| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Immunology, University of Glasgow, Glasgow, Scotland; and
Institute for Cancer Studies, University of Birmingham Medical School, Birmingham, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We now show that cross-linking of the Ag receptors on B cells induces DNA synthesis and proliferation in a PLD-dependent manner. Although phosphatidylcholine-PLD has been shown to play a central role in the transduction of proliferative responses for a wide variety of calcium-mobilizing receptors, we show that the Ag receptors are not coupled to this phospholipase, but rather to a novel putative phosphatidylinositol-specific PLD. The sIg-generated PtdOH, unlike phosphatidylcholine-derived phosphatidic acid, is converted to DAG. These data provide the first evidence that while the novel sIg-coupled PLD and resultant DAG generation may play a role in B cell proliferation, phosphatidylcholine-PLD may transduce, via phosphatidic acid, negative immunomodulatory signals in mature B lymphocytes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The human B cell lines, Daudi, EDR, and Ramos, were maintained in RPMI 1640 medium supplemented with penicillin/streptomycin, glutamine, and 10% FCS at 37°C in a 5% CO2 atmosphere. The following Abs were used in this study. Rabbit anti-mouse Ig (F(ab')2) and anti-human CD40 were purchased from Jackson ImmunoResearch (Stratech, U.K.) and Serotec (Oxford, U.K.), respectively. Hybridomas b.7.6. (anti-IgM), AMS28 (anti-IgD), Bet-2 (nonmitogenic anti-sIgM), and 34.5-3 (anti-IAd) were grown, and the appropriate mAbs purified from culture supernatants as described previously (8, 9). FGK-45 (anti-murine CD40) was donated by Drs. J. Andersson and T. Rolink (Basel Institute for Immunology, Basel, Switzerland) and was purified by S-Sepharose chromatography (Pharmacia, St. Albans, U.K.). Purified NIMR-1 (anti-Thy-1) was donated by Dr. R. M. E. Parkhouse (IAH, Pirbright, U.K.).
PMA, LPS, Ficoll-Hypaque, and Percoll were purchased from Sigma (Poole, U.K.). NHS-biotin and avidin were purchased from Pierce & Warriner (Chester, U.K.). Guinea pig complement was purchased from Sera-Lab (Crawley Down, Sussex, U.K.). Tissue culture media, including, RPMI 1640, Ham’s F-10, FCS, and HEPES, were purchased from Life Technologies (Paisley, U.K.). [9,10-N-3H]palmitic acid (spec. act., 39 Ci/mmol) was purchased from New England Nuclear-DuPont (Boston, MA). [5,6,8,9,11,12,14,15-3H]arachidonic acid (spec. act., 213 Ci/mmol), [3H]myo-inositol (spec. act., 80–120 Ci/mmol), [9,10-N-3H]myristic acid (spec. act., 49 Ci/mmol), [9,10-N-3H]oleic acid (spec. act., 10 Ci/mmol), and [1-14C]stearate (60–62 mCi/mmol) were purchased from Amersham (Aylesbury, U.K.). [3H]butanol was a gift from Dr. Neil Thompson, Wellcome Laboratories (Beckenham, U.K.). Recombinant murine IL-4 was donated by Immunex (Seattle, WA).
Murine B cell preparation
Murine B cells were prepared as described previously (8, 10) Briefly, spleens from male BALB/c mice (12–20 wk old) were excised, and cells were dispersed by being pressed through stainless steel gauze. Depletion of erythrocytes was achieved by centrifugation through a Ficoll-Hypaque cushion (400 x g for 15 min). Lymphocytes were then depleted of T cells by treatment with anti-Thy-1 mAb (NIMR-1) and guinea pig complement. B cells were further purified on a discontinuous Percoll gradient of 85–65-50% (1200 x g for 15 min at 4°C). The higher density cells recovered from the 85–65% interface were termed resting cells, and the lower density cells recovered from the 65–50% interface were termed activated cells. Immature splenic B were prepared from the spleens of 4-wk-old mice and were classified as immature because they do not induce DNA synthesis in response to anti-Ig and are thus not functionally identical with B cells from adult mice (11).
Measurement of DNA synthesis in murine B cells
Murine B cells were cultured (5 x 105 cells/200 µl) in flat-bottom microtiter wells in triplicate in RPMI 1640 medium supplemented with glutamine (2 mM), sodium pyruvate (1 mM), nonessential amino acids, ß2-ME (50 µM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 5% FCS. [3H]thymidine at 0.5 µCi/well was added at 68 h, and cultures were harvested 4 h later using an automated cell harvester (Skatron, Sterling, VA), and label incorporated into DNA was estimated by liquid scintillation counting (10).
Preparation of murine splenocyte PHA-blasts
Murine PHA-blasts were prepared essentially as described previously (12). Briefly, erythrocyte-depleted splenocytes (see above) were resuspended (107/ml) in RPMI medium supplemented with penicillin, streptomycin, glutamine, and 10% FCS and cultured overnight in the presence of PHA (50 µg/ml) at 37°C. Following incubation, the cells were separated on a discontinuous Percoll gradient of 85/65/50%. Cells were recovered from the 65–50% interface, fixed with paraformaldehyde, washed, and resuspended in RPMI medium containing 0.1% BSA and 20 mM HEPES, pH 7.4.
