From the Infectious Diseases Laboratory, Department
of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion
University of the Negev and Soroka Medical Center, Beer Sheva 84105, Israel and the § Laboratory of Host Defenses, NIAID,
National Institutes of Health, Bethesda, Maryland 20892-1886
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arachidonic acid (AA) can trigger activation of the phagocyte NADPH oxidase in a cell-free assay. However, a role for AA in activation of the oxidase in intact cells has not been established, nor has the AA generating enzyme critical to this process been identified. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected that lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid or 1,25-dihydroxyvitamin D3, indicating that cPLA2 is not involved in the differentiation process. Neither cPLA2 nor stimulated [3H]AA release were detectable in differentiated cPLA2-deficient PLB-985 cells, demonstrating that cPLA2 is the major type of PLA2 activated in phagocytic-like cells. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but the addition of exogenous AA fully restores oxidase activity. This establishes an essential requirement of cPLA2-generated AA for activation of phagocyte NADPH oxidase.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The phagocyte NADPH oxidase is a multicomponent transport chain that transfers electrons from NADPH to molecular oxygen and generates superoxide, a precursor of microbicidal oxidants important to host defense. NADPH oxidase subunits include three cytoplasmic components, p47phox, p67phox, and Rac-2, and a membrane flavocytochrome b558 composed of gp91phox and p22phox (1-9). In differentiated phagocytic cells stimulation results in translocation of the cytosolic NADPH oxidase components to the membrane where they interact with the flavocytochrome to form the activated oxidase leading to superoxide generation. The signals responsible for assembly and activation of the oxidase are not clearly defined. PLA21 activity has been implicated in a variety of responses by stimulated phagocytes, including degranulation, phagocytosis, adhesion, cell spreading, and activation of NADPH oxidase (10-16). Until recently, generation of AA had been viewed as a modulating event leading to oxidase activation but not as critical to the process. Recently we and others have used PLA2 inhibitors to implicate generation of AA as important for activation of the NADPH oxidase activity in human neutrophils (17, 18). Moreover, we have shown that AA increases the affinity of the assembled oxidase for NADPH (19). However, studies using inhibitors may not delineate the specific enzyme of a related group whose inhibition is responsible for the observed effect, and the inhibition seen may result from action on more than one enzyme.
In the last decade, several secreted and cytosolic mammalian PLA2 isozymes have been described (20-24). The existence of several types of PLA2 in phagocytic cells (25-30) complicates delineation of the PLA2 responsible for release of the AA, which impacts on NADPH oxidase activation following phagocyte stimulation. In the present study, we used the RNA antisense technique to create in the human phagocyte myeloid cell line, PLB-985, a p85 cPLA2-deficient model cell line to demonstrate the role of this enzyme in NADPH oxidase activation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture-- PLB-985 cells (31) were grown in stationary suspension culture in RPMI 1640 medium containing 10% bovine serum (Hyclone Laboratories, Inc. Logan, UT), 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 12.5 units/ml nystatin (Biological Industries, Beth Haemek, Israel) at 37 °C in a humidified of 5% CO2 atmosphere. Cell number and viability were determined by trypan blue exclusion. [3H]Thymidine incorporation was determined as described earlier (32).
Construction of Expression Vector-- Human cPLA2 cDNA (in a PMT2 vector) was generously provided by Dr. J. L. Knopf (Genetic Institute, Cambridge, MA). cPLA2 cDNA (1-530) was excised from its PMT2 vector with the restriction enzymes SalI and XbaI. The ends were filled in with Klenow fragment and ligated in the antisense direction into the XhoI site of pcDNA3 (Invitrogen, San Diego, CA), which was also filled in with Klenow to form the plasmid cPLA2 (1-530)-pcDNA3. The antisense orientation of this insert was confirmed by DNA sequence analysis.
