutes at 22°C. Where indicated, DiC10 was added, and incubation proceeded for another 15 minutes at 22°C. PMNs were then stimulated at 37°C with G-CSF (50 ng/mL) for 10 minutes, followed by FMLP (100 nM) for 5 minutes. Alternatively, PMNs were stimulated with cytochalasin B (5 µg/mL) for 3 minutes, then FMLP (100 nM) for 5 minutes. Cells were placed on ice for 5 minutes, then centrifuged. Lactoferrin and gelatinase content in the supernatants was measured by enzyme-linked immunosorbent assay (ELISA) as previously described.32 Unstimulated controls were incubated at the same temperatures and times as the stimulated samples.Superoxide PMNs were treated with lipids as described above. Before stimulation, cytochrome C and superoxide dismutase or buffer were added to give final concentrations of 75 µM/mL and 60 µg/mL, respectively, and 1 × 106 PMNs per milliliter. PMNs were stimulated as described above, and the reduction of cytochrome C in supernatants was read spectroscopically as an end-point assay.33 Unstimulated controls were incubated at the same temperatures and times as the stimulated samples.Phospholipase D PMNs were labeled with 1-O-[3H]-octadecyl-sn-glycero-3 phosphocholine (Amersham, Arlington Heights, IL) as previously described.7 Cells were treated with lipids and stimulated at 2 × 106/mL as described above, except that before stimulation cells were incubated with 1% ethanol for 5 minutes at 37°C. Cell pellets were extracted according to the method of Van Veldhoven and Bell.34 Lipid products were separated with the use of thin-layer chromatography (TLC) as previously described,6 and the [3H]-phosphatidylethanol (PEt) was removed and counted in a scintillation counter.Subcellular fractionation PMNs (2 × 106/mL) were incubated with C2-ceramide and stimulated with cytochalasin B and FMLP as described above. PMNs were then centrifuged and suspended at 25 to 40 × 106/mL in disruption buffer containing 100 mM KCl, 3 mM NaCl, 10 mM piperazine diethanesulfonic acid (pH 7.0), and 3.5 mM MgCl2. PMNs were nitrogen cavitated, layered over a 3-layer Percoll gradient, and fractionated as described by Kjeldsen et al.35 Gelatinase was determined by ELISA and latent alkaline phosphatase by the difference in activity in the presence and absence of Triton X-100.35The p47phox phosphorylation PMNs were labeled at 108/mL with H3[32P]O4, 2 mCi (74 × 106 Bq)/108 PMNs, in 30 mM Hepes (pH 7.4), 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 10 mM glucose. Cells were washed twice and suspended (2 × 106/mL) in PBS containing 1 mM Ca++ and 1 mM Mg++, with each sample containing 107 PMNs. PMNs were exposed to lipids and stimulated as described above, except that FMLP stimulation was for 1 minute. A positive control was made by stimulating one sample with 100 ng/mL phorbol myristate acetate (PMA) for 3 minutes at 37°C. PMNs were centrifuged, then lysed 30 minutes on ice in 0.8 mL buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% sodium deoxycholate, 1% Igepal CA-630, 40 mM P-nitrophenylphosphate, 1 mM phenymethyl sulfonyl fluoride (PMSF), 1 mM Na3VO4, 50 mM NaF, and 10 µg/mL each of aprotinin, leupeptin, pepstatin, and soybean trypsin inhibitor. Samples were microfuged for 5 minutes; then goat anti-p47phox was added to supernatants and incubated, rotating for 1 hour at 4°C. Rabbit antigoat antibody was added and samples were incubated overnight. Protein A-sepharose beads were added and samples incubated 1 hour. Beads were washed and boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer; then supernatants were subjected to 10% SDS-PAGE. Gels were stained, dried, and exposed to x-ray film. Samples were also processed on a 25-cm-tall 10% SDS-PAGE gel to separate p47phox from the 55-kd immunoglobulin (Ig)-G band. Proteins were transferred to polyvinylidene fluoride membranes, then probed with antibody to p47phox to show equal amounts of protein per sample.The p47phox, PKC-  or  , followed by horseradish
peroxidase-conjugated antigoat or antimouse respectively, and
chemiluminescence detection.
