1 Institut für Pharmakologie and 2 Abteilung für Nieren- und Hochdruckkrankheiten, Universitätsklinikum, D-45122 Essen, Germany
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ABSTRACT |
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Proliferation and immunoglobulin secretion of B lymphocytes are regulated by specific antigens and numerous accessory immunomodulatory factors. Lysophosphatidic acid (LPA) is a glycerophospholipid mediator that is released from activated blood platelets, attains high levels in serum, and exerts potent stimulatory effects on, e.g., neutrophils, monocytes, and T lymphocytes. LPA is also generated by a secretory, cytokine-inducible phospholipase A2 present in high concentrations in inflammatory exudates and septic states. We investigated effects of LPA on human Epstein-Barr virus-immortalized B lymphoblasts, a model for immunoglobulin-secreting B cells. Intracellular Ca2+ was determined with fura 2 and the formation of inositol 1,4,5-trisphosphate by anion-exchange chromatography. LPA stimulated an increase in inositol 1,4,5-trisphosphate levels and induced a transient rise in intracellular free Ca2+ concentration from 105 ± 17 to 226 ± 21 nM. This Ca2+ signal resulted from Ca2+ mobilization and Ca2+ influx and was subject to homologous desensitization. Pertussis toxin inhibited these responses by ~70%. Furthermore, LPA stimulated a 27.5% increase in guanosine 5'-O-(3-thiotriphosphate) binding to permeabilized B lymphoblasts, which suggests the direct activation of pertussis toxin-sensitive G proteins by LPA. LPA stimulated a strong increase in the specific phosphorylation of the mitogen-activated protein kinase (immunoblot analysis) that was prevented by the MEK inhibitor PD-98059. Finally, LPA triggered a 2-fold increase in DNA synthesis ([3H]thymidine incorporation) and a 2-fold increase in B lymphoblast number and evoked a 20- to 50-fold increase in immunoglobulin formation. By RT-PCR we detected specific mRNA transcripts for the recently cloned human LPA receptor. Thus our data suggest that LPA behaves as a B cell growth factor.
G protein-coupled receptor; signal transduction; pertussis toxin; immunoglobulin; proliferation; phospholipase A2
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INTRODUCTION |
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THE PRINCIPAL MEDIATOR of adaptive humoral immunity is the B lymphocyte by virtue of its production of specific antibodies. Antigens bind to target B cell receptors and activate antigen-specific B lymphocyte clones, thus promoting their proliferation and differentiation into antibody-secreting plasma cells. This primary immune response of B lymphocytes is accompanied by the generation of memory B cells that serve as the cellular repository of the immunologic memory. Although antigen binding to the B cell receptor is a key event in the specific activation of B cell clones, a number of additional interacting signals regulate the B lymphocyte-mediated immune response. Thus induction of antibody-secreting plasma cells and memory cells is a complex process modulated by several cytokines as well as para- and autocrine factors in a specialized environment requiring various subsets of lymphocytes and macrophages. This context of antigen challenge, soluble factors, and cell adhesion molecules determines the fate of B lymphocytes, culminating in proliferation and antibody secretion or in programmed cell death (1, 7).
Lysophosphatidic acid (1-acyl-2-hydroxy-sn-glycero-3-phosphate, LPA) is a naturally occurring water-soluble phospholipid that was originally identified as a key intermediate in de novo lipid biosynthesis. LPA is now regarded as an important lipid mediator with growth factor-like activities modulating functional responses of a variety of cell types ranging from neurons to vascular smooth muscle cells and fibroblasts (for a recent review see Ref. 30). These responses to LPA are mediated by a specific, G protein-coupled LPA receptor (or receptors), the mouse and human homologues of which have recently been cloned (2, 14). Another type of LPA receptor cloned from Xenopus laevis oocytes shares no significant homology with the human LPA receptor, which may indicate the presence of several LPA receptor isoforms (13). The human LPA receptor interacts with pertussis toxin (PTX)-sensitive and -insensitive G proteins (14).