Transfection and Selection of PLB-985 Clones-- PLB-985 cells (1 × 107) in logarithmic growth were transfected in 0.3 ml of culture medium with 20 µg of plasmid DNA (antisense or vector alone) by electroporation at 250 V and 960 microfarad in a Gene pulser Unit (Bio-Rad, Melville, NY) and selected in the presence of 0.8 mg/ml G418 (Life Technologies, Inc.) (33). At 48 h. posttransfection, the living cells were separated on a Ficoll gradient (typically 30-50% of starting number), diluted to 5 × 104 cells/ml, and plated in 96-well plates in the presence of the appropriate antibiotic for selection of clones (G418 (Life Technologies, Inc.) 0.8 mg/ml). Clones resistant to the neomycin analogue, G418, were screened by Western blot to select those that were cPLA2 protein-deficient.
Immunoblot Analysis-- For immunoblot detection of cPLA2, cells were centrifuged and sonicated in the presence of 5 mM EGTA, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 10 mM PIPES (pH 7.4), 1 mM PMSF, and 100 µM leupeptin, and membrane and cytosol fractions were separated as described (34). Under these conditions whole cell cPLA2 protein and activity were found entirely in the cytosol fraction as shown earlier (35). Alternatively, cell lysates were prepared using 1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 25 mM NaF, 10 µM ZnCl2, 1 mM PMSF, and 100 µM leupeptin. 100 µg of protein from cell cytosol were separated by electrophoresis on 7.5% polyacrylamide SDS gels and blotted to nitrocellulose. The detection of cPLA2 protein was performed using rabbit antibodies raised against a glutathione S-transferase fusion with cPLA2 as described earlier (35). This antiserum detects three protein bands in neutrophil and PLB-985 cell cytosol, only one of which co-migrates with recombinant cPLA2 and cross-reacts specifically with other cPLA2 antibodies (provided by Dr. J. Knopf, Genetics Institute Inc., Cambridge, MA) (35).2 The other (larger and smaller) bands are nonspecific proteins also detected by preimmunized serum from this rabbit. Immunoblot detection of NADPH oxidase components was performed as described earlier (34). The relative changes of the proteins was quantitated using densitometry in a reflectance mode (Hoefer Scientific Instruments, San Francisco, CA).
DTT-resistant Phospholipase A2 Activity-- PLB-985 cells (5 × 106 cells/ml) in HBSS buffer were stimulated for 2 min at 37 °C by 1 mg/ml OZ or 50 ng/ml PMA. The reaction was stopped by 10-fold dilution with cold HBSS and immediate centrifugation at 4 °C. Membrane and cytosol fractions were separated as described earlier (35). PLA2 activity was determined in the cytosols using sonicated dispersions of 1-stearoyl-2-[14C]arachidonyl phosphatidyl choline (30 µM, 50,000 dpm/assay) and sn-1,2-dioleoylglycerol (molar ratio, 2:1) in an assay mixture containing 5 mM DTT (36, 37) with some modifications as described earlier (35). Briefly, the assay mixture contained the phospholipid substrate in 80 mM KCl, 5 mM CaCl2, 5 mM DTT, 1 mg/ml bovine serum albumin, 1 mM EDTA, and 10 mM HEPES (pH 7.4). The reaction was started by the addition of 50 µg of cytosolic protein (within the linear protein range of the assay) and incubated at 37 °C in a shaking water bath for 10 min.
Release of [3H]AA-- Assays of incorporation and release of radiolabeled arachidonic acid ([3H]AA) were performed as previously reported (35). PLB-985 cells (108 cells/ml) were incubated for 30 min at 37 °C in Ca2+ and Mg2+-free PBS containing 1 µCi of [3H]AA. After appropriate washes, PLB-985 cells (107 cells/ml) were stimulated, and the release of [3H]AA was determined in the linear range of the reaction.
Induction and Determination of Differentiation--
Conditions
for dose- and time-dependent responses to induction were
explored to determine which conditions resulted in the highest
superoxide production and expression of differentiation markers after 5 days of differentiation without reducing cell viability below 95%.
