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Cooperation between the FcϵR1 and Formyl Peptide Receptor Signaling Pathways in RBLFPRCells: The Contribution of Receptor-Specific Ca2+Mobilization Responses
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
235, 812–819 (1997)
RC976874
Cooperation between the FceR1 and Formyl Peptide Receptor Signaling Pathways in RBLFPR Cells: The Contribution of Receptor-Specific Ca2/ Mobilization Responses Rebecca J. Lee,1 Don E. Lujan,* Ann L. Hall, Larry A. Sklar, Bridget S. Wilson, and Janet M. Oliver Department of Pathology and Cancer Research & Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131; and *Department of Chemistry, Northern Arizona University, Flagstaff, Arizona 86001
Received May 23, 1997
RBLFPR mast cells express the tyrosine kinase-coupled IgE receptor, FceR1, and the G-protein-coupled formyl peptide receptor, FPR. FceR1 crosslinking causes Ca2/ stores release, Ca2/ influx, Ins(1,4,5)P3 production and secretion. FPR ligation also mobilizes Ca2/, but without measurable Ins(1,4,5)P3 production or secretion. Co-stimulating the FPR and FceR1 induces more Ins(1,4,5)P3 production and secretion than FceR1 cross-linking alone. Costimulation also produces more rapid and sustained Ca2/ responses than are generated by FceR1 activation alone. We identified multiple differences between the FPR- and FceR1-coupled Ca2/ responses, including a more rapid Ca2/ spike response to FPR ligation; intracellular Ca2/ stores that are empty following FceR1 crosslinking but partially full following FPR activation; a more sustained Ca2/ influx response to FceR1 crosslinking; and the immediate inhibition of stimulated Ca2/ influx by FPR antagonists but not by monovalent ligand that terminates FceR1 crosslinking. We hypothesize that the interaction of receptor-specific Ca2/ mobilization pathways contributes to the FPR-mediated potentiation of FceR1-coupled secretion. q 1997 Academic Press
We have used RBLFPR cells that express the high affinity IgE receptor, FceR1, and the formyl peptide receptor, FPR, to explore interactions between tyrosine kinase-coupled and G-protein-coupled signaling pathways. In RBLFPR cells, as in the parental RBL-2H3 rat mast cells, cross-linking the heterotrimeric (abg2) FceR1 with multivalent antigen activates the cytoplasmic tyrosine 1
Corresponding author: J. M. Oliver, Cell Pathology Laboratory, Surge Building, UNM School of Medicine, Albuquerque, N.M. 87131. Fax: (505) 272-9135. E-mail: [email protected]. 0006-291X/97 $25.00
kinases Lyn and Syk (1,2), and triggers a signalling cascade that leads to protein tyrosine phosphorylation, inositol phospholipid hydrolysis, Ca2/ mobilization, actin polymerization, membrane ruffling, the assembly of actin plaques involved in increased adhesion and spreading, and secretion (3-11). In human neutrophils, ligating the G protein-coupled FPR with N-formyl-methionyl-leucyl phenylalanine (fMLF) and other N-formylated peptides also triggers cellular responses, including Ca2/ mobilization and changes in cell shape, polarity and adhesion (12). In RBLFPR cells, ligating the transfected FPR similarly causes Ca2/ mobilization (13) and actin polymerization2 that are inhibited by pertussis toxin (14). Because RBL2H3 cells express both Gai2 and Gai3 , but not Go (15,16), these fMLF-activated responses most likely involve FPR coupling to Gi . Although FPR activation alone produces no Ins(1,4,5)P3 and little secretion in RBLFPR cells, we found that the presence of fMLF potentiates antigeninduced Ins(1,4,5)P3 production and secretion. We hypothesized that the activation of receptor-specific Ca2/ mobilization responses might contribute to this cooperation and tested the hypothesis using single cell Ca2/ assays. We report that the Ca2/ responses activated through the FceR1 and FPR differ in the lag time to Ca2/ stores release, in the filling state of the Ca2/ stores during activation, and in the magnitude and dependence on continued cross-linking of the Ca2/ influx responses. We also show that the simultaneous activation of both receptors produces Ca2/ responses that are initiated more rapidly and sustained longer than the responses to FceR1 crosslinking alone. 2 Hall, A. L., Pfeiffer, J. R., Wilson, B. S., Oliver, J. M., and Sklar, L. A. (1997) Ligand-receptor dynamics of the human formyl peptide receptor expressed in rat basophilic leukemia cells. J. Leukocyte Biol. Accepted for publication.
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Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Materials. Fura-2AM, Pluronic F-127 and dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) were from Molecular Probes (Eugene, OR). Minimal essential medium (MEM) was from Life Technologies (Grand Island, NY). [3H]Serotonin (5-[1,2-3H(N)hydroxytryptamine binoxalate) and [3H]Ins(1,4,5)P3 were from Dupont/NEN (Boston, MA). DNP-specific IgE was purified from mouse ascites containing the H1-DNP-e-26-82 hybridoma (17). Thapsigargin and all other reagents were obtained from Sigma Chemical (St. Louis, MO). FPR transfection, cell culture and activation. RBL-2H3 cells were transfected with the plasmid pRSVFPR containing FPR DNA, a generous gift of Richard Ye (18). Briefly, FPR cDNA isolated from an HL-60 library was inserted into the plasmid pRc/RSV behind the RSV promoter. Plasmid DNA was electroporated into RBL-2H3 cells and the cells were selected for 8–12 days in G418. Surviving cells were expanded and sorted by flow cytometry of fluorescein isothiocyanate-conjugated N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK-FITC)labelled cells for the top 5% of FPR-expressors (1-31105FPR/cell). RBLFPR cells were maintained at 377C, 5% CO2 in MEM-Earles medium with 10% Hybrimax defined serum supplement (Sigma), 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 800 mg/ml G418. RBL-2H3 cells were maintained identically but without G418. One to 3 days prior to experiments, cells were plated into 24-well culture dishes (for secretion assays) or onto suspension grade plates (for Ins(1,4,5)P3 and Ca2/ assays) in G418-free medium containing 15% heat-inactivated fetal bovine serum. For FceR1 activation, cells were incubated for at least 2 hours with 1 mg/ml dinitrophenyl-specific IgE (anti-DNP IgE) to bind FceR1, then rinsed and antigen (100 ng/ml DNP-BSA) added to cross-link the IgE-FceR1 complexes. The FPR was activated with either 10 or 100 nM fMLF. All assays were performed in modified Hanks’ solution (19) containing 125 mM NaCl, 5mM KCl, 0.7 mM Na2HPO4 , 0.7 mM NaH2PO4 , 15 mM NaHCO3 , 5.5mM glucose, 0.75 mM MgCl2 , 1.8 mM CaCl2 and 0.05% BSA. In nominally Ca2/-free Hanks’-BSA, CaCl2 was omitted. Measurement of secretion. Agonist-stimulated secretion was measured as the release of preloaded [3H]serotonin during a 20-minute incubation in Hanks’-BSA solution at 377C under 5% CO2 (21). Measurement of Ins(1,4,5)P3 . Cells were washed once and suspended in Hanks’-BSA medium at 20 1 106 cells/ml. Four hundred microliter aliquots were activated with either 100 ng/ml DNP-BSA or 100 nM fMLF. Reactions were stopped by the addition of 16% trichloroacetic acid. Ins(1,4,5)P3 levels were determined using the competitive binding assay described in Deanin et al. (22). Measurement of [Ca2/]i . The intracellular concentration of free Ca2/ ([Ca2/]i) was measured in individual, fura-2AM-loaded cells using fluorescence ratio imaging microscopy. Cells were loaded with 2 mM fura-2AM in MEM at room temperature in an atmosphere of 5% CO2/95% air. Following loading, the medium was exchanged for Hanks’-BSA, the CO2 atmosphere was maintained and the temperature was increased to 357C. Solution exchanges were made with a two-syringe device that exchanges 8-10 coverslip dish volumes (45 ml total). Receptor agonists and antagonists were added by pipette (RBLFPR cells) or by solution exchange (RBL-2H3 cells). Average ratio values for each cell in a field were calculated for user-defined areas within each cell and were converted to [Ca2/]i as described in Grynkiewicz et al. (20) using ratio values measured on calibration solutions containing maximal and minimal Ca2/ concentrations. Calibrations were performed daily. All other details of [Ca2/]i measurement are described in Lee and Oliver (21).