LPA is released from aggregated platelets (4), and high concentrations of LPA bound to albumin are detectable in serum. Growth factor-stimulated fibroblasts constitute another source for LPA secretion (27, 28), and high concentrations of LPA have been detected in malignant ascites fluid (45).
Extracellular LPA can be generated by a secretory phospholipase A2 (sPLA2), which cleaves phosphatidic acid (PA) to LPA in membrane microvesicles shed from activated cells (10). Besides its constitutive expression on platelets and neutrophils, sPLA2 is induced by inflammatory cytokines in a variety of cell types, and very high concentrations of sPLA2 have been detected in inflammatory exudates of patients with rheumatoid arthritis (10). Taken together, these data provide evidence for an important role of LPA in inflammation and wound healing (28).
Human neutrophils (5), monocytes (46), and T lymphocytes (45) are activated by LPA, but information regarding whether LPA also influences B lymphocytes is lacking. Therefore, we investigated the effects of LPA on human Epstein-Barr virus (EBV)-immortalized lymphoblasts. EBV-immortalized B lymphoblasts behave as immunoglobulin-secreting lymphoblasts and have been widely used for the study of B cell growth factors (12, 38).
We report here that LPA acts as a B lymphoblast growth factor and stimulates immunoglobulin formation. Furthermore, our data reveal that LPA signaling in B lymphoblasts involves activation of PTX-sensitive G proteins, formation of inositol 1,4,5-trisphosphate (IP3), Ca2+ mobilization, and stimulation of mitogen-activated protein (MAP) kinase. B lymphoblasts express specific mRNA transcripts for the recently cloned human LPA receptor, which is, therefore, the most likely candidate for mediating LPA signals in human B lymphoblasts.
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MATERIALS AND METHODS |
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Materials.
Materials and reagents were obtained from the following sources: LPA
(1-oleoyl-sn-glycerol-3-phosphate),
dioleoyl-L--phosphatidic acid, L-
-phosphatidic acid
from egg yolk,
L-
-glycerophosphate, oleic
acid, linoleic acid, the phorbol ester
12-O-tetradecanoylphorbol-13-acetate, fatty acid-free BSA,
protease inhibitors, and detergents from Sigma Chemical (Deisenhofen,
Germany); RPMI 1640 cell culture medium, penicillin-streptomycin
solution, and fetal bovine serum from Life Technologies (Eggenstein,
Germany); platelet-activating factor (PAF) and
lyso-PAF (C16) from Calbiochem (Bad
Soden, Germany); PTX from List Laboratories (Campbell, CA); digitonin
from Merck (Darmstadt, Germany); fura 2-AM from Molecular Probes
(Eugene, OR); unlabeled guanine nucleotides and adenosine
5'-[
,
-imino]triphosphate (AppNHp) from
Boehringer (Mannheim, Germany);
[methyl-3H]thymidine
(specific activity 6.7 Ci/mmol) from Hartmann Analytics (Braunschweig,
Germany);
[35S]guanosine
5'-O-(3-thiotriphosphate)
(GTP
S, specific activity 1,200-1,400 Ci/mmol) from DuPont-NEN
(Bad Homburg, Germany);
myo-[3H]inositol
from American Radiolabeled Chemicals (St. Louis, MO); antibodies for
immunoglobulin ELISA (all immunopurified) from Tago Immunologicals
(Burlingame, CA); the specific MAP kinase kinase (MEK) inhibitor
PD-98059 and the phosphospecific p44/42 MAP kinase antibody from New
England Biolabs (Schwalbach, Germany); the peroxidase-conjugated goat
anti-rabbit antibody from Sigma Chemical; the enhanced
chemiluminescence detection kit from Amersham (Braunschweig,
Germany); RT (Superscript) from Life Technologies (Eggenstein,
Germany); and Taq polymerase, dNTPs,
XbaI, and RNasin from Fermentas (St.
