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The Journal of Immunology, 2000, 165: 2423-2431.
Copyright © 00 by The American Association of Immunologists

Activation Via Multiple Signaling Pathways Induces Down-Regulation of Platelet-Activating Factor Receptors on Human B Lymphocytes1

Qianli Zhuang, Yolande Bastien and Bruce D. Mazer2

Division of Allergy and Immunology, Montreal Children’s Hospital, McGill University/Montreal Children’s Hospital Research Institute, and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada, H2X 2P2


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Platelet-activating factor receptor (PAFR) has been identified in B cell lines and primary human B cells, but the regulation of PAFR during B cell activation has not been completely elucidated. In the present study, we have investigated the effects of B cell activation on PAFR binding parameters, PAFR mRNA and PAF-triggered intracellular calcium mobilization. The human B lymphoid cell line LA350 was shown to exhibit high levels of PAFR (48,550 ± 4,310 sites/cell) as determined by radio-ligand binding assay with PAFR antagonist [3H]WEB2086. Treatment with phorbol 12,13-dibutyrate caused a biphasic reduction of PAFR binding. The early phase was inhibited by the protein kinase C inhibitor bisindolylmaleimide I (BIM), whereas the late phase was not blocked by BIM, protein tyrosine kinase inhibitor genistein, or the mitogen-activated protein kinase/extracellular signal-related kinase inhibitor PD98059. However, staurosporine, a broad-spectrum protein kinase inhibitor, completely inhibited the late phase down-regulation. Ionomycin also decreased [3H]WEB2086 binding sites, whereas the combination of PDB and ionomycin induced a greater reduction than either agent alone. Cross-linking of B cell receptor by anti-IgM Ab also induced down-regulation of PAFR, which was abolished by genistein or PD98059, but not by BIM or staurosporine. The decrease in surface PAFR number was closely paralleled by the reduction in PAFR mRNA both in LA350 cells and human tonsillar B cells, and was associated with decreased response to PAF indicated by decreased intracellular calcium mobilization. These data show that multiple signaling pathways are involved in down-regulating PAFR expression during B cell activation and development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Platelet-activating factor (PAF)3 is one of the most potent phospholipid mediators with a broad-spectrum of biological activities (1). Among its numerous physiological and pathological actions, PAF has been shown to play an important role in inflammatory and immune responses, including B lymphocyte-mediated immunity (2, 3). PAF is produced by a large number of cells, including neutrophils, eosinophils, basophils, platelets, endothelial cells, and stromal cells (3). It has also been detected in bone marrow (4) and lymphoid tissue (5), the sites of lymphocyte development and functioning.

The biological activity of PAF is mediated through a specific G-protein-coupled receptor (PAFR) on the membrane of responsive cells, which has been identified on many hemopoietic cells, including platelets, neutrophils, monocytes, and dendritic cells (6). Our laboratory has identified that PAFR is present on B cell lines and human tonsillar B lymphocytes (7, 8, 9, 10, 11). We have reported that germinal center (GC) B cells express increased PAFR mRNA and have greater intracellular calcium ([Ca2+]i) responses compared with more mature mantle zone B cells (12). PAF stimulation of B cells induces a series of early signaling events, such as phosphatidylinositol hydrolysis, elevation of [Ca2+]i (13, 14, 15), protein phosphorylation (16, 17), and arachidonic acid release (15). These early signals culminate finally to increase Ig production (18, 19), abrogate apoptosis (20), and enhance cytokine synthesis (12).

The expression of PAFR can be regulated by a variety of physiological and pharmacological factors and may vary with differentiation and activation status. It has been shown in B cells that PAFR binding and mRNA expression can be up-regulated by TGF-ß (21, 22), IL-4, or Cowan I strain Staphylococcus aureus (10, 11). Phorbol esters have been reported to up-regulate (10, 11) or diminish (23, 24) PAFR on B lymphocytes and other cell types. In addition, there is no data on the expression of PAFR following engagement of the B cell Ag receptor (BCR). After engagement with Ag, the BCR transmits signals through several signaling pathways that result in the expression of a variety of genes associated with B cell activation and development. We have previously shown that BCR cross-linking does not induce early desensitization of PAFR signaling (13), but the long-term effects of B cell activation via the BCR on PAFR expression remain to be elucidated.