RESULTS fMLF alone stimulates little secretion but potentiates antigen-stimulated secretion in RBLFPR cells. The an-
FIG. 1. Net secretion by antigen- and fMLF-stimulated RBL2H3 and RBLFPR cells. Secretion of 3H-serotonin over a 20-minute incubation with agonist was measured as described in Methods. Cells were activated with 100 ng/ml DNP-BSA (Ag), 10 or 100 nM fMLF, or 100 ng/ml DNP-BSA plus 100 nM fMLF, as indicated. The reported percentage secretion values are the average of duplicate samples from a single experiment with spontaneous secretion subtracted. Duplicates differed by less than 10%. Similar results were obtained in 6 experiments.
tigen- and fMLF-activated secretory responses of RBL2H3 and RBLFPR cells are summarized in Figure 1. RBL-2H3 cells do not express the FPR and fMLF addition elicits no secretion. In RBLFPR cells, 10 or 100 nM fMLF stimulates little or no release of preloaded 3Hserotonin. Adding 100 ng/ml DNP-BSA to IgE-primed RBLFPR cells activates secretion. Importantly, the simultaneous addition of fMLF and antigen produces a secretory response that exceeds the sum of the individual fMLF- and antigen-stimulated responses. Potentiated secretion was observed in 6 of 6 experiments, with fMLF providing an additional 10–40% secretion over the sum of the secretory responses elicited independently by fMLF and antigen. fMLF alone does not induce Ins(1,4,5)P3 production, but potentiates antigen-stimulated Ins(1,4,5)P3 production. Antigen stimulation of both RBL-2H3 (22) and RBLFPR (Fig. 2, circles) cells leads to an elevated level of Ins(1,4,5)P3 which is maintained for at least 10 min. Adding fMLF to RBLFPR cells does not lead to increased Ins(1,4,5)P3 production detectable by our assay (Fig. 2, triangles). There was also no increase in Ins(1,4,5)P3 levels measured 10 and 15 sec after fMLF addition (not shown). However, fMLF potentiates Ins(1,4,5)P3 production activated by antigen (Fig. 2, squares). Comparison of fMLF- and antigen-stimulated Ca2/ responses. In the presence of extracellular Ca2/ (Cao2/), 10 nM fMLF elicited Ca2/ responses from 29 of 32 cells and 100 nM fMLF produced Ca2/ responses in 63 of 64 cells. Formyl peptide-activated Ca2/ responses generally consisted of a high initial [Ca2/]i spike, fol-
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(SDÅ23). These results indicate that intracellular Ca2/ stores are released more rapidly by FPR ligation than by FceR1 cross-linking.
FIG. 2. Time course of Ins(1,4,5)P3 production by antigen- and fMLF-stimulated RBLFPR cells. Ins(1,4,5)P3 levels were measured as described in Methods. Agonists, 100 ng/ml DNP-BSA, 100 nM fMLF or both, were added at time 0. The reported data are averages of two separate experiments, each performed in duplicate on the same day. Similar results were obtained in 6 additional experiments.
lowed by a decrease to a lower, but still elevated, level (Fig. 3A,B). Approximately one-third of fMLF-stimulated cells instead showed an abrupt elevation in [Ca2/]i that lacked the initial spike and that was maintained at an elevated level (see Fig. 5B for an example). FceR1 cross-linking also led, after a lag, to a rapid increase in [Ca2/]i (Fig. 3C,D). There was day-to-day variability in the magnitudes of Ca2/ responses to fMLF and antigen (compare Figs. 3B and 6A, cells activated with 10 nM fMLF on different days; and Figs. 3C and 6B, cells activated with 100 ng/ml DNP-BSA on different days). However, activation with 100 nM fMLF consistently produced larger Ca2/ responses than did 10 nM fMLF and activation with 100 ng/ml DNP-BSA produced more sustained responses than either concentration of peptide (compare Figs. 3A and 3C). fMLF stimulates both Ca2/ stores release and Ca2/ influx. In the absence of Cao2/ , fMLF addition led to a rapid increase in [Ca2/]i that was not sustained (Fig. 3B, inset). fMLF at 10nM caused Ca2/ stores release in 5 of 9 cells; 100 nM fMLF stimulated Ca2/ responses in 9 of 9 cells. The lag times from fMLF addition to Ca2/ response were comparable in the presence and absence of Cao2/ , demonstrating that the initial Ca2/ response is the result of Ca2/ stores release. The sustained elevation in [Ca2/]i following fMLF stimulation requires Ca2/ influx, as removal of Cao2/ leads to an immediate drop in [Ca2/]i (Fig. 3B). As previously described (21,23), antigen-stimulated Ca2/ responses in RBL-2H3 cells also result from an initial release of intracellular Ca2/ stores, followed by the continued influx of Cao2/ (Fig. 3D and inset). However, the average lag time for 63 cells activated with 100 nM fMLF was 8 sec (SDÅ4 sec), whereas the average lag time for 33 antigen-stimulated cells was 36 sec
Antigen stimulation of RBLFPR cells results in a persistent depletion of intracellular Ca2/ stores while FPR activation does not. RBL-2H3 cells demonstrate capacitative Ca2/ entry, defined as the influx of Ca2/ following intracellular stores depletion (24,25), and there is evidence that depleted Ca2/ stores are responsible for a substantial portion of the Ca2/ influx response to FceR1 crosslinking (26,27). If this is also the case for the FPR, then the different Ca2/ influx responses of fMLF and antigen-treated cells could reflect different filling states of their stores. To test this prediction, RBLFPR cells were activated with antigen in the presence of Cao2/ . After 90 sec, Cao2/ was removed and 100 nM thapsigargin was added to assess the filling state of the Ca2/ stores. Thapsigargin inhibits SERCA, the sarcoplasmic and endoplasmic reticulum Ca2/-ATPase responsible for filling Ca2/ stores and leads to the depletion of intracellular stores through an uncharacterized leak pathway (28,29). In Figure 4A, Ca2/ responses from two antigen-stimulated RBLFPR cells are plotted. Thapsigargin addition produced no detectable increase in [Ca2/]i in these or 9 other antigen-stimulated cells. Similar experiments in RBL-2H3 cells revealed a persistent depletion of thapsigargin-sensitive Ca2/ stores in 24 of 36 cells DNP-BSA (100 ng/ml)stimulated cells. In the remaining RBL-2H3 cells, thapsigargin addition caused a small release of Ca2/ (example not shown). In contrast, intracellular Ca2/ stores were not completely empty in any of the fMLF-stimulated RBLFPR cells tested. Fig. 4B shows the Ca2/ responses of two representative cells (of 16) activated by fMLF in the presence of Cao2/ . In fMLF-treated cells, thapsigargin addition following the removal of Cao2/ produced an increase in [Ca2/]. In Fig. 4C, plots from two representative cells (of 16) show that thapsigargin also releases Ca2/ from unstimulated cells subjected to the same Cao2/ removal protocol. Integrals of [Ca2/]i over time were computed in all RBLFPR cells for the two-minute period following thapsigargin addition. The average integral in fMLF-stimulated cells was roughly half that calculated for unstimulated cells. These data suggest that intracellular Ca2/ stores are only partially depleted during continuous fMLF stimulation while antigen stimulation leads to a maintained depletion of intracellular stores. The FPR-activated Ca2/ response requires continuous occupation of the formyl peptide receptor while antigenstimulated Ca2/ responses do not require continued FceR1 cross-linking. The FPR desensitizes rapidly in both neutrophils and RBLFPR cells (30). Consistent with this, the fMLF-stimulated Ca2/ responses are quickly terminated following the addition of an FPR antago-
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FIG. 3. fMLF- and antigen-stimulated Ca2/ responses in individual RBLFPR cells. Changes in [Ca2/]i over time in fura-2-loaded RBLFPR cells were measured as described in Methods. The number of cells observed under each condition is indicated in the text. Each plot represents the Ca2/ response of a single representative cell. Agonist was added at the arrow and was then present continuously. Ca2/ o was present as indicated by the solid bars in A through D, and was not present in the experiments plotted in the insets of B and D. A. Typical Ca2/ response 2/ to fMLF stimulation showing a high initial spike followed by a lower, sustained elevation in [Ca ]i that gradually falls toward baseline over the course of the experiment. B. The sustained elevation in [Ca2/]i in fMLF-stimulated cells depends upon influx of Ca2/ o as its removal leads to an immediate decrease in [Ca2/]i to pre-stimulation levels. In the absence of Ca2/ o (inset), fMLF activates a rapid, transient elevation in [Ca2/]i . C. Ca2/ response to antigen stimulation showing an approximately 50 s lag time, a small initial spike and an elevation in [Ca2/]i that is sustained at a higher level for a longer period of time than is typical of Ca2/ responses to fMLF stimulation. D. The sustained 2/ elevation in [Ca2/]i in antigen-stimulated cells also depends upon influx of Ca2/ o . In the absence of Cao (inset), antigen stimulation causes a transient elevation in [Ca2/]i similar to that observed in fMLF-stimulated cells, but with a longer lag time.