The p38 MAP kinase activation PMN samples (2 × 106) were treated with C2-ceramide and stimulated with G-CSF and FMLP as described above. PMNs were lysed in 1% Triton X-100, 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM PMSF, 1 mM Na3VO4, and 10 µg/mL each of aprotinin, leupeptin, pepstatin, and soybean trypsin inhibitor. Lysates were microfuged for 5 minutes and supernatants combined with SDS-PAGE buffer. Western blots were performed as described above and membranes probed with antibody against active (phosphorylated) p38. Blots were stripped and reprobed with antibody recognizing both active and inactive p38 to demonstrate equal loading.
C2-ceramide inhibition of function We hypothesized that the sphingomyelinase pathway, particularly the sphingomyelinase product ceramide, is involved in termination of oxidant production and degranulation as well as phagocytosis because of the functional linkage of these processes and previous studies showing common elements of signaling. To address this hypothesis, we first assayed oxidant production and degranulation in the presence of exogenous cell-permeable C2-ceramide to simulate accumulation of endogenous ceramide. PMNs were incubated with C2-ceramide or its inactive analog dihydro-C2-ceramide, then stimulated with G-CSF (or, in some experiments, cytochalasin B), followed by FMLP; then supernatants were assayed for superoxide, gelatinase (tertiary granule marker), or lactoferrin (specific granule marker). G-CSF-primed PMNs were used because pilot experiments showed that FMLP alone yielded low levels of oxidant release and degranulation (data not shown), and PLD activation represented by PA and DAG accumulation is low without an additional stimulus.17,37Superoxide production in G-CSF-primed, FMLP-stimulated PMNs was
inhibited in dose-dependent fashion by C2-ceramide.
Approximately 35% of superoxide release was inhibited by 3 µM
C2-ceramide, and 74% by 10 µM, reducing superoxide to
levels not significantly different from those of unstimulated PMNs
(Figure 1A). These concentrations are
similar to those reported by others as inhibiting PMN superoxide release.25-27 Superoxide was not significantly inhibited
by 10 µM dihydro-C2-ceramide. G-CSF alone did not
stimulate oxidant release, while FMLP alone stimulated little
(approximately 3 nmol/106 PMNs per 5 minutes) oxidase
activity (data not shown). Superoxide release stimulated by PMA was not
significantly inhibited by C2-ceramide (data not shown),
probably because PMA bypasses receptor-mediated signaling to directly
stimulate PKC. Similarly to the superoxide results, the release of
gelatinase and lactoferrin was inhibited by C2-ceramide but
not by dihydro-C2-ceramide, and 10 µM
C2-ceramide reduced release of both granule sets to
unstimulated levels (Figure 1B-C). To demonstrate that
C2-ceramide did not directly affect the oxidase or
measurement of granule markers, C2-ceramide was added
immediately after FMLP, and no inhibition was observed (data not
shown). Notably, the dose response was different for gelatinase and
lactoferrin. Lactoferrin release declined at low
C2-ceramide concentrations and was significantly inhibited
at concentrations as low as 1 µM (Figure 1C), while 5 µM was
necessary to significantly inhibit gelatinase release (Figure 1B). This
is consistent with observations by others that tertiary granules
degranulate more readily than specific granules38 and
suggests that tertiary granules may also be more refractory to
inhibition. When activating PMNs with cytochalasin B and FMLP,
superoxide and lactoferrin release were increased 2- to 3-fold, and
gelatinase release increased about 60% compared with G-CSF-primed,
FMLP-stimulated cells. However, C2-ceramide incubation
resulted in essentially the same inhibition and dose response (data not
shown). Previously, we determined that the time of ceramide formation
occurred together with the termination of the respiratory
burst24 and phagocytosis.7 Inhibition
by C2-ceramide suggests that these functions, like phagocytosis, may be terminated by intracellular ceramide
accumulation.