The aim of the present study was to clarify the issue of PAFR regulation in human B cells. We have investigated changes in PAFR expression and function using radio-ligand binding assays for membrane PAFR binding parameters, RT-PCR for PAFR mRNA and [Ca2+]i measurements as a biological response. These studies were performed with the LA350 human B lymphoid cell line as well as freshly isolated human tonsillar B lymphocytes. We demonstrate that PAF receptors are down-regulated during cell activation either physiologically by cross-linking of BCR, or pharmacologically by stimulating with phorbol 12,13-dibutyrate (PDB) and ionomycin. Our data also suggest that different signaling pathways may be involved in the down-regulation of PAFR during B cell activation by these two methods. Ag receptor-mediated decreases in PAFR expression may explain the observed differences between immature GC-like tonsillar B cells and mature B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

PAF (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, C-16), purchased from Biomol (Plymouth Meeting, PA), and [methyl-3H]WEB2086 (0.70 TBq/mmol), from DuPont-New England Nuclear (Boston, MA), were dissolved in ethanol and stored at -20°C. Before use, the ethanol was evaporated under nitrogen and reconstituted in binding buffer (10 mM HEPES, 4.8 mM KCl, 145 mM NaCl, 1.6 mM MgCl2, 0.6 mM NaH2PO4, 0.4 mM K2HPO4, 6 mM glucose, 0.1% BSA, pH 7.4). WEB2086, kindly supplied by Boehringer Ingelheim (Ingelheim en Rhein, Germany), was resuspended in binding buffer before use. Mouse anti-human IgM Ab (IgG-BU1 clone) was purchased from The Binding Site (San Diego, CA). PDB, ionomycin (ION), staurosporine (STA), bisindolylmaleimide I (BIM), genistein (GEN), and PD98059 were products of Calbiochem (La Jolla, CA); Indo-1-AM was purchased from Molecular Probes (Eugene, OR).

Cell culture

LA350 (kindly provided by Drs. W. Shearer and H. Rosenblatt, Baylor University, Houston, TX) is an EBV-transformed human lymphoblastoid B cell line that expresses surface IgM and IgD and secretes IgM. LA350 was maintained in complete medium consisting of RPMI 1640 (Life Technologies, Burlington, Ontario, Canada) supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT), 50 U/ml penicillin, 50 µg/ml streptomycin, 10 µg/ml L-glutamine, and 5 µg/ml sodium pyruvate (all from Life Technologies). Frozen aliquots were thawed every 8 wk to minimize interassay variability. Routinely, cells were subcultured three times a week at a concentration of 2 x 105 cells/ml. Before binding assays, cells were washed and resuspended at a density of 5 x 105 cells/ml with appropriate reagents at defined concentrations and cultured for 24 h. Cell count and viability (by trypan blue exclusion) were assessed at the time of each assay.

Fresh B lymphocytes were isolated from human tonsils discarded after surgery. The tonsils were thoroughly minced, resuspended in wash medium consisting of RPMI 1640 supplemented with 2% FCS, and 500 U/ml penicillin, 500 µg/ml streptomycin, and amphotericin B (1/500 w/v) from Life Technologies, and then layered onto a Ficoll-Paque (Phamacia Biotech, Uppsala, Sweden) gradient. Tonsillar lymphocytes were separated by rosetting with neuraminidase-treated sheep RBC and Ficoll-Paque density centrifugation. Monocytes were removed from the E-rosette negative fraction by adherence depletion; the remaining B cells were routinely demonstrated to be >97% pure on flow cytometry by CD19 staining, with <1% CD14+ and <2% CD3+.

PAFR binding assay

LA350 cells were washed and resuspended with binding buffer at a final concentration of 4 x 106 cells/ml. Reactions were performed in Eppendorf microfuge tubes in a total volume of 500 µl. Cells were incubated in duplicate at room temperature with increasing concentrations of [3H]WEB2086 (1–100 nM) in the presence or absence of 1000 times excess unlabeled WEB2086 for 2 h. The reaction was terminated by rapid vacuum filtration through Whatman (Tewksbury, MA) GF/C glass filters that had been presoaked for at least 60 min in binding buffer containing 1% (w/v) BSA, and then washed with 4 ml of ice-cold binding buffer three times. The radioactivity retained by each filter was measured by liquid scintillation counting in 6 ml of Cytoscint (ICN, Costa Mesa, CA) using a Beckman liquid scintillation counter. For competitive binding assays, cells were incubated with 5 nM [3H]WEB2086 and increasing concentrations of unlabeled PAF (from 10-11 to 10-5 M) or lyso-PAF (10-6 and 10-5 M). For some experiments, a single-point method was used in which cells were incubated with 100 nM of [3H]WEB2086 with or without 100 µM of unlabeled WEB2086, as at this concentration the binding was almost saturated. The specific binding sites thus obtained were consistently about 70% of those derived from the saturation binding assay.