nist, t-boc (tert-butoxy-phenylalanyl-leucyl-phenylalanyl-leucyl-phenylalanine), in cell populations (14) and in single cells (Fig. 5A,B). The fMLF-induced Ca2/ response in Figure 5A is typical; a high initial Ca2/ spike
that decreases rapidly to a lower, but still elevated, level. The addition of t-boc reduces this maintained elevation to baseline [Ca2/]i . T-boc also causes an immediate reduction in [Ca2/]i in cells that show the less-
FIG. 4. Antigen stimulation completely empties intracellular Ca2/ stores while fMLF stimulation does not. Panels A, B and C present results from three experiments, all performed on the same day. Each plot represents the Ca2/ response over time of a single RBLFPR cell. was present in the medium Agonist, either 100 ng/ml DNP-BSA (A) or 10 nM fMLF (B) was added as indicated by the solid arrow. Ca2/ o during the time indicated by the solid bar beneath each graph. 100 nM thapsigargin was added at the dashed arrow. The Ca2/ responses plotted in panel C are from cells that were not stimulated prior to removal of Ca2/ o and thapsigargin addition. 815
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FIG. 5. FPR-stimulated Ca2/ responses require continuous receptor occupation while antigen-stimulated Ca2/ responses do not. Panels A, B, and B inset represent Ca2/ responses over time for individual RBLFPR cells activated at the solid arrow by 100 nM fMLF. T-boc (10 mM) was added as indicated by the dashed arrow (A,B). The cell response plotted in B, inset is from an experiment done on the same day as that in B and shows the cellular response to fMLF alone. The axes of the inset are the same as those in panel B. Panels C, D, and D inset represent Ca2/ responses over time for individual RBL-2H3 cells activated with 100 ng/ml DNP-BSA at the solid arrow. In C, DNPBSA was removed by solution exchange for Ca2/-containing Hanks’ medium as indicated by the dashed bar. In D, 10 mM DNP-lysine was exchanged for DNP-BSA as indicated by the dashed arrow. The inset to panel D shows the Ca2/ response of a control cell from an experiment done on the same day as that in panel D. Two separate additions of DNP-BSA were made by solution exchange at the times indicated by the solid arrows. The axes of the inset are the same as for panel D.
typical FPR-activated response that lacks the initial Ca2/ spike (Figure 5B). Abrupt decreases in [Ca2/]i resulting from t-boc addition were observed in 19 of 22 fMLF-stimulated cells. Figure 5B (inset) shows that the fMLF-stimulated [Ca2/]i response decreases over time even without t-boc, but the decrease is much more gradual. In contrast, Ca2/ responses to 100 ng/ml DNP-BSA are not immediately diminished by the removal of antigen without (Fig. 5C) or with the addition of DNP-lysine (Fig. 5D). DNP-lysine is a monovalent hapten that displaces a portion of bound antigen, prevents the formation of new cross-links, and rapidly terminates secretion (data not shown; 31). Similar results were observed in 20 of 29 cells in which DNPBSA was removed and in 30 of 35 cells in which DNPlysine was added simultaneously with antigen removal. In the remainder of cells, [Ca2/]i decreased followed these treatments. Figure 5D (inset) shows that activated cells also show little response to subsequent applications of antigen. The simultaneous addition of fMLF and antigen produces a more sustained Ca2/ response than does either agonist alone. Stimulation with both antigen
and fMLF (Fig. 6C) produces responses that are initiated more rapidly than those induced by antigen alone (Fig. 6B) and are sustained for longer periods than those observed following either antigen (Fig. 6B) or fMLF alone (Fig. 6A). The average lag time following the simultaneous addition of antigen and fMLF was 11 sec (SDÅ9 sec; nÅ30), comparable to that measured in response to fMLF alone and much shorter than the average lag time following addition of antigen. Additionally, in approximately half of the cells, the initial Ca2/ spike was larger than that induced by fMLF or antigen alone. In 15 of 22 cells in 4 separate experiments, the subsequent sustained elevation was of similar magnitude to that observed in response to antigen alone, but was maintained at this elevated level through the point when the antigen-stimulated response typically started to decline. In the remaining cells, the elevated Ca2/ response stimulated by antigen plus fMLF was comparable to the response activated by antigen alone. DISCUSSION We have used RBLFPR cells to study signaling responses activated through the tyrosine kinase-coupled
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FIG. 6. fMLF and antigen added together produce more sustained Ca2/ responses than does either agonist alone. Plots represent changes in [Ca2/]i over time for individual RBLFPR cells from three separate experiments, all performed on the same day. Agonists, 10 nM fMLF (A), 100 ng/ml DNP-BSA (B), or 10 nM fMLF / 100 ng/ ml DNP-BSA (C), were added as indicated by the arrows. The cell plotted in B showed several oscillations in [Ca2/]i prior to the addition of antigen. Similar spontaneous oscillations in [Ca2/]i were observed in 14% of RBLFPR cells (22 of 159 cells).