DAG restoration of function We previously showed that C2-ceramide inhibits PLD activity during phagocytosis in suspended PMNs7 and during oxidant production in adherent PMNs.24 We surmised that the mechanism of ceramide inhibition of suspended PMN oxidant production and degranulation was also through its inhibition of PLD and resulting decrease in DAG production. To address this proposal, we used the DAG analog DiC10 in an attempt to reverse ceramide inhibition of functional responses. Treating PMNs with DiC10 prior to C2-ceramide treatment restored superoxide production and degranulation stimulated by G-CSF and FMLP (Figure 2). In the presence of 10 µM C2-ceramide, 10 µM DiC10 restored superoxide, gelatinase, and lactoferrin release to levels observed when no ceramide was present, and 20 to 30 µM DiC10 increased release to an even greater degree. When no C2-ceramide was present, DiC10 also increased superoxide production and degranulation in G-CSF-primed, FMLP-stimulated PMNs (Figure 2). The results with and without C2-ceramide converged at 20 to 30 µM, suggesting that a limit to the effect of DiC10 was reached. High concentrations (exceeding 30 µM) of DiC10 stimulated PMNs to release superoxide in the absence of other stimulation. However, 10 µM DiC10, a level at which function is fully restored in stimulated PMNs (Figure 2), did not significantly increase oxidant release or degranulation by itself (data not shown). DiC10 also completely restored oxidant production and degranulation in PMNs activated with cytochalasin B and FMLP (data not shown). DiC10 reversal of C2-ceramide inhibition indicates that the PLD pathway is likely to be involved in oxidant production and degranulation and that C2-ceramide inhibition of these functions is a result of its effect on PLD.
C2-ceramide inhibition of PLD activity We measured PLD activity under conditions that stimulate degranulation and oxidant production in the presence of C2-ceramide to further investigate the involvement of the sphingomyelinase pathway in the activation of PLD. PMNs were labeled with 1-O-[3H]-octadecyl-sn-glycero-3 phosphocholine and stimulated in the presence of ethanol; the accumulation of PEt, a product of PLD under these conditions, was measured. Stimulation of PMNs with G-CSF and FMLP increased PLD activity 3-fold, and activity was inhibited in a dose-dependent fashion by C2-ceramide (Figure 3). Similarly, the PLD activity of PMNs stimulated with cytochalasin B and FMLP increased about 30-fold and was inhibited to unstimulated levels by 10 µM C2-ceramide (Figure 3). These observations are consistent with the hypothesis that oxidant production and degranulation involve the sphingomyelinase and PLD pathways.
Translocation of PLD In resting PMNs, PLD is found primarily in secretory vesicles and is mobilized to the plasma membrane upon FMLP stimulation.39 We performed subcellular fractionation and examined release of secretory vesicles to determine whether C2-ceramide inhibited PLD translocation to the plasma membrane. Latent alkaline phosphatase (LAP) was used as a marker, as it is distributed mostly in the secretory vesicles with a smaller proportion in tertiary granules.32 LAP is depleted (becomes nonlatent) during FMLP activation as secretory vesicles fuse with the plasma membrane.35 Pretreatment with C2-ceramide did not significantly affect this mobilization of LAP from secretory vesicles (Figure 4A). Thus, PLD translocation was unlikely to have been impaired by ceramide. In the same experiments, C2-ceramide inhibited oxidant production (data not shown) and depletion of gelatinase from tertiary granules (Figure 4B), consistent with ceramide inhibition of gelatinase release.
Effect of phosphatase inhibitor on superoxide production Ceramide activates a serine-threonine phosphatase whose effects can be inhibited by 0.1 to 10 nM okadaic acid, an inhibitor of serine-threonine phosphatases.40 We used okadaic acid to attempt to restore superoxide production in C2-ceramide-treated PMNs. No significant reversal of ceramide inhibition was obtained with 0.1 to 300 nM okadaic acid (data not shown), leading us to conclude that an indirect effect of ceramide on protein phosphorylation was not responsible for inhibition of PLD.Translocation of ARF1 and RhoA The small GTPases ARF1 and RhoA are regulators of PLD,41 and the amounts of these proteins associated with the plasma membrane increase with stimulation in PMNs and HL-60 cells.29,42 C2-ceramide (50 µM) inhibits this translocation in HL-60 cells29; therefore, we determined whether the same mechanism operates in PMNs. A concentration of C2-ceramide (10 µM) that completely inhibits function in PMNs (Figure 1) had no effect on the amount of ARF1 and RhoA associated with the membrane (Figure 5).