RT-PCR detection of PAFR mRNA

RT-PCR for PAFR mRNA was performed as previously described (9) with some modifications. All reagents used were purchased from Life Technologies unless otherwise indicated. Total RNA was extracted from 5 x 106 LA350 cells or 15 x 106 human tonsillar B cells with Trizol. To avoid genomic DNA contamination, samples following homogenization were centrifuged at 12,000 x g for 10 min at 4°C to pellet DNA before adding chloroform. Total RNA extracted was dissolved in diethylpyrocarbonate-treated ddH2O and stored until use at -80°C. First-strand cDNA was synthesized in a 20-µl reaction volume containing 2 µg of total RNA, 100 U MMuLV reverse transcriptase, 0.5 µg oligo(dT)12–18 primer, 10 mM DTT, 1 mM of each kind of dNTP and 30 U RNase inhibitor (Pharmacia). Following denaturation for 5 min at 72°C, RNA was reverse transcribed for 1 h at 42°C, and then the RT enzyme was heat inactivated for 5 min at 94°C and samples stored at -20°C until use.

PCR was performed in a 50-µl reaction mixture containing 3 µl synthesized cDNA product, 5 µl 10x PCR buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl), 1.5 mM MgCl2, 0.2 mM each dNTP, 1.5 U Taq polymerase and 0.5 µM of each primer. Primer sequences were: PAFR sense (5'-CGGACATGCTCTTCTTTGATCA-3'), PAFR antisense (5'-GTCTAAGACACAGTTGGTGCTA-3') (9); ß-actin sense (5'-CCTTCCTGGGCATGGAGTCCT-3'), ß-actin antisense (5'-GGAGCAATGATCTTGATCTTC-3') (25). Amplifications were performed in a PTC-100 Programmable Thermal Controller (MJ Research, Waltham, MA) using the following programs: for PAFR, an initial cycle of 94°C for 5 min, 62°C for 3 min, and 72°C for 2 min, followed by 27 cycles of 94°C for 60 s, 60°C for 90 s, and 72°C for 90 s; for ß-actin, an initial cycle of 94°C for 3 min, 50°C for 5 min, and 72°C for 5 min, followed by 25 cycles of 94°C for 60 s, 60°C for 60 s, and 72°C for 90 s. The number of cycles was selected to remain in the linear phase of PCR amplification. Ten microliters of the PCR products were applied on a 1.5% agarose gel and visualized by ethidium-bromide staining. Densitometric analysis was performed by the FluroChem 8000 Imaging System (Alpha Innotech, San Leandro, CA).

Measurement of intracellular calcium levels

Intracellular calcium levels were measured as previously described (26, 27). Briefly, cells were resuspended in complete medium at a concentration of 1 x 107/ml, and were loaded with 1 µM of Indo-1-AM for 45 min at 37°C. Cells were then washed and resuspended at a concentration of 4 x 106/ml in serum-free medium (27). One milliliter of loaded cells was spun to pellet and resuspended in 2 ml HEPES buffer containing 140 mM NaCl, 2 mM KCl, 10 mM glucose, 1 mM MgCl2, and 1 mM CaCl2. Cytosolic calcium levels were monitored using a Shimadzu RF-5000 spectrofluorometer (Shimadzu, Japan).

Data analysis

Graphics and statistical analysis were performed using GraphPad PRISM software (GraphPad Software, San Diego, CA). Maximum binding sites (Bmax) and dissociation constant (Kd) were determined through Scatchard plots. Significance testing was conducted by use of Student’s t test or one-way ANOVA with post Newman-Keuls test. Significance was considered at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LA350 cells express a high level of PAFR

LA350 is an EBV-transformed IgM-secreting human B lymphoid line. In previous studies, we and others have extensively characterized LA350 responses to PAF stimulation, including [Ca2+]i mobilization, phosphatidylinositol hydrolysis, MAPK activation, and early activation gene expression (13, 14, 16). In addition, ligation of the BCR does not induce apoptosis, making LA350 an ideal model for the study of PAFR regulation in the context of physiologic B cell activation.

Saturation binding assays were conducted on LA350 cells (4 x 106/ml) incubated in duplicate with various concentrations of the radiolabeled PAF antagonist [3H]WEB2086 with or without unlabeled WEB2086. As shown in Fig. 1GoA, the binding was concentration-dependent, saturable, and conformed to a single hyperbola. Nonspecific binding increased linearly as a function of radioligand concentration but at much lower rate, accounting for ~15% of the total binding at the concentration of Kd. Scatchard transformation of the specific binding data yielded a linear plot, indicating a homogeneous population of binding sites (Fig. 1GoB). In five separate experiments, the mean equilibrium Kd, representing the affinity of binding sites, was 16.19 ± 6.85 nM, and the mean Bmax, representing the total number of binding sites, was 6799 ± 604 dpm (161 ± 14 fmol)/2 x 106 cells. Assuming a 1:1 stoichiometry between receptor and ligand, this Bmax corresponds to 48,550 ± 4,310 binding sites per LA350 cell. The specificity of PAF binding to LA350 cells was confirmed by competition studies utilizing unlabeled PAF as well as lyso-PAF, the inactive precursor and metabolite of PAF. PAF (10 pm to 10 µM) competed with [3H]WEB2086 for the PAFR binding sites on LA350 cells, whereas lyso-PAF did not compete for these sites even at 10 µM (Fig. 1GoC). In addition, the competition curve descended from 90% to 10% of specific binding over an 81-fold increase in the concentration of PAF. The Hill coefficient (nH) yielded by GraphPad PRISM is -0.9, not significantly different from unity, which also supports the presence of a single class of binding sites.