Optimal concentration of 5 × 108 M
1,25(OH)2D3 or 10
6 M
RA were added to 2 × 105 cells/ml PLB-985 cells at
their logarithm growth phase. 1,25(OH)2D3 was
kindly provided by Dr. Uskokovich from Hoffmann La-Roche Inc. (Nutley,
NJ). Mac-1 antigen determination was detected by indirect immunofluorescence as described earlier (38).
Superoxide Anion Measurements--
The production of superoxide
anion (O2) by intact cells was measured as the superoxide
dismutase inhibitable reduction of ferricytochrome c (34).
Cells were suspended (5 × 105 cells/well) in 100 µl
of HBSS containing 150 µM ferricytochrome c
and activated by the addition of the appropriate stimulus. The reduction of ferricytochrome c was followed by a change of
absorbance at 550 nm every 2 min over a 20-min time course using a
Thermomax microplate reader (Molecular Devices, Menlo Park, CA). The
maximal rates of superoxide generation were determined using a
extinction coefficient E550 = 21 mM
1 cm
1. OZ was prepared as
follows: 20 mg zymosan was incubated with 1 ml of pooled human serum
(lipopolysaccharide-free) for 1 h at 37 °C and washed three
times with HBSS.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
PLB-985 cells were transfected with human cPLA2 cDNA engineered in the antisense orientation in the pcDNA3 expression vector, which also contains a neomycin resistance element. Cell cytosolic fractions from clones resistant to the neomycin analogue, G418, were screened by Western blot to select those that were cPLA2 protein-deficient. Three pcDNA3-cPLA2 antisense transfected clones were completely deficient of cPLA2 protein (PLB-D) (Fig. 1A). Several other clones were shown to be partially deficient, containing residual amounts of cPLA2 protein (PLB-R). Both the parent PLB-985 line (PLB) and a G418-resistant clone transfected with the empty pcDNA3 vector (PLB-V) were used as controls. As shown in Fig. 1A, PLB-V produced high levels of cPLA2 protein, and this amount was indistinguishable from that present in the parent PLB cells.
|
Cytosol fractions of the various PLB-derived cell lines were examined for the presence of DTT-resistant enzymatic activity characteristic of p85 cPLA2 using 1-stearoyl-2-[1-14C]arachidonyl phosphatidylcholine as a substrate. As shown in Fig. 1B, basal cPLA2 activity resistant to inhibition by 5 mM DTT detected in unactivated PLB-V cytosol was similar to that seen in the parent PLB cells (1.25 ± 0.2 and 1.46 ± 0.3 pmol/mg protein/min, respectively). By contrast PLB-D cells that lack cPLA2 protein had no detectable DTT-resistant cPLA2 activity, and PLB-R that express reduced levels of cPLA2 protein demonstrated reduced levels of cPLA2 activity in cytosol (0.71 ± 0.4 pmol/mg protein/min). In other experiments (not shown), the cPLA2 activity and protein detected in Triton-extracted cell lysates were identical to that observed in the cytosol fractions shown in Fig. 1.