Translocation of PKC DAG, the downstream product of PLD, is an activator of PKC; therefore, we tested the effect of C2-ceramide on PKC-,
an isomer associated with oxidant production and p47phox
phosphorylation.43 During PMN activation, PKC-
translocates from the cytosol to the plasma membrane.43 In
addition, there is a small amount of PKC- in PMNs, and this kinase
regulates PLD in some systems.28 Therefore, we also looked
for a ceramide effect on translocation of PKC-. The association of
either protein kinase with the plasma membrane was not affected by
C2-ceramide treatment (data not shown). This observation
was consistent with the lack of effect by C2-ceramide on
oxidant production stimulated by directly activating PKC with phorbol ester.
Phosphorylation of p47phox and p38 MAP kinase Although ceramide inhibition of PLD may be a sufficient explanation for inhibition of the oxidase, we also wished to investigate other possible targets of ceramide. C2-ceramide inhibits phosphorylation and activation of ERK1 and ERK2 during PMN FMLP activation or phagocytosis,1 while sphingosine inhibits translocation of PKC- and Raf-1.4 Therefore,
we studied phosphorylation of 2 proteins known to be a part of NADPH
oxidase activation: p47phox and p38 MAP kinase. Phosphorylation of
p47phox occurs during oxidant production.18 The
p47phox phosphorylation was enhanced by PMN activation with G-CSF and
FMLP, and C2-ceramide did not inhibit this phosphorylation
(Figure 6A). Figure 6B demonstrates equal amounts of p47phox among the samples in Figure 6A. Translocation of
p47phox to the plasma membrane is also associated with oxidase activation. Similarly, C2-ceramide had no inhibitory effect
on the amount of p47phox in the plasma membrane (Figure 6C). The p38
MAP kinase, which is capable of in vitro phosphorylation of p47phox,
was phosphorylated, indicating activation during G-CSF and FMLP
stimulation (Figure 7). The p38
phosphorylation was not inhibited by C2-ceramide.
Therefore, NADPH oxidase inhibition was not a direct result of ceramide
effects on p47phox or p38 MAP kinase, but could be attributed to
inhibition of PLD activity.
We have demonstrated in this study that C2-ceramide inhibited both PMN degranulation and NADPH oxidase activity. In turn, the DAG analog DiC10 restored function when used at concentrations found incapable of activating PMNs themselves, indicating that the ceramide effect on PLD was bypassed by replacement of its downstream product DAG. DAG probably then contributed to granule-membrane fusion. PLD activity, stimulated either by G-CSF and FMLP or by cytochalasin B and FMLP, was inhibited by the same concentrations of C2-ceramide shown to inhibit oxidase function and degranulation. Similarly, C2-ceramide inhibits PLD activity stimulated by PMN phagocytosis of IgG-opsonized erythrocytes or in adherent PMNs stimulated by FMLP.7,24 C2-ceramide inhibits FMLP-stimulated PLD activity in PMN-like differentiated HL-60 cells as well as in a cell-free system.29 Others have linked PLD activation with oxidant production and degranulation.6,8,16,44 Therefore, we concluded that C2-ceramide inhibition of these functions is through inhibition of PLD. PLD is activated by small GTPases of the ARF and Rho families,
perhaps synergistically with other activators such as PKC- C2-ceramide inhibition of function is consistent with a negative modulatory role of endogenous ceramide. Previously, we determined that ceramide formation correlated with the termination of the respiratory burst in adherent PMNs24 and of phagocytosis of opsonized red cells by PMNs.7 When PMNs in suspension were either activated with FMLP or primed with FMLP followed by challenge with antibody-coated red cells, plasma membrane-associated neutral sphingomyelinase activity rose significantly at a time when both the respiratory burst23 and phagocytosis7 were terminated, respectively. In this model, we postulate that sphingomyelinase is activated by agonists along with, or soon after, effectors of phagocytosis, oxidant