Leon-Rot, Germany). All other chemicals were from commercial sources
and of the highest purity available. LPA was routinely dissolved in
water or serum-free cell culture medium. L-
-Glycerophosphate, oleic
acid, linoleic acid, PAF, and lyso-PAF were dissolved in water or ethanol as recommended by the supplier. These stock solutions were further diluted in NaCl-HEPES buffer containing 0.25% BSA. Sphingosine 1-phosphate (SPP) was obtained from
Biomol (Hamburg, Germany) and was dissolved in methanol and further
diluted in NaCl-HEPES buffer containing 0.25% (wt/vol) BSA.
B lymphoblast cell lines. Preparation of peripheral blood lymphocytes and the immortalization procedure using the EBV have been detailed elsewhere (34). The experiments presented here were conducted on four different B lymphoblast cell lines derived from four healthy men. The immunologic characteristics of those B lymphoblasts have been reported elsewhere (35). Briefly, all B lymphoblast cell lines were of polyclonal origin and did not differ with regard to expression of IgM, IgA, IgD, or IgG. The supernatants of each B lymphoblast cell line contained IgG, IgA, and IgM. All cell lines expressed the B cell markers CD19, CD22, CD23, and CD38 as well as the proliferation markers CD25 and CD30. T cell markers, e.g., CD3, were completely absent.
Cells were passaged biweekly and were routinely maintained at a concentration of 5-10 × 105 cells/ml in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2 in air. Because serum contains high amounts of LPA bound to BSA (30), B lymphoblasts were subcultured into serum-free medium (21) at a density of 2 × 105 cells/ml for the investigation of LPA-induced cell proliferation, DNA synthesis, and immunoglobulin formation. This serum-free medium is essentially protein free, with the exception of 5 µg/ml iron-saturated transferrin, 10 µg/ml insulin, and 1 mg/ml fatty acid-free BSA. For the measurement of Ca2+ signals, B lymphoblasts were subcultured at a density of 2 × 105/ml in serum-free RPMI 1640 medium 24 h before the experiment.Measurement of cytoplasmic free Ca2+. Cytoplasmic free Ca2+ ([Ca2+]i) in B lymphoblasts was measured with the fluorescent Ca2+ indicator fura 2, as detailed previously (36). Lymphoblasts were incubated in serum-free RPMI 1640 medium at a density of 1 × 107 cells/ml with 2 µM fura 2-AM for 30 min at 37°C. Thereafter, lymphoblasts were pelleted, resuspended at a density of 5 × 107 cells/ml, and stored for another 30 min at 37°C to allow for complete hydrolysis of the acetoxymethyl ester. Aliquots (~1 × 107 cells) were pelleted in a microcentrifuge, resuspended in 2 ml of prewarmed (37°C) NaCl-HEPES buffer consisting of (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 D-glucose, and 20 HEPES-Cl, pH 7.4, and finally transferred to a thermostated (37°C) cuvette. Fluorescence was measured in a spectrofluorometer (model LS5B, Perkin-Elmer) equipped with a fast filter device. Excitation wavelengths rapidly alternated from 340 to 380 nm, and emission was continuously monitored at 495 nm. After lysis of the cells with digitonin, calibration of the ratio of intensities measured at excitation wavelengths of 340 and 380 nm in terms of [Ca2+]i was performed as described previously (36). For experiments in Ca2+-free medium, EGTA was added to a final concentration of 5 mM ~10 s before addition of agonist.
Analysis of inositol phosphate formation. Formation of inositol phosphates was quantified as described previously (36). Briefly, lymphoblasts were labeled with myo-[3H]inositol (5 µCi/ml) in serum- and inositol-free RPMI 1640 for 24 h. Cells were washed with PBS, and aliquots were stimulated with 100 nM LPA for different periods of time at 37°C as indicated. Reactions were terminated by addition of 500 µl of 1.2 M HCl and then by addition of 3 ml of chloroform-methanol-HCl (200:100:0.75, vol/vol/vol). After phase separation by centrifugation at 2,500 g for 10 min, the aqueous supernatants containing the water-soluble inositol phosphates were separated into an IP3- and an inositol monophosphate-inositol bisphosphate (IP1 + IP2) fraction by column chromatography on AG 1-X8 formate anion-exchange resin (200-400 mesh, Bio-Rad, Munich, Germany) and quantified by liquid scintillation counting (36).