Flow cytometry
Cells to be examined for annexin V expression were washed in PBS and incubated with propidium iodide and/or annexin V-FITC conjugate in defined calcium and magnesium concentrations according to the instructions provided by Boehringer Mannheim (Lewes, East Sussex, U.K.). Cells were acquired (10,000 events) and immediately analyzed with a Becton Dickinson FACScan (Mountain View, CA) using LYSIS II software for analysis.
Analysis of phospholipid labeling
B cells were resuspended in RPMI 1640 medium containing penicillin, streptomycin, glutamine, and FCS; plated out (0.5 x 107 cells in a final volume of 300 µl) into 24-well plates; and incubated with [3H]palmitate, [3H]arachidonate, [3H]myristate, or [3H]oleate (3 µCi/well) at 37°C for either 4 or 18 h. Methanol (500 µl) was then added to each well, and the disrupted cell suspension was transferred to glass vials. Lipid extracts were prepared by a Bligh-Dyer extraction as described previously (11) and were resolved by TLC on silica gel 60 plates (20 x 20 cm, 250 µm thick; Merck, Darmstadt, Germany) that were activated by prerunning in 1 mM EDTA, air-drying, and heating at 120°C for 1 h before use. Plates were developed in a paper-lined tank, pre-equilibrated with the solvent chloroform/acetone/methanol/glacial acetic acid/water (80/30/26/24/14, v/v/v/v/v). Phospholipids were detected by iodine vapor, and the associated radioactivities corresponding to [3H]PtdIns, -PtdCho, -PtdEth, -PtdSer, and -PtdOH standards were determined by scraping and liquid scintillation counting (11).
PLD assay
PLD activity was measured by transphosphatidylation as described previously (13). To identify PtdCho-specific PLD activity, cells were prelabeled with [3H]palmitate to preferentially label the PtdCho phospholipid pool (11). The specificity of the assay in B cells was defined by showing that PMA stimulated generation of [3H]PtdBut in cells cultured with butan-1-ol (maximal at 0.1–0.3% butan-1-ol) but not butan-2-ol (results not shown). Cells labeled for 4 h were resuspended (5 x 107/ml) and cultured in RPMI 1640 medium, supplemented with 10% dialyzed FCS and the appropriate label (1 µCi/106 cells). Cells labeled for 18 h were resuspended (107/ml) and cultured in RPMI medium, supplemented with penicillin, streptomycin, glutamine, 10% dialyzed FCS, and the appropriate label (1 µCi/106 cells) at 37°C in a 5% CO2 atmosphere. Labeled cells were then washed and resuspended (3 x 107/ml) in RPMI 1640 containing 20 mM HEPES, 0.1% BSA, and 0.3% butan-1-ol and equilibrated at 37°C for 15 min. To measure the total phospholipid-PLD activity, unlabeled cells were resuspended (3 x 107/ml) in RPMI 1640 medium containing 20 mM HEPES (pH 7.4), 0.1% BSA, and [3H]butan-1-ol (20 µCi/ml) and then incubated at 37°C for 15 min before stimulation. Reactions (in glass vials) were initiated by the addition of cells (3 x 106) to the appropriate stimulus prepared in butanol-containing medium (final volume, 150 µl). Where stock reagents were prepared in solvents such as DMSO, appropriate vehicle controls were added to the unstimulated control cells. After the indicated time, incubations were terminated, and lipid samples were prepared by a Bligh-Dyer extraction (14) for resolution by TLC in the organic phase of a solvent comprising 2,2,4-trimethylpentane/ethyl acetate/acetic acid/water (5/11/2/10, v/v/v/v) using an unlined, unequilibrated chromatography tank. [3H]PtdBut bands, identified by unlabeled or [14C]PtdBut standards, were scraped and quantitated by liquid scintillation counting (14).
Inositol phospholipid levels
Resting B cells were labeled with [3H]myo-inositol (2.5 µCi/106 cells) for 4 h at 37°C and stimulated with the appropriate ligand, and the reactions were quenched by the Bligh-Dyer two-phase separation. Aliquots of the organic phase were dried down in vacuo and applied to oxalic acid-pretreated silica gel 60 plates. Phospholipids were then separated by TLC, and [3H]PtdIns, [3H]PtdInsP, and [3H]PtdInsP2 were located by visualization of unlabeled standards. Radioactivity was determined by scraping and liquid scintillation counting (15).
Mass measurement of DAG
Resting B cells were stimulated with the appropriate ligand, and the reactions were quenched by Bligh-Dyer two-phase separation. Aliquots of the lower organic phase were dried in vacuo, the lipids were solubilized in a Triton X-100/phosphatidylserine mixture, and DAG mass was determined using the DAG kinase assay as previously described (16). The generated PtdOH was separated on silica TLC plates (Merck 5714; 5 x 20 cm 60F254), developed in chloroform/methanol/acetic acid (38/9/4.5. v/v/v) to within 5 mm of the top of the plate. The PtdOH bands (relative to standards) were located by autoradiography or phosphorimaging and were scraped into scintillation vials, scintillant was added, and the associated radioactivity was determined by liquid scintillation counting.