production, and degranulation. In suspended PMNs, phosphorylation, kinase and lipase activation, and molecular translocation begin within 1 minute after stimulation. Maximum rates of phagocytosis, oxidase activity, and degranulation are achieved within 5 minutes, after which these processes slow or cease.1,4,7,26,36,38,45 Ceramide, the product of sphingomyelinase, begins to accumulate in the cell with a time lag as compared with the initiators of function7,24 and, reaching significant levels several minutes later, may result in PLD inhibition and termination of function. The results in the present study demonstrate differential C2-ceramide inhibition of organelle release: lactoferrin release is most potently inhibited by ceramide, gelatinase less so, and secretory vesicles not at all. This is the reverse order of controlled exocytosis shown by Sengelov et al,38 where PMNs were stimulated by FMLP or manipulation of cytosolic calcium, and secretory vesicles were most readily released, followed by tertiary (gelatinase) and secondary (lactoferrin) granules. These observations suggest that easily mobilizable granules may be more difficult to inhibit, perhaps relating to different signaling pathways, levels of PLD involvement, or targets of ceramide inhibition involved in granule mobilization. Various studies have shown that ERK1 and ERK2 are involved in PMN
oxidase activation, possibly by phosphorylating
p47phox.3,45,46 C2-ceramide inhibits ERK
activation in phagocytosing PMNs.7 Additionally,
C2-ceramide treatment of PMNs inhibits activation of
p21-activated kinases, which may phosphorylate p47phox.21 However, we found that C2-ceramide did not substantially
inhibit p47phox phosphorylation, suggesting that ERK or p21-activated kinase is responsible for little or no phosphorylation of p47phox. Indeed, mutation of ERK target serines 345 and 348 to alanines has no
significant effect on oxidase activity,18 and MEK
inhibitor PD098059 does not consistently block superoxide production
(unpublished observations, April 1999, and Kuroki and
O'Flaherty47). Our data do not rule out the
possibility that p47phox is inactivated by inhibiting ERK
phosphorylation of a particular residue while other residues are
normally phosphorylated, but even so, this would be consistent with a
downstream effect of PLD inhibition. Translocation of PKC- C2-ceramide did not inhibit phosphorylation and activation of p38 MAP kinase. The evidence for p38 involvement in NADPH oxidase activation includes inhibition of superoxide production by SB203580 (unpublished observations, April 1999, and Rane et al22) and in vitro phosphorylation of p47phox by p38 MAP kinase.2 Thus, p38 regulation of the oxidase appears to be separate from the PLD pathway inhibited by ceramide. Although C2-ceramide did not noticeably affect
phosphorylation or translocation of p47phox, PKC- The data presented suggest that accumulation of endogenous ceramide may participate in the termination of oxidant release and degranulation as well as phagocytosis. Exogenous C2-ceramide may inhibit release of reactive oxygen intermediates to the extracellular environment, while not necessarily inhibiting assembly of oxidase in the phagolysosome or on granule membranes. However, C2-ceramide also prevents formation of the phagolysosome by inhibiting ingestion and possibly also granule membrane fusion with the phagolysosome, believed to be mediated by DAG produced by PA phosphohydrolase from the PLD product PA.30
Submitted February 13, 2001; accepted October 17, 2001.
Supported by National Institutes of Health grants AI20065 (L.A.B.) and DK41487 and DK39255 (J.A.S.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Laurence A. Boxer, University of Michigan, Dept of Pediatrics, Hematology/Oncology, L2110 Women's Hospital, Box 0238, 1500 E Medical Center Dr, Ann Arbor, MI 48109; e-mail: laboxer{at}med.umich.edu.
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Abstract
Introduction
, and 
receptor signaling.
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