Measurement of GTPS binding to
digitonin-permeabilized B lymphoblasts.
[35S]GTP
S binding
to digitonin-permeabilized B lymphoblasts was assayed as described
previously (36). Lymphoblasts were harvested, washed with PBS, and
resuspended in an ice-cold NaCl-triethanolamine buffer consisting of
(in mM) 150 NaCl, 5 MgCl2, 1 EDTA,
and 50 triethanolamine-HCl, pH 7.4. B lymphoblasts (~1 × 106) were added to a
thermoequilibrated (30°C) reaction mixture (final volume 200 µl)
consisting of NaCl-triethanolamine buffer containing 10 µM digitonin,
10 µM GDP, and 100 µM AppNHp. After a 15-min permeabilization
period, LPA or its solvent was added, and after another 60 s the
binding reaction was started by addition of 10 nM
[35S]GTP
S (0.1 µCi/tube) and conducted for 10 min at 30°C. Protein-bound GTP
S
was separated by rapid filtration through nitrocellulose filters
followed by intensive rinsing with 12.5 ml of ice-cold washing buffer
(50 mM Tris · HCl, pH 7.5, and 5 mM
MgCl2). Nonspecific binding was
defined as the fraction of bound
[35S]GTP
S not
competed for by 10 µM unlabeled GTP
S. Measurements were carried
out in triplicate.
Preparation of cell extracts and measurement of MAP kinase
activity.
One hour before the assay, lymphoblasts were subcultured at a density
of 3 × 106 cells/ml into
fresh serum-free RPMI 1640 medium. Cells (~1 × 107) were incubated with 100 nM
LPA for the indicated periods of time and rapidly spun down in a
microcentrifuge (10,000 g, 15 s), and
the cell pellet was homogenized into ice-cold lysis buffer consisting
of (in mM) 50 -glycerophosphate, pH 7.3, 2.0 MgCl2, 1.0 EGTA, 1.0 dithiothreitol, and 1.0 phenylmethylsulfonyl fluoride and 100 µM
Na3VO4,
0.5% (vol/vol) Triton X-100, 20 µM pepstatin A, 20 µM leupeptin,
and 10 µg/ml aprotinin. Lysates were kept on ice for 15 min.
Thereafter, nonlysed debris was removed by centrifugation at 10,000 g for 20 min at 4°C. Protein
concentration was quantified according to Bradford (3), with bovine IgG
as standard.
Analysis of [3H]thymidine incorporation, cell proliferation, and immunoglobulin synthesis. B lymphoblasts were seeded at a density of 2 × 105/ml in serum-free medium (21) and cultivated for 4 days in the presence or absence of LPA. Cells were counted daily with a CASY cell analyzer device (Schärfe, Reutlingen, Germany). Cell culture supernatants were harvested daily for immunoglobulin quantification. For determination of DNA synthesis, B lymphoblasts were subcultured in parallel to the experiments described above in 24-well dishes under otherwise identical experimental conditions. On day 2 after LPA stimulation, cells were prepulsed with 1 µCi of [methyl-3H]thymidine for 18 h. Cells were filtered through glass fiber filters, which were extensively washed with ice-cold PBS. Incorporated radioactivity was determined by liquid scintillation counting. Immunoglobulin levels were quantified by ELISA, as described previously (24, 35).