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  		FIGURE 1. [3H]WEB2086 binding to LA350 cells. A, Saturation isotherm for [3H]WEB2086 binding. Cells (2 x106/500 µl) were incubated with [3H]WEB2086 (0.5–100 nM) at 22°C for 2 h. The specific binding (SB) was calculated as total binding (TB) minus nonspecific binding (NSB) determined in the presence of 1000-fold excess unlabeled WEB2086. B, Scatchard transformation of the data in A. Representative of five identical experiments. C, Competition of [3H]WEB2086 binding to LA350 cells. Cells were incubated at 22°C for 2 h in binding buffer containing 5 nM [3H]WEB2086 without or with PAF (10-11 to 10-6 M) or lyso-PAF (10-6 to 10-5 M). The results are expressed as percentage of the control (without competitor) from duplicate determinations. A replicate experiment gave identical results.

 
PDB and ION down-regulate PAFR on LA350 cells.

We subsequently investigated the modulation of PAFR following LA350 cell activation. The combination of calcium ionophore and phorbol ester strongly activates B cells, and both agents have been reported to modulate the PAFR (23, 28, 29). In the present study, we found that both PDB and ION caused down-regulation of PAF binding sites on LA350 cells. Binding assays were performed after 24 h of culture with 10 nM PDB, 0.5 µM ION, or PDB plus ION at the same concentrations. As shown in Fig. 2GoA, both PDB and ION significantly decreased the number of [3H]WEB2086 binding sites compared with control untreated cells. Addition of these two agents in combination induced greater inhibition than either agent alone (by 57.65 ± 4.80%, 37.44 ± 12.55%, and 74.29 ± 2.09% for PDB, ION, and PDB plus ION, respectively, n = 3). In contrast, no significant difference was observed in the Kd for all conditions (Fig. 2GoB). These experiments indicate that down-regulation of PAFR by PDB and ION is due to a change in receptor density rather than receptor affinity.



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  		FIGURE 2. Regulation of [3H]WEB2086 binding to LA350 cells by PDB and ION. Cells were incubated at 37°C for 24 h in the absence (CON) or presence of PDB (10 nM), ION (0.5 µM) or both, then harvested for [3H]WEB2086 binding. Bmax (A) and Kd (B) values were calculated for each condition from three independent experiments. *, p < 0.01 vs CON; {wedge}, p < 0.05 vs PDB/ION. C, The time course of PDB- or ION-induced down-regulation of [3H]WEB2086 binding on LA350 cells. Cells were incubated with or without 10 nM PDB or 0.5 µM ION for the indicated time periods, and then assayed with single-point method as described in Materials and Methods. The results are expressed as percentages of the control and are representative of two independent experiments.

 
The time course of PDB- or ION-induced PAFR down-regulation was similar (Fig. 2GoC). Both caused a time-dependent reduction of PAF binding sites, which was maximal at 24 h of incubation, and almost completely recovered at 48 h. It was notable that PDB caused an early phase decrease of PAFR binding, which was observed following 5 min of incubation, peaking at 30 min, and recovering after 2 h. ION did not show this biphasic effect.

It has been suggested that the late decrease in PAFR surface expression was due to decreased expression of PAFR mRNA (23). We performed RT-PCR to measure the mRNA level of PAFR before and after cell activation. We optimized the number of cycles for PAFR and ß-actin amplification to ensure that the PCR were in the linear phase. Minor changes in template RNA could thus be distinguished by densitometric analysis, and was comparable to those obtained using our semiquantitative radioactive RT-PCR (12). We found that both PDB and ION markedly diminished PAFR mRNA (Fig. 3GoA). The effect of PDB was most significant at 12 h, and mRNA recovered to normal levels at 24 h (Fig. 3GoB). Incubation with medium alone up to 48 h did not appreciably change the PAFR mRNA. No PCR product was detected when reverse transcriptase was omitted, indicating that there was no genomic DNA contamination in the RT-PCR template (9). The prolonged latency between the recovery of PAFR mRNA and the recovery of membrane binding sites is most likely due to translation and posttranslational modification. However, we cannot exclude posttranscriptional regulation, such as increased mRNA degradation (23), mRNA storage, or decelerated translation.