Lipid species analysis
Cells were stimulated as described above, and lipids were extracted into chloroform/methanol as previously described (17). Internal standard (1 µg 1, 2–12:0/12:0 DAG) and marker (1 µg 1, 2 + 1, 3–20:0/20:0 DAG) were added to each sample before lipid extraction. Following derivatization with 3,5-dinitrobenzoyl-chloride (50 mg/ml in dry pyridine, 60°C, 15 min) and solid phase extraction, the diradylglycerol derivatives were separated by HPLC, firstly into separate classes on a Kromasil silica column (5 µm, 2.1 x 250 mm) using a solvent gradient of 100% hexane/cyclohexane/diethyl ether/propan-2-ol (49/49/2/0.1, v/v/v/v), changing to 100% cyclohexane/diethyl ether/propan-2-ol (85/15/0.1, v/v/v) over 45 min at 0.5 ml/min and then into the individual molecular species on a Spherisorb S5ODS2 (Sigma-Aldrich, Poole, Dorset, U.K.) reverse phase column (5 µm, 3.2 x 250 mm), using a gradient of acetonitrile/propan-2-ol (9/1, v/v), changing to acetonitrile/propan-2-ol (1/1, v/v) over 45 min at 0.5 ml/min with detection at 254 nm. Identification was through the use of authentic standards and by reference to previously determined relative retention time data (17).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To investigate a role for PLD activities in Ag receptor-mediated
B cell proliferation, we have taken advantage of the
underlying principle of the definitive PLD transphosphatidylation
assay. In this, the primary alcohol, butan-1-ol acts as a
preferential nucleophilic acceptor for the phosphatidyl-moiety
resulting from PLD-mediated cleavage of phospholipids to
generate the inactive, nonmetabolizable product, PtdBut. Butan-1-ol
abrogated anti-Ig-stimulated [3H]thymidine
incorporation in murine resting splenic B cells (Fig. 1
A). In contrast, butan-1-ol
did not substantially block the [3H]thymidine uptake
resulting from stimulation of murine splenic B cells with LPS (50
µg/ml; Fig. 1B). Nonspecific effects of butanol were ruled
out by use of the stereoisoform, butan-2-ol, which cannot act as
acceptor for the phosphatidyl moiety (Fig. 1).
|
A).
Furthermore, cell death resulting from apoptosis (annexin V/propidium
iodide double-positive cells) of B cells stimulated with anti-Ig in
the presence of butan-1-ol, but not butan-2-ol, was almost complete
(range, 75–95%) within 24 h (Fig. 2B). Evidence that
such butan-1-ol-mediated apoptosis is not simply the result of a cell
stress-induced event but, rather, reflects specific targeting of a B
cell survival/proliferative signal was provided by an additional series
of experiments, which showed that culture with IL-4 (Fig. 2C) could almost completely prevent the induction of cell
death by apoptosis. Culture with anti-CD40 could also prevent such
butan-1-ol-induced apoptotic cell death, but to a lesser degree
(results not shown). Thus, taken together, these data suggest that one
or more PLD activities play an important role in the sIg-mediated
induction of survival and proliferation of B cells.
|
Ag receptor coupling to PtdCho-PLD activity was determined
by measurement of stimulated [3H]PtdBut
formation in [3H]fatty acid-labeled B cells.
Labelling studies demonstrated that after 4-h incubation,
[3H]palmitate, [3H]myristate,
[3H]oleate, and [3H]arachidonate were all
preferentially incorporated into PtdCho, indicating that the
[3H]PtdBut species generated from such
[3H]fatty acid-labeled B cells were derived from PtdCho
(11, 14). Cross-linking of the Ag receptors on murine splenic B cells
(sIg), using a mitogenic concentration (50 µg/ml) of anti-Ig Abs
(F(ab')2), did not stimulate the formation of
[3H]PtdBut over a 15-s to 60-min period from cells
prelabeled with any of the above [3H]fatty acids (Fig. 3A and results not shown).
Stimulation of [3H]palmitate-labeled murine resting
splenic B cells with a range of anti-Ig concentrations (0.05–500
µg/ml) or with anti-µ or anti-
mAbs (50 µg/ml) did not
stimulate PtdCho-PLD activity (Fig. 3, B and C,
and results not shown). Moreover, stimulation of murine B cells with
other polyclonal activators, such as LPS, also failed to stimulate
PtdCho-PLD (Fig. 3B). Similar results were obtained
following cross-linking of the Ag receptors on in vivo activated murine
splenic B cells and the human lymphoblastoid cell lines, Daudi and
Ramos, under conditions in which anti-Ig induced release of
[3H]InsPs from [3H]inositol-labeled cells,
indicating that these cells were still responsive to stimulation via
sIg (results not shown).
|
In contrast, PMA, which activates PtdCho-PLD in most cell types,
strongly stimulated [3H]PtdBut formation from
[3H]palmitate- and [3H]arachidonate-labeled
B cells in a linear manner for up to 60 min (Fig. 3
), indicating the
presence of stimulatable PtdCho-PLD activity in murine B
cells. These findings therefore suggest that it is unlikely that 1) the
Ag receptors on B cells are coupled to PtdCho-PLD in a cell
cycle-dependent manner or 2) PtdCho-PLD plays a role in the maintenance
of Ag-driven B cell proliferation.