RT PCR. Total cellular RNA from B lymphoblasts was prepared by guanidinium isothiocyanate-phenol-chloroform extraction (6). cDNA was synthesized from 2 µg of RNA with 100 ng of random hexadeoxynucleotide primers by RT reaction (Superscript, Life Technologies) according to the manufacturer's protocol. The sequences for the oligonucleotide primers were derived from the sequence for the human LPA receptor (2) and were 5'-CCACACACGGATGAGCAACC-3' and 5'-GCAGAGGATCTGCCTAAAGG-3' for sense- and antisense primer, respectively. The predicted PCR product is 517 bp long and includes a single restriction site for Xba I that cleaves the amplicon into two fragments of 374 and 143 bp, respectively. PCR were performed with a Taq polymerase kit (Fermentas, St. Leon-Rot, Germany). After an initial denaturing step at 95°C for 5 min, 35 cycles at 94°C for 30 s, 58°C for 45 s, and 72°C for 45 s followed. The specificity of the resulting PCR products was verified by restriction fragment analysis with Xba I. Absence of contaminating DNA was checked by control reactions with RNA that had not been reverse transcribed. DNA fragments were separated in 1.5% agarose gels, with Alu-digested pBR322 plasmid as marker.
Statistics and presentation of data. If not otherwise indicated, all experiments were performed in triplicate and values are means ± SE. Data were compared by unpaired two-tailed Student's t-tests, and differences were regarded statistically significant at P < 0.05.
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RESULTS |
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LPA stimulates [Ca2+]i increases, phosphoinositol formation, and G protein activation. First, we analyzed the effect of LPA on [Ca2+]i in human B lymphoblasts. In the presence of 1 mM extracellular Ca2+, baseline [Ca2+]i averaged 105 ± 17 nM (n = 28). On addition of 100 nM LPA, [Ca2+]i increased to 226 ± 21 nM (n = 28). These LPA-evoked Ca2+ signals consisted of an initial, transient peak followed by a decline to a sustained plateau (Fig. 1A). On chelation of extracellular Ca2+ by addition of EGTA, LPA still evoked an initial transient Ca2+ peak, whereas the sustained plateau was no longer present (Fig. 1B). From these experiments it can be concluded that LPA-evoked changes in [Ca2]i originate from Ca2+ release from intracellular stores as well as from transmembrane Ca2+ influx. The effect of LPA on Ca2+ mobilization was concentration dependent, the minimum concentration required being ~1 nM (Fig. 1C). Maximum responses were elicited by 1 µM LPA, and the EC50 was calculated at 42 ± 5 nM (Fig. 1C). LPA-stimulated [Ca2+]i increases were subject to homologous desensitization; i.e., a second addition of agonist did not induce a second increase in [Ca2+]i (Fig. 1A). This suggests that LPA mediates its effects via a specific membrane receptor. The desensitization was not caused by depletion of intracellular Ca2+ stores, since LPA did not abrogate the ability of PAF to subsequently increase [Ca2+]i (data not shown). PAF, an ether-linked phospholipid mediator, is a well-characterized activator of B lymphoblasts (24-26, 36). Metabolites of PAF (ether-linked LPA) have been shown to interact with the potential LPA receptor (17). PAF (100 nM) elicited increases in [Ca2+]i that were on average 30-40% larger than those evoked by LPA (Fig. 1D). Treatment of B lymphoblasts with PAF or LPA did not result in mutual cross desensitization (Fig. 1D), which suggests that the effects of LPA and PAF are mediated by different membrane receptors.
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LPA induces MAP kinase activation, DNA synthesis, and immunoglobulin formation in B lymphoblasts. Activation of MAP kinase was investigated by immunoblot analysis of lysates from LPA- and PAF-stimulated B lymphoblasts with a phosphospecific MAP kinase antibody, i.e., an antibody that recognizes the phosphorylated and, thus, activated MAP kinase isoforms p42 and p44. As shown in Fig. 5, LPA induced a strong phosphorylation of both MAP kinase isoforms within 2 min. The increase in MAP kinase phosphorylation varied from 3- to 17-fold compared with nonstimulated controls depending on cell line and experiment (n = 5), as estimated from densitometric analysis of the immunoblots. The degree of MAP kinase activation by LPA was comparable to that evoked by PAF (Fig. 5). On treatment of B lymphoblasts for 30 min with 50 µM PD-98059, the specific MEK inhibitor, MAP kinase activation in response to LPA and PAF was completely blocked, which strongly supports the notion that LPA stimulates the typical MAP kinase cascade consisting of p21ras, Raf kinase, MEK, and MAP kinase. Pretreatment of B lymphoblasts with PTX (50 ng/ml) for 16 h completely blunted the LPA-induced MAP kinase activation (Table 1).