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  		FIGURE 3. Effects of PDB/ION and anti-IgM ({alpha}IgM) Ab on the expression of PAFR mRNA in LA350 cells. A, LA350 cells were incubated for 12 h with medium alone (CON, lane 1), 10 nM PDB (lane 2), 0.5 µM ION (lane 3), or both (lane 4). B, LA350 cells were incubated with medium alone (lane 1), or with 10 nM PDB for indicated times: 2 h (lane 2), 6 h (lane 3), 12 h (lane 4), 24 h (lane 5), 36 h (lane 6). C, LA350 cells were incubated with medium alone (lane 1), or anti-IgM Ab (2 µg/ml) for indicated times: 2 h (lane 2), 6 h (lane 3), 12 h (lane 4), 24 h (lane 5), 36 h (lane 6). A–C, Following incubation, total RNA was isolated, and RT-PCR using human PAFR and ß-actin primers was performed. The upper panel of each figure indicates a representative experiment, indicating the PCR products under the various conditions. PCR product were quantified by densitometry; the relative amount of PAFR mRNA was expressed as a ratio compared with that of ß-actin and shown in the lower panels. Representative data from two separate experiments are shown.

 
BCR cross-linking down-regulates PAFR on LA350 cells

BCR cross-linking on LA350 cells by anti-IgM Ab has been shown to activate LA350 cells as evidenced by the hydrolysis of phosphatidylinositol, increase of cytosolic Ca2+, and secretion of IgM (13, 30). We therefore examined the effect of anti-IgM Ab on PAFR binding. LA350 cells were treated with medium or anti-IgM Ab (2 µg/ml) for 24 h, and then [3H]WEB2086 binding studies were performed. As shown in Fig. 4Go, this treatment decreased the number of binding sites by 40%, from 48,060 ± 6,862 per cell in the untreated cells to 28,890 ± 1,983 per cell in the anti-IgM Ab-treated cells (p < 0.05, n = 4) (Fig. 4GoA). Similar to PDB and ION, there was no significant difference in receptor affinity between untreated (Kd = 16.16 ± 3.19 nM) and anti-IgM Ab-treated cells (Kd = 13.00 ± 2.78 nM, NS, n = 4) (Fig. 4GoB). Time course studies revealed a uniphasic decrease in binding sites that reached the nadir after a 24-h incubation, and remained suppressed for at least 48 h (Fig. 4GoC). In accordance with this, PAFR mRNA was also decreased in a time-dependent manner following anti-IgM Ab treatment, and remained at low levels even after 36 h (Fig. 3GoC).



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  		FIGURE 4. Regulation of [3H]WEB2086 binding to LA350 cells by anti-IgM Ab ({alpha}IgM). Cells were incubated for 24 h without (CON) or with anti-IgM Ab (2 µg/ml), then harvested for [3H]WEB2086 binding. Four independent experiments were done and the mean ± SEM of Bmax (A) and Kd (B) are shown. *, p < 0.01 vs CON. C, Time course of anti-IgM Ab effect on [3H]WEB2086 binding to LA350 cells. Cells were incubated with or without 2 µg/ml anti-IgM Ab for indicated time periods, then harvested for binding assay using the single-point method as described in Materials and Methods. The results are expressed as percentages of control and are representative of two separate experiments.

 
PDB/ION and BCR cross-linking down-regulate PAFR on fresh human B cells

We have demonstrated that PAFR mRNA is expressed in freshly isolated human B lymphocytes from tonsil and that PAFR ligation increases [Ca2+]i and Ig production in these cells (9, 12). We investigated whether the down-regulation of PAFR by PDB/ION stimulation or BCR engagement on LA350 B lymphoid cell line could be extended to primary B lymphocytes. Fresh human B cells were isolated from tonsils with >97% purity. B cells express PAFR, as demonstrated by [3H]WEB2086 binding, albeit 10- to 20-fold lower than LA350 cells (data not shown). As in our previous studies comparing B cells to the Ramos cell line (12), baseline PAF mRNA in fresh human B cells was lower than in LA350 cells. Similar to LA350 cells, we found that treatment with PDB, ION, or anti-IgM Ab for 12 or 24 h decreased PAFR mRNA in isolated tonsillar B lymphocytes (Fig. 5Go). We did not find any appreciable change of PAFR mRNA in these cells following incubation with medium alone for up to 48 h (data not shown).