PtdCho-PLD does not play a role in T cell-dependent B cell activation
Stimulation of B cells with anti-Ig Abs probably represents polyclonal activation by type 2 T cell-independent Ags that typically are large molecules of repeating epitopes capable of effectively cross-linking sIg (1). However, the vast majority of soluble Ags are T cell dependent, and hence, both cytokine-directed (e.g., IL-4) and B-T cell contact-mediated (e.g., MHC class II, CD40) signals are required, in addition to those via the Ag receptors, for full B cell activation (1, 18). We therefore determined whether sIg or any of these additionally recruited receptor-mediated signals were coupled to PtdCho-PLD activity during T cell-dependent B cell activation.
Stimulation of murine B cells with IL-4 (1–1000 U/ml), either alone or in combination with anti-Ig (5–50 µg/ml), failed to induce PtdCho-PLD activation over a 60-min period (results not shown) despite costimulation with anti-Ig and IL-4 being mitogenic for B cells (19) (results not shown). Moreover, priming (1–16 h) of B cells with anti-Ig (50 µg/ml), IL-4 (100 U/ml), anti-Ig (5 µg/ml) plus IL-4 (100 U/ml), or LPS (50 µg/ml) failed to induce coupling of sIg to PtdCho-PLD (results not shown).
Secondly, we addressed whether mimicking B-T cell cooperation by ligation of MHC class II molecules by anti-Iad Abs would result in PtdCho-PLD activation. Cambier and co-workers have previously shown that class II molecules on murine B cells were differentially coupled to adenylyl cyclase or phospholipase C activation depending on the activation status of the B cells, i.e., resting B cells or cells primed by pretreatment (16 h) with IL-4 plus anti-Ig (18). We therefore investigated the coupling of class II molecules to PtdCho-PLD activity, either alone or in combination with anti-Ig (50 µg/ml), IL-4 (100 U/ml), or anti-Ig (5 µg/ml) plus IL-4 (100 U/ml), on freshly prepared or IL-4/anti-Ig-primed cells. In these experiments, the class II molecules on [3H]palmitate-labeled B cells were stimulated by either cross-linking of biotinylated anti-Iad Abs (anti-Iad mAbs 39-10-8 and 34.5-3; final concentration, 100 µg/ml) by avidin (25 µg/ml) or incubation on plates precoated with anti-Iad Abs. No activation of PtdCho-PLD could be detected under any of these conditions (results not shown).
Finally, to determine whether ligation of B cell surface Ags such as CD40 by T cell counterstructures during B-T cooperation induced PtdCho-PLD activation, [3H]palmitate-labeled resting B cells and B cells preincubated with anti-Ig plus IL-4 were incubated with fixed PHA-splenic T cell blasts in the presence and the absence of IL-4 (100 U/ml). No PtdCho-PLD activity was detected (results not shown). Furthermore, stimulation of resting murine B cells or the human B cell line, EDR, with appropriate anti-CD40 Abs did not induce PtdCho-PLD activity in these cells (results not shown).
PtdCho-PLD does not play a role in transducing early growth arrest signals in immature B cells
Since stimulation of resting immunocompetent B cells under mitogenic conditions did not stimulate PtdCho-PLD activity despite the potential to couple to one or more such activities, we determined whether the Ag receptors were coupled to PtdCho-PLD activity under conditions that induced B cell anergy and/or apoptosis. However, ligation of the Ag receptors on [3H]palmitate-labeled immature splenic B cells or the WEHI 231 immature B lymphoma cell line under conditions that induce anergy in immature splenic B cells (11) (results not shown) and growth arrest and apoptosis in WEHI 231 cells (11, 20) did not induce stimulation of PtdCho-PLD activity. In addition, no stimulation of PtdCho-PLD activity could be detected following stimulation of WEHI 231 cells with LPS (results not shown).
Ag receptors on B cells are coupled to a non-PtdCho-specific PLD during mitogenic B cell activation
The DNA synthesis studies performed in the presence and the
absence of butan-1-ol (Fig. 1) suggested that the Ag receptors were
coupled to one or more phospholipase D activities under conditions of
mitogenic stimulation. That the Ag receptors on B cells were indeed
coupled to such a PLD activity was shown by experiments in which B
cells were mitogenically stimulated in the presence of
[3H]butan-1-ol (20 µCi/ml) to trap
[3H]PtdBut products of all PLD activities regardless of
phospholipid substrate specificity (Fig. 4). Stimulation of B cells with
anti-Ig (50 µg/ml) under these conditions induced a rapid
accumulation of [3H]PtdBut (Fig. 4A), which
reached a maximum over a period of approximately 10 min. As expected,
PMA stimulation of B cells also resulted in the accumulation of
[3H]PtdBut (Fig. 4A). Thus, the Ag receptors
on murine splenic B cells are coupled to one or more PLD activities. In
contrast, stimulation of sIg on WEHI-231 B cells under conditions that
lead to growth arrest and apoptosis did not induce the generation of
[3H]PtdBut when these B cells were cultured in the
presence of [3H]butanol (Fig. 4B).