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Specific mRNA transcripts for the human LPA receptor are expressed in B lymphoblasts. A mouse LPA receptor has recently been cloned (14), and the human homologue has also been identified (2). Specific mRNA for this receptor has been detected in thymus and spleen by Northern blot analysis (2, 14). Therefore, we studied whether the mRNA of the human LPA receptor is also expressed in B lymphoblasts. RNA from B lymphoblasts was prepared and reverse transcribed, and specific transcripts were amplified by PCR. As demonstrated in Fig. 7, human B lymphoblasts express specific mRNA transcripts for the human LPA receptor.
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DISCUSSION |
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LPA, the simplest natural phospholipid, acts as an intercellular messenger molecule with a multiplicity of biological effects (for reviews see Refs. 27 and 30). Here we describe LPA as a modulator of immune responses supporting cell growth and immunoglobulin formation in four different human EBV-immortalized B lymphoblast lines. This raises several questions regarding 1) the signaling pathways of LPA, 2) the mechanisms that finally trigger B lymphoblast proliferation, and 3) the potential physiological significance of these observations.
Addition of LPA to human EBV-immortalized B lymphoblasts evokes the
formation of IP3, which is
accompanied by a transient increase in
[Ca2+]i.
The threshold concentration for LPA to evoke
Ca2+ signals is as low as 1 nM,
with maximum responses observed at ~1 µM. This is in good agreement
with results from LPA-binding and -labeling studies, which demonstrated
high-affinity binding sites on intact fibroblasts with dissociation
constants and EC50 values in the
nanomolar range (39, 44). LPA-induced
[Ca2+]i
increases consisted of Ca2+
mobilization from intracellular stores as well as transmembrane Ca2+ influx. Thus the
Ca2+ responses induced by LPA in B
cells resemble those evoked by LPA in fibroblasts (19). LPA-stimulated
Ca2+ signals were subject to
homologous desensitization in human lymphoblasts, whereas cross
desensitization with PAF did not occur. This strongly suggests that PAF
and LPA do not share a common receptor. Other bioactive phospholipids
such as SPP and PA neither evoked
Ca2+ signals nor interfered with
consecutive signals elicited by PAF or LPA. This underscores the belief
that SPP and LPA transduce their actions via different receptors (30)
and that the biological actions of PA are not mediated by the LPA
receptor (30). Furthermore, precursors of LPA such as
-glycerophosphate, linoleic acid, and oleic acid did not induce
Ca2+ signals in these cells.
These data are in accord with the results from the recent cloning of putative mouse and human LPA receptors (14). Specific transcripts of this typical heptahelical receptor (Vzg-1/edg-2/rec 3.1) have been demonstrated in spleen and thymus (2, 14). We also detected transcripts of this receptor by RT-PCR in EBV-immortalized B lymphoblasts. Available data on the substrate specificity of Vzg-1 (i.e., no response to SPP and PA) (14) are in agreement with our results. Furthermore, this receptor (Vzg-1) elicits PTX-sensitive (inhibition of cAMP generation) and PTX-insensitive effects (cell rounding) (14), which supports the hypothesis that the LPA receptor couples to more than one G protein (30). Taken together, Vzg-1 appears a likely candidate for mediating the LPA signals observed in B lymphoblasts. Because the X. laevis LPA receptor does not share homology with the mammalian LPA receptor (13), the existence of further lysophospholipid or LPA receptor isoforms appears possible.