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  		FIGURE 5. Effects of PDB/ION and anti-IgM Ab ({alpha}IgM) on the expression of PAFR mRNA in human tonsillar B cells. A, Freshly isolated human tonsillar B cells were incubated with medium alone (lane 1), 10 nM PDB (lane 2), 0.5 µM ION (lane 3), or both (lane 4) for 12 h. B, Tonsillar B cells were incubated with medium alone (lane 1) or 2 µg/ml anti-IgM Ab for 24 h. After incubation, total RNA was extracted, RT-PCR for PAFR and ß-actin was performed. PCR product was analyzed as described in Materials and Methods. The histograms demonstrate the relative amounts of PAFR mRNA, expressed as the ratio to ß-actin mRNA. Representative data from two separate experiments are shown.

 
PDB and anti-IgM Ab induce down-regulation of PAFR through different signaling pathways

B lymphocyte activation following cross-linking of BCR involves a series of signaling pathways, involving both protein tyrosine kinases (PTK) and serine/threonine kinases (31). PDB exerts its effect primarily through activation of PKC, but may also involve molecules such as protein kinase D (PKD) (32), PTK (33, 34), and MAP kinase (35, 36). We attempted to elucidate the signaling pathways involved in PAFR down-regulation by PDB and anti-IgM Ab by assessing the effects of protein kinase inhibitors including BIM, a highly selective PKC inhibitor (37); STA, a broad spectrum protein kinase inhibitor (38, 39); GEN, a PTK inhibitor (40); and PD98059, a MEK inhibitor (41). As shown in Fig. 6Go, none of the inhibitors showed significant effect on the basal level of [3H]WEB2086 binding at the concentrations used. To our surprise, whereas STA completely blocked PDB induced down-regulation (Fig. 6GoB), no significant effect of BIM was observed (Fig. 6GoA) even at a concentration of 1 µM (data not shown). GEN and PD98059 did not have any significant effect on PDB-induced down-regulation (Fig. 6Go, C and D). With respect to anti-IgM Ab-induced down-regulation, neither BIM nor STA had any significant effect (Fig. 6Go, A and B), even at extremely high concentrations (1 µM BIM or 500 nM STA, data not shown). However, GEN and PD98059 blocked the anti-IgM Ab effect significantly. Following addition of GEN, the number of [3H]WEB2086 binding sites recovered from 59.0 ± 4.3% to 86.0 ± 2.4% of control (Fig. 6GoC), indicating that PTK is involved in the down-regulation caused by BCR cross-linking. Pretreatment with PD98059 also restored the binding sites from 61.4 ± 5.4% to 87.8 ± 6.4% of control (Fig. 6GoD), implicating the MAPK pathway in this down-regulation.



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  		FIGURE 6. Effects of protein kinase inhibitors on PDB or anti-IgM ({alpha}IgM) Ab-induced down-regulation of [3H]WEB2086 binding to LA350 cells. LA350 cells were incubated with 100 nM BIM (A), 50 nM STA (B), 50 µM GEN (C), or 10 µM PD98059 (D) for 2 h before incubation with 10 nM PDB or 2 µg/ml anti-IgM Ab for 24 h. Binding assays were performed with the single-point method as described. Values shown are percentage of the control (CON) and are mean ± SEM of three separate experiments. *, p < 0.05 vs CON; {wedge}, p < 0.05 vs PDB; #, p < 0.05 vs anti-IgM Ab.

 
Down-regulation of PAFR is correlated with decreased PAF-induced [Ca2+]i mobilization.

Changes in PAFR numbers have been shown to correlate well with changes in the biological response to PAF (29, 42). To determine whether there was a functional decrease in PAFR responsiveness following B cell activation, we have measured changes in [Ca2+]i in Indo-1-loaded LA350 cells following PDB or anti-IgM Ab treatment. Addition of 10-7 M PAF (Fig. 7GoA), but not lyso-PAF (Fig. 7GoB) at the same concentration, led to a rapid increase of [Ca2+]i, peaking within 1 min, followed by a secondary plateau and a slow return to baseline. As we have shown before (13), pretreatment with anti-IgM Ab (2 µg/ml) for 5 min did not inhibit PAF-induced {Delta}[Ca2+]i (Fig. 7GoE). In contrast, pretreatment with PDB (10 nM) for 5 min almost completely inhibited the PAF-triggered calcium response (Fig. 7GoC). This inhibition could be blocked by the PKC-specific inhibitor BIM (Fig. 7GoD). Treatment with PDB and anti-IgM Ab for 24 h also resulted in a marked inhibition of PAF-induced [Ca2+]i elevation (Fig. 7Go, F and G).