|
), the level of
free [3H]ethanol amine detected was 7560 ± 440 dpm
relative to a control value of 7880 ± 400 dpm. Likewise, the
values for [3H]ethanolamine phosphate formation were
6961 ± 307 and 7076 ± 500 dpm, respectively. In contrast,
[3H]inositol labeling studies demonstrated that
anti-Ig Abs induced a substantial PtdIns hydrolysis, with kinetics
consistent with the formation of [3H]PtdBut, in mature
(Fig. 4C), but not immature (Fig. 4D), murine
splenic B cells, suggesting that PtdIns is probably the substrate for
the sIg-coupled PLD activity found in mature B cells. Role for PtdCho-PLD in the transduction of antiproliferative signals in mature B cells
ATP has been widely reported to exhibit immunomodulatory
properties on human B cell activation and lymphocyte proliferation
(21, 22, 23, 24). ATP exerts its effects on human B cells via
P2-purinoceptors and is coupled to PtdInsP2-PLC
and calcium mobilization (21). In addition, this class of receptors has
been shown to be coupled to PtdCho-PLD activation in a number of cell
types (reviewed in 25 ; we therefore determined whether
P2-purinoceptors could modulate murine B cell responses and
are coupled to PtdCho-PLD activation in murine B cells. We found that
ATP inhibits anti-Ig-mediated DNA synthesis in murine B cells in a
dose-dependent manner (Fig. 5A). Stimulation of
[3H]palmitate-labeled murine resting splenic B cells with
ATP at concentrations that inhibited sIg-mediated DNA synthesis induced
generation of [3H]PtdBut, with maximal activation
occurring at approximately 200 µM ATP (Fig. 5B).
|
A) or
Daudi and Ramos B cells (results not shown) with pertussis toxin led to
an increased basal level of [3H]PtdBut generation,
suggesting that one or more PtdCho-PLD activities could be under the
negative control of a Gi-coupled receptor on B
cells. Despite the elevated basal level of [3H]PtdBut
generation resulting from pertussis toxin treatment, ATP further
stimulated [3H]PtdBut formation (Fig. 6A). In
addition, pretreatment of murine B cells with selective tyrosine kinase
inhibitors, such as genistein and tyrphostin, under conditions that
inhibit sIg-stimulated tyrosine phosphorylation (results not shown) led
to an enhanced ATP-mediated PtdCho-PLD signal (Fig. 6B), suggesting that the ATP-coupled PtdCho-PLD pathway may
be under the negative control of a tyrosine kinase(s).
|
Since there was a difference in the phospholipid substrate of the
sIg- and P2-coupled PLDs, we considered it possible that
there would be a distinction between the PtdOH or DAG products
generated in response to stimulation of murine B cells with anti-Ig
or ATP. The sIg-mediated generation of DAG is biphasic and comprises an
early phase believed to correspond to sIg-coupled
PtdInsP2 hydrolysis followed by a second phase
that is maximal within 10 min and is sustained for at least 30 min (27)
(results not shown). Although the results in this paper suggest that
this late phase generation of DAG is not due to either PLD- or
PLC-mediated hydrolysis of PtdCho or PtdEth, it was possible that at
least some of the sIg-mediated DAG signal was derived, via PtdOH
phosphohydrolase activity, from PtdOH generated by the novel
sIg-coupled PLD activity. This was tested by determining the mass level
of DAG generated over a 30-min period in response to anti-Ig in B
cells preincubated with butanol (0.3%) to trap any potential PtdOH
species formed. To our surprise, DAG generation at 1 min, which was
previously thought to be due to hydrolysis of PtdInsP2, was
almost fully blocked by preincubation with butanol and was therefore
derived from PtdOH (Fig. 7A).
Taken together with the results presented in Fig. 4A, which
show that sIg couples to one or more PLD activities within 30 s,
these data suggest that a PLD is predominantly responsible for early
sIg-mediated DAG signals in B cells. However, only about 50% of the
DAG generated at 10 min appears to be derived from PtdOH (Fig. 7B). Although IP3 production appears to be
desensitized within 2 min of stimulation, high levels (up to about 20%
total [3H]inositol-labeled lipids) of total InsPs
continue to be generated in a linear manner for at least 2 h
following anti-Ig stimulation of B cells (27), and thus, the late
and sustained phase DAG may be produced by both PLC- and PLD-dependent
mechanisms of inositol lipid hydrolysis. In contrast, similar studies
in WEHI-231 cells showed that sIg-stimulated DAG generation was not
blocked by preincubation with butanol (Fig. 7C and results
not shown), suggesting that in these immature B cells the DAG generated
under conditions leading to growth arrest is not derived via a
PLD-derived mechanism.
|
).
Despite ATP stimulating PtdCho-PLD in murine B cells and reports that
it stimulates PtdInsP2 hydrolysis in at least human B cells
(21), ATP did not induce DAG generation at any of the time points
tested (1, 15, or 30 min; Fig. 7D and results not shown).