IgE receptor, FceR1, and the G-protein-coupled chemotactic peptide receptor, FPR. Only antigen, and not fMLF, induces measurable Ins(1,4,5)P3 synthesis and secretion. However, fMLF potentiates antigen-induced Ins(1,4,5)P3 synthesis and secretion. Since the FceR1 and the FPR are both Ca2/-mobilizing receptors, we hypothesized that interactions between receptor-specific Ca2/ mobilization responses might contribute to this cooperation. This was tested by characterizing the
Ca2/ responses of cells stimulated with antigen and fMLF, alone and in combination. We first determined that the average lag time from fMLF addition to Ca2/ response is significantly shorter than the average lag time measured in antigen-stimulated cells. Short lag times to response were reported previously for RBL-2H3 cells transfected with the m1 muscarinic acetylcholine receptor (RBL-2H3(m1); 32) and may be characteristic of lag times to Ca2/ response induced by G protein-linked receptors. Receptor-activated Ca2/ stores release is typically mediated by Ins(1,4,5)P3 generated by phospholipase C (PLC), either the tyrosine kinase-activated PLCg isoforms or the G protein-coupled PLCb isoforms. In RBL-2H3 cells, FceR1 cross-linking causes the tyrosine phosphorylation and activation of both PLCg1 (8,9) and PLCg2.3 PLCb3 is the predominant PLCb isoform in RBL-2H3 cells and preliminary experiments suggested its activation by fMLF in RBLFPR cells (unpublished results, in collaboration with K. Caldwell and L. Gaudet). Thus, different kinetics of activation of PLC isoforms could explain the different lag times to Ca2/ stores release. Our inability to measure FPR-stimulated Ins(1,4,5)P3 synthesis by our radioreceptor assay complicates this interpretation. However, bulk assays may not reveal local Ins(1,4,5)P3 increases that presumably determine Ca2/ stores release. Further studies showed that the status of the intracellular stores is also different between fMLF- and antigen-stimulated cells. Antigen-stimulated cells contain little or no thapsigargin-releasable (stored) Ca2/, whereas fMLF-activated cells contain approximately one half as much thapsigargin-releasable Ca2/ as unstimulated cells. These results establish that antigen leads to a maintained depletion of intracellular Ca2/ stores, whereas Ca2/ stores remain partially full, or can refill, in the continued presence of fMLF. The persistent Ca2/ stores of fMLF-stimulated cells could represent a fMLF-insensitive but antigen-sensitive compartment. However, the initial Ca2/ response to fMLF is at least as robust as the response to antigen, suggesting that Ca2/ stores release is essentially complete in both cases. Thus we hypothesise that, once Ca2/ stores are released, antigen-stimulated cells more effectively maintain them in an empty state. This difference could occur because antigen-treated cells produce more Ins(1,4,5)P3 than do fMLF-stimulated RBLFPR cells, so that any Ca2/ pumped into the stores is immediately released through the open Ins(1,4,5)P3 receptor/ Ca2/ channel. Alternatively, SERCA, the Ca2/ pump responsible for Ca2/ re-uptake into stores, may be in3 Barker, S. A., Caldwell, K. K., Pfeiffer, J. R., Oliver, J. M., and Wilson, B. S. Wortmannin-sensitive phosphorylation, translocation and activation of PLCg1, but not PLCg2, in antigen-stimulated RBL2H3 mast cells. Submitted for publication.
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hibited by signals from the FceR1 (as proposed in 21) but not from the FPR. Comparison of fMLF- and antigen-stimulated Ca2/ responses additionally revealed that fMLF-stimulated Ca2/ influx is, in general, smaller than antigen-stimulated influx. This is the predicted result if capacitative Ca2/ entry, Ca2/ influx following intracellular stores depletion (33), is responsible for the bulk of fMLF as well as agonist-stimulated Ca2/ influx. We have demonstrated that the thapsigargin-sensitive intracellular Ca2/ stores are empty for many minutes in antigenstimulated RBL-2H3 cells, creating conditions that favor Ca2/ influx. In contrast, our observation that fMLF causes only partial depletion of thapsigargin-sensitive Ca2/ stores provides a reasonable explanation for the smaller magnitude of the fMLF-stimulated Ca2/ influx response. Studies of Ca2/ responses to receptor antagonists revealed another difference between the FceR1 and FPR signaling pathways. [Ca2/]i levels in fMLF-treated cells decrease almost immediately to baseline upon the addition of the FPR antagonist t-boc that prevents further occupation of the FPR. These results indicate that FPRmediated Ca2/ entry requires continuous signaling. In contrast, antagonizing the cross-linking of FceR1 with 10 mM DNP-lysine did not return the antigen-stimulated elevation in [Ca2/]i to basal levels during the 6 minutes of our assay, although excess DNP-lysine did immediately inhibit secretion. These results suggest that FceR1 cross-linking activates Ca2/ influx via a pathway that does not require continuous receptor crosslinking. The evidence (above) linking sustained antigen-induced Ca2/ influx to the empty state of the Ca2/ stores provides an obvious mechanism for continued Ca2/ influx without continued receptor signalling. Consistent with our hypothesis, Zhang and McCloskey (27) showed that DNP-lysine addition to antigen-stimulated cells causes a relatively slow reduction in the antigen-stimulated Ca2/ current, thought to be the capacitative Ca2/ entry current, measured using the perforated patch clamp method. On the other hand, our results appear to contradict work by Fewtrell and Sherman (34) showing an immediate inhibition of DNPBSA-induced 45Ca influx by the addition of DNP-lysine to quin2-buffered RBL-2H3 cells; by Maeyama et al. (35) showing that DNP-lysine causes a rapid drop in fluorescence of quin2-labelled cells; and by Weetall et al. (36) showing that adding monovalent hapten to cells activated with a bivalent antigen causes a rapid decrease in [Ca2/]i indicated by indo-1 fluorescence. The two fluorimetric studies were performed under conditions of suboptimal stimulation (less than 10% receptor occupancy in one case; a weak antigen in the other). Continued receptor crosslinking may be more important for maintaining stimulated Ca2/ influx under these conditions than under our conditions of maximal stimulation. Our time-resolved microscopic assays of
[Ca2/]i levels in single, adherent fura-2-labeled cells are not easily comparable with Fewtrell and Sherman’s radio-isotopic study of 45Ca2/ influx in suspensions of quin2-loaded cells. Importantly, simultaneous FPR and FceR1 activation produces Ca2/ influx responses that begin sooner and are more sustained than those in cells activated by antigen alone. These data support the hypothesis that antigen and fMLF activate receptor-specific Ca2/ mobilization responses in the RBLFPR transfectants. We speculate that the more rapid and sustained Ca2/ responses of co-stimulated cells may in turn potentiate Ca2/-dependent signaling responses that include the activation of PLC isoforms responsible for Ins(1,4,5)IP3 synthesis (37) and secretion. Rather than potentiating antigen-induced responses, the RBLFPR cell line generated independently by Ali et al. (13) responded to fMLF with Ca2/ mobilization, Ins(1,4,5)P3 production and secretion. When we measured fMLF-stimulated secretion in cytochalasin Btreated cells, the conditions used by Ali and colleagues, net secretion was still less than 6% (data not shown). Our RBLFPR transfectants expressed 1-3 1 105 FPR/ cell, while Ali et al. report approximately 9 1 104 FPR/ cell. We are unable to explain why fMLF addition to our RBLFPR cells prepares these cells for enhanced antigenmediated functional responses whereas it directly elicits responses in another line. We note, however, that occupying the G protein-coupled adenosine receptors of RBL-2H3 cells also causes no secretion but enhances antigen-induced secretion (38). In summary, we have demonstrated that mast cell responses to activating the tyrosine kinase-coupled FceR1 can be enhanced through the simultaneous activation of the G protein-coupled FPR. Our results implicate receptor-specific mechanisms that regulate Ca2/ release from stores, Ca2/ re-uptake into stores, and Ca2/ influx in the FPR-mediated potentiation of FceR1coupled responses. ACKNOWLEDGMENTS We thank A. Marina Martinez for technical assistance. The ratio imaging microscope and flow cytometer used in this study are shared instruments maintained in the Cytometry and Microscopy shared facilities of the UNM Cancer Research and Treatment Center. This work was supported in part by NIH Grants GM50562 (BSW), HL56384 and GM49814 (JMO), AI19032 and RR01315 (LAS), and AI35997 (R. Posner, Northern Arizona University, for DEL).
REFERENCES 1. Eiseman, E., and Bolen, J. B. (1992) Nature 355, 8–80. 2. Hutchcroft, J. E., Geahlen, R. L., Deanin, G. G., and Oliver, J. M. (1992) Proc. Natl. Acad. Sci. USA 89, 9107–9111. 3. Beaven, M. A., Moore, J. P., Smith, G. A., Hesketh, T. R., and Metcalfe, J. C. (1984) J. Biol. Chem. 259, 7137–7142.