In all cells investigated so far, LPA signals are transmitted to
intracellular effectors by heterotrimeric G proteins (4, 8, 43). This
obviously also applies to B lymphoblasts. First, we observed that LPA
induced a significant stimulation of GTPS binding to permeabilized
lymphoblasts. Second, LPA-evoked
Ca2+ signals, as well as
IP3 formation, LPA-stimulated
GTP
S binding, and MAP kinase activation, were strongly inhibited by
PTX. Thus our data provide further evidence for the participation of
Gi proteins in LPA signaling. In
contrast to the results presented here and findings from
X. laevis oocytes (8), Jalink et al. (19) reported that IP3 formation
and Ca2+ mobilization were
completely PTX insensitive in rat fibroblasts, suggesting the
predominant involvement of a Gq/11
protein in LPA-induced activation of PLC. On the other hand, Carr et
al. (4) showed that LPA stimulated the cholera toxin-catalyzed
[32P]ADP ribosylation
of G
i in Rat-1 fibroblasts. In
human skin fibroblasts, LPA-stimulated
Ca2+ signals are inhibited by
~50% on pretreatment of cells with PTX (32). Thus the LPA receptor
may couple to different G proteins depending on cell type.
We could demonstrate the expression of the PTX-sensitive G subunits
G
i-2 and
G
i-3 in membranes from human B
lymphoblasts, whereas other PTX-sensitive G proteins
(G
o,
G
i-1) could not be detected
(unpublished observations). Hence, two likely pathways result in
PTX-sensitive increases in
[Ca2+]i.
G
subunits released from activated
G
i may directly stimulate a
PLC-
isoform (33) or may, together with adaptor proteins and
nonreceptor tyrosine kinases, activate a PLC-
isoform. This latter
pathway has been proposed for PAF-mediated increases in [Ca2+]i
in B lymphoblasts (22). Further experiments will have to distinguish
between these possibilities in LPA signaling.
In B lymphoblasts the MAP kinase cascade is activated in response to
PAF stimulation or cross-linking of membrane IgM (11, 41). Addition of
LPA to Rat-1 fibroblasts or COS cells results in PTX-sensitive
activation of ras (16, 43) and the Raf
kinase (16). This finally culminates in stimulation of MAP kinase, presumably via activation of MEK (15, 16). Again, free G subunits
of PTX-sensitive G proteins appear to initiate this pathway and may
constitute a common mechanism for a parallel activation of PLC-
and
MAP kinase. Preincubation of B lymphoblasts with the MEK inhibitor
PD-98059 completely blocked MAP kinase activation by LPA in human B
lymphoblasts.
The most relevant of our findings is that LPA induces B lymphoblast proliferation and immunoglobulin formation. Thus LPA can be regarded as a B cell growth factor. These effects resemble the growth-promoting actions of LPA on T lymphocytes (45). However, Tigyi et al. (40) observed antiproliferative effects of LPA on Sp2 myeloma cells. Thus LPA may exert antagonistic effects on different subsets or differentiation stages of lymphocytes. Interestingly, the antiproliferative effect of LPA on Sp2 myeloma cells was accompanied by an LPA-triggered increase in cAMP (40). Aside from these myeloma cells, all other cell types investigated so far displayed reduced or unchanged cAMP levels on stimulation with LPA (18, 42). Thus the coupling of LPA receptor(s) to different G proteins or the expression of various adenylyl cyclase isoforms may vary between these cell types.
The exact mechanisms by which LPA affects B lymphoblast growth are not
clear. Hence, one may speculate whether LPA is a growth factor in a
strict sense or whether LPA mediates its effects by triggering the
release of autocrine growth factors. There are considerable variations
in the reported concentrations of LPA for stimulation of
Ca2+ signals, PLC, or MAP kinase
activation compared with the published concentrations required to
initiate cell growth (27, 30, 42). Hence, LPA is two to three orders of
magnitude less potent in stimulating fibroblast or vascular smooth
muscle cell proliferation than in its ability to elicit
Ca2+ signals in these cells. This
is not the case for B lymphoblasts. LPA (100 nM) promoted B lymphoblast
proliferation. In our proliferation assays, cells were cultured in
serum-free medium. Hence, LPA degradation or scavenging of LPA to serum
constituents may be negligible under our conditions. On the other hand,
we cannot exclude the possibility that nonmitogenic concentrations of
LPA suffice to stimulate the secretion of autocrine growth factors in B
lymphoblasts, which in turn stimulate B lymphoblast proliferation. This
may resemble the effects of LPA on keratinocytes, the proliferation of
which is initiated by an LPA-triggered autocrine release of
transforming growth factor (31).