Likewise, PMA did not induce any significant generation of DAG over
this time course (Fig. 7A and results not shown). However,
following pretreatment with butanol, both PMA and ATP stimulated
substantial levels of DAG production (Figs. 7, A and
D), suggesting that in B cells, PtdCho-derived PtdOH is not
converted to DAG and, indeed, appears to act to suppress DAG levels
perhaps by inducing DAG kinase or lipase activities or even, in an
analogous manner to PMA (28), by inhibiting PtdInsP2-PLC. Anti-Ig and ATP induce distinct species of PtdOH and DAG in B cells
The finding that sIg and purinoceptors are coupled to the
generation of distinct species of PtdOH and DAG was confirmed by
experiments analyzing the DAG species generated in B cells following
stimulation with either anti-Ig or ATP. Following isolation of the
diradylglycerols from control and stimulated cells, separation of the
three diradylglycerol classes (DAG, alkylacylglycerol, and
alkenylacylglycerol) showed that the cells contained very little
alkylacylglycerol or alkenylacylglycerol and that the levels of these
diradylglycerols did not vary in response to any stimulation. However,
a complex pattern of changes was observed in the DAGs. As we have
observed with other cell types (17, 29), the majority of DAG species
obtained were saturated/mono- or diunsaturated: in particular, there
were high levels of 14:0/16:1, 16:0/16:0, 14:0/18:1, 18:0/18:2,
16:0/18:1n-9, 16:0/18:0, and 18:0/18:0. Together these species made up
approximately 52% of the total basal levels of DAG; however, there was
also a significant resting level (9%) of an 18:0/20:4n-6 DAG species.
Stimulation of the cells with anti-Ig caused an increase in DAG
mass that was blocked by inclusion of butanol, as was also observed
using the DAG kinase assay (Fig. 7, A and B).
Analysis of the sIg-stimulated DAG species following inclusion of
butanol revealed a dramatic reduction in the increase in the combined
levels of 18:0/18:2 and 16:0/18:1n-9 species (control, 9% of the total
DAG; control plus butan-1-ol, 6.8%; sIg, 20%; sIg plus butan-1-ol,
5.9%) and a small, but not statistically significant, reduction in the
18:0/20:4n-6 species. No increase in the relative levels of DAG species
was observed in response to stimulation of murine B cells with ATP or
PMA, results consistent with those obtained by the DAG mass assay (Fig. 7, A and C). The surprising increases in DAG mass
observed in response to ATP or PMA in the presence of butanol in the
DAG kinase assay (Fig. 7, A and C) were, however,
confirmed by DAG species analysis: for example, in the control cells
the level of 16:0/16:0 increased from 6.8 ± 0.1 to 9.0 ±
1.2% in the presence of butanol, while in the ATP-stimulated cells the
combined levels of 18:0/18:2 and 16:0/18:1n-9 were increased from
4.2 ± 0.2 to 6.8 ± 1.7% by the alcohol, and in the
PMA-stimulated cells butanol increased the level of 18:0/20:4n-6 from
3.7 ± 1.0 to 7.1 ± 1.9%.
These results clearly show that anti-Ig and ATP/PMA induce differential effects on both the mass of DAG and the individual species of DAG generated. Moreover, they indicate that these stimuli use distinct lipid signaling pathways to generate their respective DAG species. Indeed, given that anti-Ig and ATP, but not PMA, similarly stimulate PtdInsP2-PLC and that butanol blocks sIg-coupled, but not ATP/PMA-coupled, DAG generation in B cells, these results demonstrate that sIg and the purinoceptors couple to distinct PLD signaling pathways in murine B cells. These results also support our contention that the lipid products of stimulated PLD activity are predominantly saturated/monounsaturated or saturated/disunsaturated rather than polyunsaturated (29).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, 2, and 4). However, we have found, in contrast to the situation with
the Ag receptors on T cells (14), that the Ag receptors on mature B
cells are not coupled to the classical PtdCho-PLD following mitogenic
stimulation with anti-Ig Abs (Fig. 3). Moreover, stimulation of B
cells under conditions designed to mimic T cell-dependent activation of
B cells failed to induce PtdCho-PLD activation (results not shown). In
contrast, ATP, which inhibits murine B cell activation, stimulates
PtdCho-PLD activation (Figs. 5 and 6 and results not shown). However,
PtdCho-PLD does not appear to transduce the early sIg-mediated signals
leading to anergy or apoptosis of immature B cells (results not shown).
The Ag receptors on mature resting murine B cells, however, are coupled
to a PtdIns-PLD following mitogenic stimulation with anti-Ig Abs
(Fig. 4). The potential importance of this novel sIg-coupled PLD
pathway to mitogenic signaling is underscored by the facts that
blockage of this pathway results in B cell death by apoptosis (Fig. 2)
and that sIg is not coupled to this pathway in immature B cells or the
immature B cell line, WEHI-231 (Figs. 4 and 7), which undergoes growth
arrest and apoptosis following ligation of sIg. Consistent with this,
an undefined PLD activity was recently similarly reported to be coupled
to mitogenic stimulation of the Ag receptors on EBV-transformed human B
cells (31), and PtdOH has previously been shown to be rapidly generated
(within 1 min) following ligation of sIg on human and murine B cells
(32). Moreover, our labeling studies, which ruled out a role for a
PtdEth-specific PLD and suggested PtdIns to be the mitogenic PLD
substrate in murine B cells (Fig. 4), are consistent with studies on
human B cells that suggested that formation of
[3H]glycerol-labeled PtdOH was not derived from
[3H]glycerol-labeled PtdCho or PtdEth but, rather, might
have resulted from sequential activation of an acyltransferase and an
undefined PLD (32). Evidence that sIg is coupled to a PtdIns-PLD rather
than to the glycosylphosphatidylinositol (GPI)-PLD previously shown to
play a role in cytokine signaling (33, 34) is provided by both our
labeling and our species analysis studies. GPI-PLD activity is
characterized by the formation of 14:0/14:0 dimyristoyl-DAG (33);
however, there was no formation of [3H]myristate-labeled
PtdBut or dimyristoyl-DAG, determined either by species analysis or by
reference to standards in the DAG kinase assay. Moreover, in the TLC
solvent system used to resolve phospholipids in this study,
glycosylphosphatidylinositol migrates in the PtdInsP2
region and is thus clearly separated from PtdIns (33). As we detect
loss of label in the PtdIns region of the TLC plate, it is not a
GPI-lipid that is being hydrolyzed (Fig. 4). Furthermore, a precedent
for a role for PtdIns-PLD in cellular activation has been set by the
report that bradykinin stimulates a rapid and transient PtdIns-PLD,
rather than a PtdCho-PLD, response in Madin-Darby canine kidney cells
(35).