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4. Beaven, M. A., Rogers, J., Moore, J. P., Hesketh, T. R., Smith, G. A., and Metcalfe, J. C. (1984) J. Biol. Chem. 259, 7129–7136. 5. Pfeiffer, J. R., Seagrave, J. C., Davis, B. H., Deanin, G. G., and Oliver, J. M. (1985) J. Cell Biol. 101, 2145–2155. 6. Pribluda, V. S., and Metzger, H. (1987) J. Biol. Chem. 262, 11449–11454. 7. Stump, R. F., Oliver, J. M., Cragoe, E. J., Jr., and Deanin, G. G. (1987) J. Immunol. 139, 881–886. 8. Park, D. J., Min, H. K., and Rhee, S. G. (1991) J. Biol. Chem. 266, 24237–24240. 9. Li, W., Deanin, G. G., Margolis, B., Schlessinger, J., and Oliver, J. M. (1992) Mol. Cell. Biol. 12, 3176–3182. 10. Pfeiffer, J. R., and Oliver, J. M. (1994) J. Immunol. 152, 270– 279. 11. Oliver, J. M., Pfeiffer, J. R., and Wilson, B. S. (1997) in IgE Receptor (FceR1) Function in Mast Cells and Basophils (Hamawy, M. M., Ed.), pp. 139–172, R. G. Landes Company, Austin. 12. Thelen, M., Dewald, B., and Baggiolini, M. (1993) Physiol. Rev. 73, 797–821. 13. Ali, H., Richardson, R. M., Tomhave, E. D., Didsbury, J. R., and Snyderman, R. (1993) J. Biol. Chem. 268, 24247–24254. 14. Ali, H., Richardson, R. M., Tomhave, E. D., Dubose, R. A., Haribabu, B., and Snyderman, R. (1994) J. Biol. Chem. 269, 24557– 24563. 15. Hide, M., Ali, H., Price, S. R., Moss, J., and Beaven, M. A. (1991) Mol. Pharm. 40, 473–479. 16. Matsuoka, M., Kaziro, Y., Asano, S., and Ogata, E. (1993) Am. J. Med. Sci. 306, 89–93. 17. Liu, R.-T., Bohn, J. W., Fenry, E. L., Yanamoto, H., Molinaro, C. A., Sherman, L. A., Klinman, N. R., and Katz, D. H. (1980) J. Immunol. 124, 2728–2736. 18. Prossnitz, E. R., Quehenberger, O., Cochrane, C. G., and Ye, R. D. (1991) Biochem. Biophys. Res. Commun. 168, 471–476. 19. Becker, E. L. (1972) J. Exp. Med. 135, 376–387.
20. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440–3450. 21. Lee, R. J., and Oliver, J. M. (1995) Mol. Biol. Cell 6, 825–839. 22. Deanin, G. G., Cutts, J. L., Pfeiffer, J. R., and Oliver, J. M. (1991) J. Immunol. 146, 3528–3535. 23. Millard, P. J., Ryan, T. A., Webb, W. W., and Fewtrell, C. (1989) J. Biol. Chem. 264, 19730–19739. 24. Wong, A., Cook, M. N., Foley, J. J., Sarau, H. M., Marshall, P., and Hwang, S. M. (1991) Biochemistry 30, 9346–9354. 25. Cleveland, P. L., Millard, P. J., Showell, H. J., and Fewtrell, C. M. S. (1993) Cell Calcium 14, 1–16. 26. Fasolato, C., Hoth, M., and Penner, R. (1993) J. Biol. Chem. 268, 20737–20740. 27. Zhang, L., and McCloskey, M. A. (1995) J. Physiol. 483, 59–66. 28. Takemura, H., Hughes, A. R., Thastrup, O., and Putney, J. W., Jr. (1989) J. Biol. Chem. 264, 12266–12271. 29. Inesi, G., and Sagara, Y. (1992) Arch. Biochem. Biophys. 298, 313–317. 30. Omann, G. M., Oades, Z. G., and Sklar, L. A. (1985) Biotechniques 3, 508–513. 31. Seagrave, J. C., Deanin, G. G., Martin, J. C., Davis, B. H., and Oliver, J. M. (1987) Cytometry 8, 287–295. 32. Jones, S. V. P., Choi, O. H., and Beaven, M. A. (1991) FEBS Letts. 289, 47–50. 33. Putney, J. W., Jr. (1986) Cell Calcium 7, 1–12. 34. Fewtrell, C., and Sherman, E. (1987) Biochemistry 26, 6995– 7003. 35. Maeyama, K., Hohman, R. J., Ali, H., Cunha-Melo, J. R., and Beaven, M. A. (1988) J. Immunol. 140, 3919–3927. 36. Weetall, M., Holowka, D., and Baird, B. (1993) J. Immunol. 150, 4072–4083. 37. Rhee, S. G. (1994) in Signal-Activated Phospholipases (Liscovitch, M., Ed.), pp. 1–12, R. G. Landes Co., Austin. 38. Ali, H., Cunha-Melo, J. R., Saul, W. F., and Beaven, M. A. (1990) J. Biol. Chem. 265, 745–753.
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Cooperation between the FceR1 and Formyl Peptide Receptor Signaling Pathways in RBLFPR Cells: The Contribution of Receptor-Specific Ca2/ Mobilization Responses Rebecca J. Lee,1 Don E. Lujan,* Ann L. Hall, Larry A. Sklar, Bridget S. Wilson, and Janet M. Oliver Department of Pathology and Cancer Research & Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131; and *Department of Chemistry, Northern Arizona University, Flagstaff, Arizona 86001
Received May 23, 1997
RBLFPR mast cells express the tyrosine kinase-coupled IgE receptor, FceR1, and the G-protein-coupled formyl peptide receptor, FPR. FceR1 crosslinking causes Ca2/ stores release, Ca2/ influx, Ins(1,4,5)P3 production and secretion. FPR ligation also mobilizes Ca2/, but without measurable Ins(1,4,5)P3 production or secretion. Co-stimulating the FPR and FceR1 induces more Ins(1,4,5)P3 production and secretion than FceR1 cross-linking alone. Costimulation also produces more rapid and sustained Ca2/ responses than are generated by FceR1 activation alone. We identified multiple differences between the FPR- and FceR1-coupled Ca2/ responses, including a more rapid Ca2/ spike response to FPR ligation; intracellular Ca2/ stores that are empty following FceR1 crosslinking but partially full following FPR activation; a more sustained Ca2/ influx response to FceR1 crosslinking; and the immediate inhibition of stimulated Ca2/ influx by FPR antagonists but not by monovalent ligand that terminates FceR1 crosslinking. We hypothesize that the interaction of receptor-specific Ca2/ mobilization pathways contributes to the FPR-mediated potentiation of FceR1-coupled secretion. q 1997 Academic Press
We have used RBLFPR cells that express the high affinity IgE receptor, FceR1, and the formyl peptide receptor, FPR, to explore interactions between tyrosine kinase-coupled and G-protein-coupled signaling pathways. In RBLFPR cells, as in the parental RBL-2H3 rat mast cells, cross-linking the heterotrimeric (abg2) FceR1 with multivalent antigen activates the cytoplasmic tyrosine 1
Corresponding author: J. M. Oliver, Cell Pathology Laboratory, Surge Building, UNM School of Medicine, Albuquerque, N.M. 87131. Fax: (505) 272-9135. E-mail: [email protected]. 0006-291X/97 $25.00
kinases Lyn and Syk (1,2), and triggers a signalling cascade that leads to protein tyrosine phosphorylation, inositol phospholipid hydrolysis, Ca2/ mobilization, actin polymerization, membrane ruffling, the assembly of actin plaques involved in increased adhesion and spreading, and secretion (3-11). In human neutrophils, ligating the G protein-coupled FPR with N-formyl-methionyl-leucyl phenylalanine (fMLF) and other N-formylated peptides also triggers cellular responses, including Ca2/ mobilization and changes in cell shape, polarity and adhesion (12). In RBLFPR cells, ligating the transfected FPR similarly causes Ca2/ mobilization (13) and actin polymerization2 that are inhibited by pertussis toxin (14). Because RBL2H3 cells express both Gai2 and Gai3 , but not Go (15,16), these fMLF-activated responses most likely involve FPR coupling to Gi . Although FPR activation alone produces no Ins(1,4,5)P3 and little secretion in RBLFPR cells, we found that the presence of fMLF potentiates antigeninduced Ins(1,4,5)P3 production and secretion. We hypothesized that the activation of receptor-specific Ca2/ mobilization responses might contribute to this cooperation and tested the hypothesis using single cell Ca2/ assays. We report that the Ca2/ responses activated through the FceR1 and FPR differ in the lag time to Ca2/ stores release, in the filling state of the Ca2/ stores during activation, and in the magnitude and dependence on continued cross-linking of the Ca2/ influx responses. We also show that the simultaneous activation of both receptors produces Ca2/ responses that are initiated more rapidly and sustained longer than the responses to FceR1 crosslinking alone. 2 Hall, A. L., Pfeiffer, J. R., Wilson, B. S., Oliver, J. M., and Sklar, L. A. (1997) Ligand-receptor dynamics of the human formyl peptide receptor expressed in rat basophilic leukemia cells. J. Leukocyte Biol. Accepted for publication.