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  		FIGURE 7. Effects of PDB and anti-IgM Ab on PAF-induced [Ca2+]i mobilization in LA350 cells. LA350 cells were loaded with Indo-1AM for 30 min and then stimulated with (A) 10-7M PAF, (B) 10-7M lyso-PAF, (C) 10 nM PDB followed by 10-7M PAF, (D) 100 nM BIM, 10 nM PDB, and 10-7 M PAF sequentially, (E) 2 µg/ml anti-IgM Ab followed by 10-7M PAF. For some experiments, LA350 cells were first incubated with (F) 10 nM PDB or (G) 2 µg/ml anti-IgM Ab for 24 h and then harvested for the measurement of PAF triggered [Ca2+]i elevation. The figure is representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ubiquitous presence and pluripotent effects of PAF in both pathological and physiological processes necessitate tight regulation of its action. There are at least two levels of control defined: the enzymatic cleavage of PAF and the down-regulation of PAFR. The inactivation of PAF by acetylhydrolase is well studied and may be an important step in limiting inflammatory responses (43). Relatively less is known about the regulation of PAFR. Control of PAFR expression during an inflammatory response is essential to counterbalance the effect of the local production of this lipid mediator. There are data regarding homologous desensitization gleaned from studies on PAFR following PAF binding (44, 45, 46, 47). PAF itself induces phosphorylation of serine/threonine residues in the carboxyl-terminal tail of PAFR, causing receptor internalization and desensitization (46, 47). Phorbol ester is another extensively studied regulator of PAFR. Although most studies suggest that PKC activation negatively modulates PAFR expression or its response to PAF (23, 24, 48, 49), other reports indicate opposite effects (11, 28). Some cytokines, inflammatory mediators, and hormones have also been shown to modulate PAFR in various cell types (6). Understanding of PAFR regulation can provide insight into the potential role of PAF in the context of other stimuli that influence cell activation and function.

We have demonstrated previously that PAF may contribute to the optimal functioning of human B cells. PAF can rescue B cells from anti-IgM Ab-induced apoptosis (20), enhance the secretion of Ig (18, 19), and up-regulate IL-4 synthesis in human tonsillar B cells (12). These data suggest that PAF may play an important role in B cell development and function. B cells exist in the immune tissues at various stages of maturation, which exhibit different biological characteristics. Indeed, we have shown that the expression of PAFR mRNA and the response to PAF is more pronounced in GC B cells than in mantle zone B cells (12).

In the present study, we have studied the expression of PAFR mRNA and binding sites in the LA350 B cell line as well as human tonsillar B cells. The PAFR density in LA350 is higher than any other previously reported PAFR-expressing B cell line except for ASK.0 (8). In both fresh and immortalized B lymphocytes, there is a decrease in PAFR expression following phorbol ester and/or ION stimulation, and importantly following BCR cross-linking by anti-IgM Ab. Activation via either pharmacological or more physiological stimuli diminished PAFR density significantly without altering receptor affinity. The changes identified by ligand binding assays were well paralleled by PAFR mRNA studies in both the LA350 cell line and human fresh B cells. PDB and/or ION as well as anti-IgM Ab all markedly diminished PAFR mRNA; whereas the PAFR mRNA restored to baseline at 24 h following PDB treatment, no such recovery was observed in anti-IgM Ab-treated cells. Finally, the down-regulation of PAFR expression during B cell activation has a functional consequence as evidenced by decreased [Ca2+]i mobilization triggered by PAF. This is likely to be significant, as several authors have shown that the functional response of PAFR correlates well with receptor number on responsive cells, and that modulation of PAFR number is accompanied by an altered responses to PAF (12, 21, 42, 50).

Divergent time courses of PAFR suppression were observed with the different activation signals; PDB decreased PAFR transiently, whereas BCR stimulation led to more prolonged suppression on LA350 cells. We thus determined that diminution and recovery of PAFR mRNA and surface expression was controlled by distinct signaling pathways. The effect of PKC on the regulation of G-protein coupled receptors has been well studied (23, 24, 48, 49). It has been revealed that putative Ser/Thr phosphorylation sites are present in the cytoplasmic tail of PAFR (51). PKC activation can phosphorylate these residues, resulting in the interaction with ß-arrestin and uncoupling from the G-protein, leading to the desensitization and receptor internalization (52). This may explain the early phase effect of PDB observed in our study, as the inhibition of PAF-triggered [Ca2+]i elevation by PDB could be blocked by pretreatment with BIM, a specific PKC inhibitor. The late phase effect of PDB in our study seems to involve a different mechanism, because the decrease in PAFR expression caused by prolonged incubation with PDB could not be prevented by BIM, but could be prevented by STA, a broad spectrum protein kinase inhibitor. Similarly, Yue et al. (48, 49) also observed that the PKC inhibitor H-7 could not completely block PDB inhibition of PAF-induced [Ca2+]i mobilization and phosphatidyl inositol turnover in neuronal cell lines. The late phase effect of PDB may be mediated through the activation of PKC isoform(s), which can be blocked by STA but not BIM, or protein kinases other than PKC that can be inhibited by STA may be involved. Recently, molecules such as PKD have been identified as new candidates for phorbol ester induction (32, 53), and may be responsible for the late phase PAFR down-regulation.