We therefore propose that while Ag-driven survival and proliferative signals in mature B cells are transduced at least in part by a PtdIns-PLD activity, PtdCho-PLD plays an antagonistic role in transducing antiproliferative signals in mature, immunocompetent B cells. This is the first report, to our knowledge, that PtdCho-PLD pathways may play a role in the negative regulation of cellular proliferation.
Distinct PtdCho-PLD subtypes have been identified in mammalian cells
that appear to differ in their coupling to cell surface receptors,
subcellular localization, and regulation (2, 3, 4, 30). For example, the
two major classes of PtdCho-PLD activity that have been well
characterized differ in their cofactor requirements for low m.w.
GTPases and PKC, while an oleate-dependent activity remains rather ill
defined. Although we have no definitive information regarding the
coupling of the ATP-stimulated PtdCho-PLD or, indeed, the novel
sIg-coupled PLD in B cells, our data on the regulation of PtdCho-PLD
activity by pharmacological agents have highlighted some novel and
interesting features of PtdCho-PLD regulation in B cells. In contrast
to studies reporting that pertussis toxin and tyrosine kinase
inhibitors inhibited agonist-stimulated PLD in neutrophils,
fibroblasts, and RBL cells (reviewed in 2 , in murine B cells,
pretreatment with pertussis toxin (4 h) and the tyrosine
kinase-selective inhibitors, genistein and tyrphostin (1 h), increased
both basal and ATP (enhanced in an additive manner)-stimulated
PtdCho-PLD activity (Fig. 6). A negative regulatory role for
Gi-like G proteins is further supported by our finding that
pertussis toxin pretreatment similarly stimulates basal PtdCho-PLD
activity in Daudi and Ramos B cells (results not shown). These results
with pertussis toxin and tyrosine kinase inhibitors therefore suggest
that in B cells PtdCho-PLD activation may be antagonistic to cell
growth and, hence, subject to negative control resulting from the
cross-talk between Gi-like G proteins and PTKs
produced by cross-linking of the B cell receptor and proposed to be
involved in sIg coupling to PtdInsP2-PLC and downstream
activation events (36, 37). A role for PtdCho-PLD in the transduction
of negative immunomodulatory signals could therefore contribute to the
pertussis toxin-mediated inhibition of sIgM-coupled up-regulation of
c-fos mRNA expression and DNA synthesis in primary B cells
(37).
The finding that the Ag receptors on B cells are not coupled to
PtdCho-PLD was initially rather surprising, since many of the receptors
known to couple to PtdCho-PLD are, like the Ag receptors,
calcium-mobilizing receptors and capable of transducing mitogenic
signals (2, 3, 4, 30). Also in common with many studies on a wide range of
cell types (2), PMA (presumably acting via PKC) (Fig. 3) stimulated one
or more PtdCho-PLD activities in B cells. These results are perhaps at
first sight inconsistent with sIg coupling to PKC, but not with
PtdCho-PLD activation following mitogenic stimulation of B cells via
the Ag receptors (38). However, in B cells, PMA may stimulate one or
more PKC isoforms not recruited following ligation of sIg. Our data
also suggest that PtdCho-PLD activity is under negative
control resulting from the cross-talk between
Gi-like G protein(s) and tyrosine kinase-mediated
signals (Fig. 6) following ligation of the Ag receptors on B cells
(36, 37, 38). Taken together with our findings that ATP and pertussis
toxin, which inhibit sIg-mediated DNA synthesis, can also stimulate
PtdCho-PLD activity (Fig. 6 and results not shown), our data suggest
that PtdCho-PLD may be antagonistic for sIg-driven B cell activation
and that cross-talk between Ag receptor-coupled G protein and tyrosine
kinase-mediated signals may indeed act to suppress PtdCho-PLD activity.
Interestingly, sIg is not coupled to PtdCho-PLD in immature B cells,
and thus, PtdCho-PLD does not appear to transduce early sIg-directed
anergic and/or apoptotic signals in immature B cells (results not
shown).
The immunoregulatory role of P2-purinoceptors on B cells is unclear, but it is well established that ATP suppresses lymphocyte cytotoxicity and proliferation (22, 23, 24). Previous studies have demonstrated that ATP-specific P2Z receptors are expressed on human B cells and coupled to PtdInsP2-PLC and mobilization of calcium (21). In addition, ATP-stimulation of P2-purinoceptors has been shown to induce the expression of c-fos, c-myc, IL-2R, and transferrin receptors in human B cells (21). However, since sIg is coupled to the generation of similar signals in B cells these data suggested that the proliferative effects mediated via sIg and/or the antiproliferative effects of ATP were probably due to additional transduction events. P2-purinoceptors have been shown to be coupled to PtdCho-PLD in a number of cell types (25). Thus, the coupling of the P2-purinoceptors to PtdCho-PLD may at least in part provide a biochemical mechanism for the transduction of ATP-mediated antiproliferative signals in murine B cells.