An important characteristic of the PLB cell line is that it can be
induced to differentiate toward a mature phagocyte phenotype in
response to a variety of differentiating agents (31). In the present
study, 106 M RA was used to induce maturation
to a granulocyte-like phenotype, whereas 5 × 10
8
M 1,25-dihydroxyvitamin D3 (D3) was
used to induce maturation to a monocyte-like phenotype (34). Of
importance for the current study is that common features of the
differentiated phenotype acquired during differentiation with both
induction regimens included expression of CD11b (Mac-1 antigen) at the
cell surface and expression of subunits required for assembly of active
NADPH oxidase. As shown in Fig. 2A, the
percentage of parent PLB cells expressing Mac-1 antigen at the cell
surface measured by indirect immunofluorescence was less than 8% in
undifferentiated cells but increased to over 70% by day 5 with either
RA or D3. Of note is that the pattern of Mac-1 antigen
expression by PLB-V cells or PLB-D cells during induction of
differentiation was indistinguishable from that seen with PLB
cells. Similarly, the patterns of expression of the subunit components of NADPH oxidase during differentiation of PLB-V, PLB-D, or
PLB cells in response to RA or D3 were indistinguishable,
as shown by the Western blots in Fig. 2 (B and
C). Rac-2 was detected in all the PLB-derived cell lines and
did not change during differentiation (not shown). Because
cPLA2 could be important for cell growth, we examined DNA
synthesis and proliferation using [3H]thymidine
incorporation and serial cell counts, respectively. We found that
parent PLB, PLB-V, and PLB-D had similar [3H]thymidine
incorporation and cell doubling times in the uninduced state and
following differentiation showed a similar decline in these measures as
maturation occurred (not shown). These analyses of phenotype changes
during differentiation indicate that neither the presence of plasmid
sequence (PLB-V) nor loss of cPLA2 (PLB-D) had any effect
on proliferation or differentiation-associated expression of Mac-1
antigen or NADPH oxidase components.
|
Differentiation was not associated with any change in the constitutive level of cPLA2 protein (Fig. 3A) or enzymatic activity in cytosol fractions (Fig. 3B) during maturation of parent PLB or PLB-V cells. These results are expected because a recent report (39) indicates that constitutive expression of cPLA2 does not change during differentiation of HL60 cells, a human promyelocytic cell line. PLB-D remained devoid of measurable cPLA2 protein or activity following differentiation (Fig. 3).
|
As shown in Table I, we compared activation of the NADPH oxidase in differentiated PLB, PLB-V, PLB-R, and PLB-D in response to both soluble and particulate stimuli. PLB and PLB-V show similar levels of superoxide generation when similar differentiation conditions and stimulants are compared. The differentiated PLB-R, which expresses some cPLA2 but less than that seen in differentiated PLB or PLB-V, generated superoxide at levels 50-60% of that seen for differentiated PLB or PLB-V for each stimulus. Differentiated PLB-D did not generate superoxide in response to any of the stimuli tested despite the fact that there were normal levels of expression of the NADPH oxidase components. Thus, the level of cPLA2 expression appeared to have a profound effect on NADPH oxidase generation of superoxide.
|
Because cPLA2 would most likely mediate its action on superoxide production by the NADPH oxidase in stimulated cells by release of AA, its role in the liberation of AA was studied. We prelabeled undifferentiated and 5 day D3-differentiated PLB with [3H]AA and then measured release of [3H]AA following stimulation with PMA or OZ (Fig. 4A). D3-differentiated PLB and PLB-V showed a 10-fold increase in [3H]AA release in response to PMA or OZ relative to that seen with the similarly stimulated undifferentiated cells. The differentiated PLB-D showed a striking difference in that the level of stimulated [3H]AA release remained at the same low level as that seen in undifferentiated cells. This suggests that in differentiated phagocytes the major portion of the pulse of AA release associated with PMA or OZ stimulation is mediated by cPLA2. It also suggests that the failure of differentiated PLB-D to produce superoxide is related to a failure to produce this large pulse of AA release following stimulation.
|
We tested this latter hypothesis by providing exogenous free AA to D3-differentiated PLB-D at the time of stimulation of the oxidase by PMA as analyzed in the kinetic assay of superoxide generation shown in Fig. 4B. Addition of 25 µMAA to the differentiated PLB-D fully restored the superoxide generating capacity to these cells relative to rates of superoxide generation by parent PLB cells stimulated with PMA. Not shown is that addition of free AA alone in the range of 10-25 µM without PMA did not induce any superoxide production. Furthermore, the effect of AA was shown to be specific, because the addition of linoleic or oleic acids (up to 100 µM) to PMA-stimulated PLB-D cells did not restore oxidase activity. This demonstrates that it is the AA generated by cPLA2 that is essential to superoxide generation by the activated NADPH oxidase.