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MATERIALS AND METHODS Materials. Fura-2AM, Pluronic F-127 and dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) were from Molecular Probes (Eugene, OR). Minimal essential medium (MEM) was from Life Technologies (Grand Island, NY). [3H]Serotonin (5-[1,2-3H(N)hydroxytryptamine binoxalate) and [3H]Ins(1,4,5)P3 were from Dupont/NEN (Boston, MA). DNP-specific IgE was purified from mouse ascites containing the H1-DNP-e-26-82 hybridoma (17). Thapsigargin and all other reagents were obtained from Sigma Chemical (St. Louis, MO). FPR transfection, cell culture and activation. RBL-2H3 cells were transfected with the plasmid pRSVFPR containing FPR DNA, a generous gift of Richard Ye (18). Briefly, FPR cDNA isolated from an HL-60 library was inserted into the plasmid pRc/RSV behind the RSV promoter. Plasmid DNA was electroporated into RBL-2H3 cells and the cells were selected for 8–12 days in G418. Surviving cells were expanded and sorted by flow cytometry of fluorescein isothiocyanate-conjugated N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK-FITC)labelled cells for the top 5% of FPR-expressors (1-31105FPR/cell). RBLFPR cells were maintained at 377C, 5% CO2 in MEM-Earles medium with 10% Hybrimax defined serum supplement (Sigma), 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 800 mg/ml G418. RBL-2H3 cells were maintained identically but without G418. One to 3 days prior to experiments, cells were plated into 24-well culture dishes (for secretion assays) or onto suspension grade plates (for Ins(1,4,5)P3 and Ca2/ assays) in G418-free medium containing 15% heat-inactivated fetal bovine serum. For FceR1 activation, cells were incubated for at least 2 hours with 1 mg/ml dinitrophenyl-specific IgE (anti-DNP IgE) to bind FceR1, then rinsed and antigen (100 ng/ml DNP-BSA) added to cross-link the IgE-FceR1 complexes. The FPR was activated with either 10 or 100 nM fMLF. All assays were performed in modified Hanks’ solution (19) containing 125 mM NaCl, 5mM KCl, 0.7 mM Na2HPO4 , 0.7 mM NaH2PO4 , 15 mM NaHCO3 , 5.5mM glucose, 0.75 mM MgCl2 , 1.8 mM CaCl2 and 0.05% BSA. In nominally Ca2/-free Hanks’-BSA, CaCl2 was omitted. Measurement of secretion. Agonist-stimulated secretion was measured as the release of preloaded [3H]serotonin during a 20-minute incubation in Hanks’-BSA solution at 377C under 5% CO2 (21). Measurement of Ins(1,4,5)P3 . Cells were washed once and suspended in Hanks’-BSA medium at 20 1 106 cells/ml. Four hundred microliter aliquots were activated with either 100 ng/ml DNP-BSA or 100 nM fMLF. Reactions were stopped by the addition of 16% trichloroacetic acid. Ins(1,4,5)P3 levels were determined using the competitive binding assay described in Deanin et al. (22). Measurement of [Ca2/]i . The intracellular concentration of free Ca2/ ([Ca2/]i) was measured in individual, fura-2AM-loaded cells using fluorescence ratio imaging microscopy. Cells were loaded with 2 mM fura-2AM in MEM at room temperature in an atmosphere of 5% CO2/95% air. Following loading, the medium was exchanged for Hanks’-BSA, the CO2 atmosphere was maintained and the temperature was increased to 357C. Solution exchanges were made with a two-syringe device that exchanges 8-10 coverslip dish volumes (45 ml total). Receptor agonists and antagonists were added by pipette (RBLFPR cells) or by solution exchange (RBL-2H3 cells). Average ratio values for each cell in a field were calculated for user-defined areas within each cell and were converted to [Ca2/]i as described in Grynkiewicz et al. (20) using ratio values measured on calibration solutions containing maximal and minimal Ca2/ concentrations. Calibrations were performed daily. All other details of [Ca2/]i measurement are described in Lee and Oliver (21).
RESULTS fMLF alone stimulates little secretion but potentiates antigen-stimulated secretion in RBLFPR cells. The an-
FIG. 1. Net secretion by antigen- and fMLF-stimulated RBL2H3 and RBLFPR cells. Secretion of 3H-serotonin over a 20-minute incubation with agonist was measured as described in Methods. Cells were activated with 100 ng/ml DNP-BSA (Ag), 10 or 100 nM fMLF, or 100 ng/ml DNP-BSA plus 100 nM fMLF, as indicated. The reported percentage secretion values are the average of duplicate samples from a single experiment with spontaneous secretion subtracted. Duplicates differed by less than 10%. Similar results were obtained in 6 experiments.
tigen- and fMLF-activated secretory responses of RBL2H3 and RBLFPR cells are summarized in Figure 1. RBL-2H3 cells do not express the FPR and fMLF addition elicits no secretion. In RBLFPR cells, 10 or 100 nM fMLF stimulates little or no release of preloaded 3Hserotonin. Adding 100 ng/ml DNP-BSA to IgE-primed RBLFPR cells activates secretion. Importantly, the simultaneous addition of fMLF and antigen produces a secretory response that exceeds the sum of the individual fMLF- and antigen-stimulated responses. Potentiated secretion was observed in 6 of 6 experiments, with fMLF providing an additional 10–40% secretion over the sum of the secretory responses elicited independently by fMLF and antigen. fMLF alone does not induce Ins(1,4,5)P3 production, but potentiates antigen-stimulated Ins(1,4,5)P3 production. Antigen stimulation of both RBL-2H3 (22) and RBLFPR (Fig. 2, circles) cells leads to an elevated level of Ins(1,4,5)P3 which is maintained for at least 10 min. Adding fMLF to RBLFPR cells does not lead to increased Ins(1,4,5)P3 production detectable by our assay (Fig. 2, triangles). There was also no increase in Ins(1,4,5)P3 levels measured 10 and 15 sec after fMLF addition (not shown). However, fMLF potentiates Ins(1,4,5)P3 production activated by antigen (Fig. 2, squares). Comparison of fMLF- and antigen-stimulated Ca2/ responses. In the presence of extracellular Ca2/ (Cao2/), 10 nM fMLF elicited Ca2/ responses from 29 of 32 cells and 100 nM fMLF produced Ca2/ responses in 63 of 64 cells. Formyl peptide-activated Ca2/ responses generally consisted of a high initial [Ca2/]i spike, fol-
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(SDÅ23). These results indicate that intracellular Ca2/ stores are released more rapidly by FPR ligation than by FceR1 cross-linking.