Although the mechanisms underlying the contrasting biological responses to stimulation of differential PLD activities in B cells (and other cell types) are currently unknown, our results show that while at least some of the sIg-stimulated PtdOH species are metabolized to enhance the DAG signal, PtdOH species derived from PtdCho in response to ATP are not converted to DAG, and indeed appear to suppress DAG that would be expected to be generated from other signaling sources, such as PtdInsP2. These findings not only support a central role for DAG in the transduction of proliferative signals in B cells but are also consistent with recent reports that PtdCho- and PtdIns-specific PLD activities generate distinct PtdOH species (differing in their fatty acid composition) in different cellular compartments and with potentially differential downstream effector mechanisms. The importance of this is demonstrated by the findings that saturated/monounsaturated, PLD-derived DAGs are unable to activate PKC in porcine aortic endothelial cells (29), and only the steroyl-arachidonyl-species of PtdOH are capable of stimulating the activity of GTPase-inhibiting protein and inhibiting the activity of GTPase-activating protein (2, 3, 4, 39). Moreover, the fate of such distinct PtdOH and DAG species and their downstream effectors may therefore provide a biochemical rationale for our observations that while the putative PtdIns-PLD plays a role in mitogenic signaling in B cells, PtdCho-specific PLD(s) may be involved in the transduction of antiproliferative signals via P2-purinoceptors, and possibly other immunomodulatory receptors, in mature, immunocompetent B cells.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Margaret M. Harnett, Department of Immunology, University of Glasgow, Glasgow, Scotland G11 6NT. E-mail address:
3 Abbreviations used in this paper: sIg, surface Ig; PTK, protein tyrosine kinase; PLC, phospholipase C; PtdIns, phosphatidylinositol; DAG, diacylglycerol; PLD, phospholipase D; PtdOH, phosphatidic acid; PKC, protein kinase C; PtdCho, phosphatidylcholine; PtdEth, phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdBut, phosphatidylbutanol; GPI, glycosylphosphatidylinositol.
Received for publication May 4, 1998. Accepted for publication August 26, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Biol. Chem. 272:3860.This article has been cited by other articles:
|
|
 |
|
  S. R. Shaikh and M. Edidin Polyunsaturated fatty acids, membrane organization, T cells, and antigen presentation Am. J. Clinical Nutrition, December 1, 2006; 84(6): 1277 - 1289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Diaz, S. Mebarek-Azzam, A. Benzaria, M. Dubois, M. Lagarde, G. Nemoz, and A.-F. Prigent Disruption of Lipid Rafts Stimulates Phospholipase D Activity in Human Lymphocytes: Implication in the Regulation of Immune Function J. Immunol., December 15, 2005; 175(12): 8077 - 8086. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Moreno-Garcia, L. N. Lopez-Bojorques, A. Zentella, L. A. Humphries, D. J. Rawlings, and L. Santos-Argumedo CD38 Signaling Regulates B Lymphocyte Activation via a Phospholipase C (PLC)-{gamma}2-Independent, Protein Kinase C, Phosphatidylcholine-PLC, and Phospholipase D-Dependent Signaling Cascade J. Immunol., March 1, 2005; 174(5): 2687 - 2695. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Katz, C. Lord, C. A. Ford, S. B. Gauld, N. A. Carter, and M. M. Harnett Bcl-xL antagonism of BCR-coupled mitochondrial phospholipase A2 signaling correlates with protection from apoptosis in WEHI-231 B cells Blood, January 1, 2004; 103(1): 168 - 176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Diaz, A. Berquand, M. Dubois, S. Di Agostino, C. Sette, S. Bourgoin, M. Lagarde, G. Nemoz, and A.-F. Prigent The Mechanism of Docosahexaenoic Acid-induced Phospholipase D Activation in Human Lymphocytes Involves Exclusion of the Enzyme from Lipid Rafts J. Biol. Chem., October 11, 2002; 277(42): 39368 - 39378. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Salh, K. Assi, S. Huang, L. O'Brien, U. Steinbrecher, and A. Gomez-Munoz Dissociated ROS production and ceramide generation in sulfasalazine-induced cell death in Raw 264.7 cells J. Leukoc. Biol., October 1, 2002; 72(4): 790 - 799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Gauld, D. Blair, C. A. Moss, S. D. Reid, and M. M. Harnett Differential Roles for Extracellularly Regulated Kinase-Mitogen-Activated Protein Kinase in B Cell Antigen Receptor-Induced Apoptosis and CD40-Mediated Rescue of WEHI-231 Immature B Cells J. Immunol., April 15, 2002; 168(8): 3855 - 3864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Katz, M. R. Deehan, S. Seatter, C. Lord, R. D. Sturrock, and M. M. Harnett B Cell Receptor-Stimulated Mitochondrial Phospholipase A2 Activation and Resultant Disruption of Mitochondrial Membrane Potential Correlate with the Induction of Apoptosis in WEHI-231 B Cells J. Immunol., January 1, 2001; 166(1): 137 - 147. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