Our finding that phorbol ester down-regulates PAFR is in agreement with several previous observations from human monocytes (23), neutrophils (29), and human neurohybrid cell lines (48, 49). Nguer et al. (11) reported that PMA increased PAFR mRNA and surface expression in human tonsillar B cells. They also found that PDB/ION increased PAF binding to human peripheral blood B cells (10). There are several possibilities accounting for these discrepancies. The PDB concentration Nguer et al. used (1 ng/ml, i.e., 1.98 nM) was lower than the concentration we used (10 nM). The effect of phorbol ester on PAFR regulation has shown to be dose-dependent; at lower concentrations (1.6 nM) PMA increases PAFR binding, whereas at higher concentrations (16 nM) it decreases PAFR binding on the same cells (54, 55). Additionally, Nguer et al. reported PAFR regulation following 48 h of treatment with phorbol ester, without assessing the effect at intermediate times. As shown in our study, the down-regulated PAFR by PDB/ION was transient, returning to baseline at 48 h.

In anti-IgM Ab-induced PAFR down-regulation, neither BIM nor STA showed any significant effect, excluding the involvement of PKC. In contrast, the PTK inhibitor genistein was able to counteract the action of anti-IgM Ab; moreover, the MEK antagonist PD98059 was also effective in inhibiting PAFR down-regulation following BCR ligation. In contrast, neither of these pathways is involved in the action of PDB. We therefore conclude that anti-IgM Ab inhibits PAFR expression mainly through protein tyrosine phosphorylation and the Ras/Raf/MAPK pathway. This is rational, as PTK recruitment is the principal mechanism initiated in the cascade of BCR signaling (31). This is the first study to demonstrate a role for the MAPK pathway in the regulation of PAFR expression. We are in the process of determining how the BCR signaling pathway contributes to the persistent down-regulation of PAFR.

Activation of B cells via the BCR is a key step in B cell differentiation and activation. Although mature B cells usually respond to BCR engagement by multiplying and differentiating into Ab secreting plasma cells or memory B cells, ligation of BCR on immature B cells can lead to the deletion of the cells by apoptosis. In the context of an infectious or inflammatory stimulus, immature B cells that are stimulated via BCR may receive second signals that allow for maturity toward plasma cells or memory B cells. Because BCR ligation by Ag does not desensitize PAFR, it can receive a synergistic signal from PAF released from locally inflamed tissues. This signal may be important in rescuing the immature B cells from apoptosis (20). However, because long-term ligation of BCR will down-regulate PAFR both the receptor number and its functional response, PAF can only act when it is present early in this process. Indeed, our previous study found that PAF could rescue Ramos cells from apoptosis only if it was added within 1 h of anti-IgM Ab treatment. The fact that BCR activation induces down-regulation of PAFR is also in accordance with our previous studies suggesting that the PAFR expression and PAF responsiveness decreases along with B cell maturation (12). Taken together, these studies present an important example of potential cross-talk between Ag signaling and inflammatory mediators present in lymph nodes and other areas of immune response.


   Acknowledgments
 
We thank Dr. Jian-hua Zhang for technical assistance. We thank Drs. Michael Ward, Barbara Tollozcko, and Williams S. Powell for sharing their reagents. We also thank Dr. Christine McCusker for helpful advice and critique for the manuscript.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada, the Fonds de la Recherche en Santé du Quebec, the Costello Foundation, and the McGill University/Montreal Children’s Hospital Research Institute. Back

2 Address correspondence and reprint requests to Dr. Bruce D. Mazer, Meakins Christie Laboratories, 3626 St. Urbain Street, Montreal, Quebec, Canada H2X 2P2. Back

3 Abbreviations used in this paper: PAF, platelet-activating factor; BCR, B cell receptor; BIM, bisindolylmaleimide I HCl; GC, germinal center; GEN, genistein; ION, ionomycin; MAPK, mitogen-associated protein kinase; MEK, MAPK extracellular signal-related kinase kinase; PAFR, PAF receptor; PDB, phorbol 12,13-dibutyrate; PKC, protein kinase C; PKD, protein kinase D; PTK, protein tyrosine kinase; STA, staurosporine; [Ca2+]i, intracellular calcium concentration. Back

Received for publication June 7, 1999. Accepted for publication June 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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