FIG. 2. Time course of Ins(1,4,5)P3 production by antigen- and fMLF-stimulated RBLFPR cells. Ins(1,4,5)P3 levels were measured as described in Methods. Agonists, 100 ng/ml DNP-BSA, 100 nM fMLF or both, were added at time 0. The reported data are averages of two separate experiments, each performed in duplicate on the same day. Similar results were obtained in 6 additional experiments.
lowed by a decrease to a lower, but still elevated, level (Fig. 3A,B). Approximately one-third of fMLF-stimulated cells instead showed an abrupt elevation in [Ca2/]i that lacked the initial spike and that was maintained at an elevated level (see Fig. 5B for an example). FceR1 cross-linking also led, after a lag, to a rapid increase in [Ca2/]i (Fig. 3C,D). There was day-to-day variability in the magnitudes of Ca2/ responses to fMLF and antigen (compare Figs. 3B and 6A, cells activated with 10 nM fMLF on different days; and Figs. 3C and 6B, cells activated with 100 ng/ml DNP-BSA on different days). However, activation with 100 nM fMLF consistently produced larger Ca2/ responses than did 10 nM fMLF and activation with 100 ng/ml DNP-BSA produced more sustained responses than either concentration of peptide (compare Figs. 3A and 3C). fMLF stimulates both Ca2/ stores release and Ca2/ influx. In the absence of Cao2/ , fMLF addition led to a rapid increase in [Ca2/]i that was not sustained (Fig. 3B, inset). fMLF at 10nM caused Ca2/ stores release in 5 of 9 cells; 100 nM fMLF stimulated Ca2/ responses in 9 of 9 cells. The lag times from fMLF addition to Ca2/ response were comparable in the presence and absence of Cao2/ , demonstrating that the initial Ca2/ response is the result of Ca2/ stores release. The sustained elevation in [Ca2/]i following fMLF stimulation requires Ca2/ influx, as removal of Cao2/ leads to an immediate drop in [Ca2/]i (Fig. 3B). As previously described (21,23), antigen-stimulated Ca2/ responses in RBL-2H3 cells also result from an initial release of intracellular Ca2/ stores, followed by the continued influx of Cao2/ (Fig. 3D and inset). However, the average lag time for 63 cells activated with 100 nM fMLF was 8 sec (SDÅ4 sec), whereas the average lag time for 33 antigen-stimulated cells was 36 sec
Antigen stimulation of RBLFPR cells results in a persistent depletion of intracellular Ca2/ stores while FPR activation does not. RBL-2H3 cells demonstrate capacitative Ca2/ entry, defined as the influx of Ca2/ following intracellular stores depletion (24,25), and there is evidence that depleted Ca2/ stores are responsible for a substantial portion of the Ca2/ influx response to FceR1 crosslinking (26,27). If this is also the case for the FPR, then the different Ca2/ influx responses of fMLF and antigen-treated cells could reflect different filling states of their stores. To test this prediction, RBLFPR cells were activated with antigen in the presence of Cao2/ . After 90 sec, Cao2/ was removed and 100 nM thapsigargin was added to assess the filling state of the Ca2/ stores. Thapsigargin inhibits SERCA, the sarcoplasmic and endoplasmic reticulum Ca2/-ATPase responsible for filling Ca2/ stores and leads to the depletion of intracellular stores through an uncharacterized leak pathway (28,29). In Figure 4A, Ca2/ responses from two antigen-stimulated RBLFPR cells are plotted. Thapsigargin addition produced no detectable increase in [Ca2/]i in these or 9 other antigen-stimulated cells. Similar experiments in RBL-2H3 cells revealed a persistent depletion of thapsigargin-sensitive Ca2/ stores in 24 of 36 cells DNP-BSA (100 ng/ml)stimulated cells. In the remaining RBL-2H3 cells, thapsigargin addition caused a small release of Ca2/ (example not shown). In contrast, intracellular Ca2/ stores were not completely empty in any of the fMLF-stimulated RBLFPR cells tested. Fig. 4B shows the Ca2/ responses of two representative cells (of 16) activated by fMLF in the presence of Cao2/ . In fMLF-treated cells, thapsigargin addition following the removal of Cao2/ produced an increase in [Ca2/]. In Fig. 4C, plots from two representative cells (of 16) show that thapsigargin also releases Ca2/ from unstimulated cells subjected to the same Cao2/ removal protocol. Integrals of [Ca2/]i over time were computed in all RBLFPR cells for the two-minute period following thapsigargin addition. The average integral in fMLF-stimulated cells was roughly half that calculated for unstimulated cells. These data suggest that intracellular Ca2/ stores are only partially depleted during continuous fMLF stimulation while antigen stimulation leads to a maintained depletion of intracellular stores. The FPR-activated Ca2/ response requires continuous occupation of the formyl peptide receptor while antigenstimulated Ca2/ responses do not require continued FceR1 cross-linking. The FPR desensitizes rapidly in both neutrophils and RBLFPR cells (30). Consistent with this, the fMLF-stimulated Ca2/ responses are quickly terminated following the addition of an FPR antago-
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FIG. 3. fMLF- and antigen-stimulated Ca2/ responses in individual RBLFPR cells. Changes in [Ca2/]i over time in fura-2-loaded RBLFPR cells were measured as described in Methods. The number of cells observed under each condition is indicated in the text. Each plot represents the Ca2/ response of a single representative cell. Agonist was added at the arrow and was then present continuously. Ca2/ o was present as indicated by the solid bars in A through D, and was not present in the experiments plotted in the insets of B and D. A. Typical Ca2/ response 2/ to fMLF stimulation showing a high initial spike followed by a lower, sustained elevation in [Ca ]i that gradually falls toward baseline over the course of the experiment. B. The sustained elevation in [Ca2/]i in fMLF-stimulated cells depends upon influx of Ca2/ o as its removal leads to an immediate decrease in [Ca2/]i to pre-stimulation levels. In the absence of Ca2/ o (inset), fMLF activates a rapid, transient elevation in [Ca2/]i . C. Ca2/ response to antigen stimulation showing an approximately 50 s lag time, a small initial spike and an elevation in [Ca2/]i that is sustained at a higher level for a longer period of time than is typical of Ca2/ responses to fMLF stimulation. D. The sustained 2/ elevation in [Ca2/]i in antigen-stimulated cells also depends upon influx of Ca2/ o . In the absence of Cao (inset), antigen stimulation causes a transient elevation in [Ca2/]i similar to that observed in fMLF-stimulated cells, but with a longer lag time.