NADPH oxidase activation requires translocation of the cytosolic subunits to the cell membrane following stimulation (1-9). As shown in the Western blot in Fig. 4C, stimulation of differentiated PLB-D resulted in plasma membrane association by the three cytoplasmic components of the NADPH oxidase that was indistinguishable from that seen in differentiated PLB-V cells. It is of note that addition of AA to differentiated PLB-D at the time of PMA stimulation did not augment the normal levels of cytosolic NADPH oxidase subunits associated with the plasma membrane (Fig. 4C, last two lanes). This suggests that AA is acting in some final step on the assembled oxidase to promote or maintain the enzyme in an active state rather than acting to induce assembly of the oxidase. Such a model is consistent with previous observations showing that PLA2 inhibitors do not affect p47phox membrane translocation or phosphorylation (17) and that PLA2 inhibitors work even when added after oxidase activation (18).
Our studies are the first to delineate the effects of complete and specific inhibition of p85 cPLA2 protein expression and strongly implicates the p85 cPLA2 as the PLA2 isozyme responsible for the majority of the pulsed release of AA following stimulation of phagocytes. It also provides the first unequivocal demonstration that AA release is an absolute requirement for superoxide generation by the NADPH oxidase regardless of the stimulus. A number of previous studies have suggested that AA release in stimulated neutrophils or monocytes is mediated by p85 cPLA2 and that this AA release is important for a variety of functional responses. Specifically, it has been shown that the decreased expression of cPLA2 in monocytes treated with antisense cPLA2 oligonucleotide resulted in significant inhibition of [3H]AA release induced by monocyte chemotactic protein-1 (40). Furthermore, treatment of human monocytes with bacterial lipopolysaccharide caused an increase in the 85-kDa PLA2 protein and activity that coincided with increased release of PGE2 (41). This increase in PGE2 formation could be prevented by treatment of the monocytes with an antisense oligonucleotide directed at the 85-kDa PLA2 mRNA, which decreased cPLA2 levels. Bacterial lipopolysaccharide primes human neutrophils for enhanced release of AA and causes phosphorylation of cPLA2 (42). In monocytes a correlation has been demonstrated between monocyte colony-stimulating factor-induced phosphorylation events, increased expression of 85-kDa PLA2, and increased PGE2 release (43). It was recently suggested (44) that cPLA2 is the enzyme that induces the release of AA in a permeabilized neutrophil model.
In conclusion, the development of differentiated PLB-D cell lines that lack any cPLA2 expression demonstrates that this protein is not required for proliferation or differentiation processes but that its enzymatic production of AA is an essential requirement for activation of the phagocyte NADPH oxidase. Although AA has been used extensively to activate the NADPH oxidase in a cell-free system (34), it was not known whether its effect in vitro had a correlate in the intact cell. Because our previous studies suggest that AA markedly enhances the affinity of the NADPH oxidase for the NADPH substrate (19), our current observations are most consistent with a model in which cPLA2-generated AA might be a co-factor acting in the intact phagocytic cell to enhance the affinity of the assembled NADPH oxidase for NADPH.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. J. L. Knopf for providing us with cPLA2 cDNA and antibody against cPLA2. We also thank M. C. Dinauer and S. J. Chanock for providing the PLB-985 cells and for advice on the transfection procedure.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 972-7-6403186; Fax: 972-7-6467477; E-mail: ral{at}bgumail.bgu.ac.il.
1 The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; AA, arachidonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; HBSS, Hanks' balanced salt solution; OZ, opsonized zymosan; PMA, phorbol myristate acetate; RA, retinoic acid; D3, 1,25-dihydroxyvitamin D3; PAGE, polyacrylamide gel electrophoresis.
2 R. Dana and R. Levy, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|