LPA-triggered autocrine release of B cell growth factors could also explain the different inhibitory potencies of PTX on LPA-stimulated "early signals" and B lymphoblast proliferation. Because PTX does not completely inhibit LPA-evoked Ca2+ signals, the residual increase in [Ca2+]i may suffice to trigger the autocrine secretion of B cell growth factors (1, 8, 12, 30). PTX-insensitive G proteins may eventually mediate the effects of such B cell growth factors.
Finally, it should be considered that PTX might directly stimulate the
proliferation of T and B lymphocytes (20). This effect has been
attributed to the B oligomer of the toxin and does not involve ADP
ribosylation of G protein -subunits by the A protomer. Kolb et al.
(20) reported an increased B cell proliferation on addition of PTX. We
also observed in some experiments that PTX inconsistently evoked minor
increases in cell number compared with untreated controls (unpublished
observations). Thus PTX could directly stimulate B cell proliferation
to a minor extent, thereby partially masking the inhibitory effect of
this toxin on LPA-induced proliferation. A final conclusion regarding
whether PTX-sensitive G proteins actually mediate the growth-promoting
effects of LPA is difficult to draw from experiments in which cells are
exposed to PTX for prolonged periods of time.
Finally, we have to address the question of potential physiological roles for LPA in the immune system. Although immune cells were initially regarded to be unresponsive toward LPA stimulation (29), we provide evidence that LPA can influence B lymphoblast function in an EBV-transformed cell culture model. Likewise, Xu et al. (45) reported that LPA stimulates interleukin-2 release and proliferation in Jurkat T lymphocytes. Furthermore, high-affinity binding sites for LPA have been identified in spleen (39), and Vzg-1 transcripts have been detected in spleen, thymus, and EBV lymphoblasts (2, 14). B lymphocyte proliferation and immunoglobulin formation are regulated in a complex network of antigen activation and accessory stimuli, e.g., cytokines, adhesion molecules, hormones, and neurotransmitters (1, 8), that govern the fate of the respective B lymphoblast to proliferation, immunoglobulin synthesis, or apoptosis. High concentrations of LPA are released from activated platelets and fibroblasts. Furthermore, one enzyme generating LPA, i.e., sPLA2, is found in high concentrations in inflammatory exudates, and sPLA2 is induced by proinflammatory cytokines (10), leading to high concentrations of sPLA2 in septic states. Although data from in vitro studies are difficult to compare with the situation in vivo, one could envisage that LPA is released at sites of local inflammation (in response to cell injury, during wound healing, or from growing tumors) or generalized inflammation (septic states or rheumatoid arthritis). In these settings, LPA may influence the function of T and B lymphocytes as well as monocytes. This could, in concert with other cytokines and specific antigens, enhance B lymphoblast immune responses, e.g., leading to enhanced proliferation of specifically primed B lymphocytes. These issues require further investigation, which will be facilitated by the recent cloning of LPA receptors, since now investigations on the differential expression and regulation of this receptor in various subsets of immune cells are feasible.
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ACKNOWLEDGEMENTS |
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The expert technical assistance of Sabine Jakoby, Iris Manthey, Martina Michel, Gerlinde Siffert, and Anne-Marie Sprungmann is gratefully acknowledged.
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FOOTNOTES |
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This study was supported by the Deutsche Forschungsgemeinschaft, the Interne Forschungsförderung Universitátsklinikum Essen program of the University Hospital in Essen, and the Novartis Stiftung für Therapeutische Forschung.
Address for reprint requests: D. Rosskopf, Institut für Pharmakologie, Universitätsklinikum Essen, Hufelandstr. 55, D-45147 Essen, Germany.
Received 11 June 1996; accepted in final form 13 February 1998.
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