nist, t-boc (tert-butoxy-phenylalanyl-leucyl-phenylalanyl-leucyl-phenylalanine), in cell populations (14) and in single cells (Fig. 5A,B). The fMLF-induced Ca2/ response in Figure 5A is typical; a high initial Ca2/ spike
that decreases rapidly to a lower, but still elevated, level. The addition of t-boc reduces this maintained elevation to baseline [Ca2/]i . T-boc also causes an immediate reduction in [Ca2/]i in cells that show the less-
FIG. 4. Antigen stimulation completely empties intracellular Ca2/ stores while fMLF stimulation does not. Panels A, B and C present results from three experiments, all performed on the same day. Each plot represents the Ca2/ response over time of a single RBLFPR cell. was present in the medium Agonist, either 100 ng/ml DNP-BSA (A) or 10 nM fMLF (B) was added as indicated by the solid arrow. Ca2/ o during the time indicated by the solid bar beneath each graph. 100 nM thapsigargin was added at the dashed arrow. The Ca2/ responses plotted in panel C are from cells that were not stimulated prior to removal of Ca2/ o and thapsigargin addition. 815
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FIG. 5. FPR-stimulated Ca2/ responses require continuous receptor occupation while antigen-stimulated Ca2/ responses do not. Panels A, B, and B inset represent Ca2/ responses over time for individual RBLFPR cells activated at the solid arrow by 100 nM fMLF. T-boc (10 mM) was added as indicated by the dashed arrow (A,B). The cell response plotted in B, inset is from an experiment done on the same day as that in B and shows the cellular response to fMLF alone. The axes of the inset are the same as those in panel B. Panels C, D, and D inset represent Ca2/ responses over time for individual RBL-2H3 cells activated with 100 ng/ml DNP-BSA at the solid arrow. In C, DNPBSA was removed by solution exchange for Ca2/-containing Hanks’ medium as indicated by the dashed bar. In D, 10 mM DNP-lysine was exchanged for DNP-BSA as indicated by the dashed arrow. The inset to panel D shows the Ca2/ response of a control cell from an experiment done on the same day as that in panel D. Two separate additions of DNP-BSA were made by solution exchange at the times indicated by the solid arrows. The axes of the inset are the same as for panel D.
typical FPR-activated response that lacks the initial Ca2/ spike (Figure 5B). Abrupt decreases in [Ca2/]i resulting from t-boc addition were observed in 19 of 22 fMLF-stimulated cells. Figure 5B (inset) shows that the fMLF-stimulated [Ca2/]i response decreases over time even without t-boc, but the decrease is much more gradual. In contrast, Ca2/ responses to 100 ng/ml DNP-BSA are not immediately diminished by the removal of antigen without (Fig. 5C) or with the addition of DNP-lysine (Fig. 5D). DNP-lysine is a monovalent hapten that displaces a portion of bound antigen, prevents the formation of new cross-links, and rapidly terminates secretion (data not shown; 31). Similar results were observed in 20 of 29 cells in which DNPBSA was removed and in 30 of 35 cells in which DNPlysine was added simultaneously with antigen removal. In the remainder of cells, [Ca2/]i decreased followed these treatments. Figure 5D (inset) shows that activated cells also show little response to subsequent applications of antigen. The simultaneous addition of fMLF and antigen produces a more sustained Ca2/ response than does either agonist alone. Stimulation with both antigen
and fMLF (Fig. 6C) produces responses that are initiated more rapidly than those induced by antigen alone (Fig. 6B) and are sustained for longer periods than those observed following either antigen (Fig. 6B) or fMLF alone (Fig. 6A). The average lag time following the simultaneous addition of antigen and fMLF was 11 sec (SDÅ9 sec; nÅ30), comparable to that measured in response to fMLF alone and much shorter than the average lag time following addition of antigen. Additionally, in approximately half of the cells, the initial Ca2/ spike was larger than that induced by fMLF or antigen alone. In 15 of 22 cells in 4 separate experiments, the subsequent sustained elevation was of similar magnitude to that observed in response to antigen alone, but was maintained at this elevated level through the point when the antigen-stimulated response typically started to decline. In the remaining cells, the elevated Ca2/ response stimulated by antigen plus fMLF was comparable to the response activated by antigen alone. DISCUSSION We have used RBLFPR cells to study signaling responses activated through the tyrosine kinase-coupled
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FIG. 6. fMLF and antigen added together produce more sustained Ca2/ responses than does either agonist alone. Plots represent changes in [Ca2/]i over time for individual RBLFPR cells from three separate experiments, all performed on the same day. Agonists, 10 nM fMLF (A), 100 ng/ml DNP-BSA (B), or 10 nM fMLF / 100 ng/ ml DNP-BSA (C), were added as indicated by the arrows. The cell plotted in B showed several oscillations in [Ca2/]i prior to the addition of antigen. Similar spontaneous oscillations in [Ca2/]i were observed in 14% of RBLFPR cells (22 of 159 cells).
IgE receptor, FceR1, and the G-protein-coupled chemotactic peptide receptor, FPR. Only antigen, and not fMLF, induces measurable Ins(1,4,5)P3 synthesis and secretion. However, fMLF potentiates antigen-induced Ins(1,4,5)P3 synthesis and secretion. Since the FceR1 and the FPR are both Ca2/-mobilizing receptors, we hypothesized that interactions between receptor-specific Ca2/ mobilization responses might contribute to this cooperation. This was tested by characterizing the
Ca2/ responses of cells stimulated with antigen and fMLF, alone and in combination. We first determined that the average lag time from fMLF addition to Ca2/ response is significantly shorter than the average lag time measured in antigen-stimulated cells. Short lag times to response were reported previously for RBL-2H3 cells transfected with the m1 muscarinic acetylcholine receptor (RBL-2H3(m1); 32) and may be characteristic of lag times to Ca2/ response induced by G protein-linked receptors. Receptor-activated Ca2/ stores release is typically mediated by Ins(1,4,5)P3 generated by phospholipase C (PLC), either the tyrosine kinase-activated PLCg isoforms or the G protein-coupled PLCb isoforms. In RBL-2H3 cells, FceR1 cross-linking causes the tyrosine phosphorylation and activation of both PLCg1 (8,9) and PLCg2.3 PLCb3 is the predominant PLCb isoform in RBL-2H3 cells and preliminary experiments suggested its activation by fMLF in RBLFPR cells (unpublished results, in collaboration with K. Caldwell and L. Gaudet). Thus, different kinetics of activation of PLC isoforms could explain the different lag times to Ca2/ stores release. Our inability to measure FPR-stimulated Ins(1,4,5)P3 synthesis by our radioreceptor assay complicates this interpretation. However, bulk assays may not reveal local Ins(1,4,5)P3 increases that presumably determine Ca2/ stores release. Further studies showed that the status of the intracellular stores is also different between fMLF- and antigen-stimulated cells. Antigen-stimulated cells contain little or no thapsigargin-releasable (stored) Ca2/, whereas fMLF-activated cells contain approximately one half as much thapsigargin-releasable Ca2/ as unstimulated cells. These results establish that antigen leads to a maintained depletion of intracellular Ca2/ stores, whereas Ca2/ stores remain partially full, or can refill, in the continued presence of fMLF. The persistent Ca2/ stores of fMLF-stimulated cells could represent a fMLF-insensitive but antigen-sensitive compartment. However, the initial Ca2/ response to fMLF is at least as robust as the response to antigen, suggesting that Ca2/ stores release is essentially complete in both cases. Thus we hypothesise that, once Ca2/ stores are released, antigen-stimulated cells more effectively maintain them in an empty state. This difference could occur because antigen-treated cells produce more Ins(1,4,5)P3 than do fMLF-stimulated RBLFPR cells, so that any Ca2/ pumped into the stores is immediately released through the open Ins(1,4,5)P3 receptor/ Ca2/ channel. Alternatively, SERCA, the Ca2/ pump responsible for Ca2/ re-uptake into stores, may be in3 Barker, S. A., Caldwell, K. K., Pfeiffer, J. R., Oliver, J. M., and Wilson, B. S. Wortmannin-sensitive phosphorylation, translocation and activation of PLCg1, but not PLCg2, in antigen-stimulated RBL2H3 mast cells. Submitted for publication.
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hibited by signals from the FceR1 (as proposed in 21) but not from the FPR. Comparison of fMLF- and antigen-stimulated Ca2/ responses additionally revealed that fMLF-stimulated Ca2/ influx is, in general, smaller than antigen-stimulated influx. This is the predicted result if capacitative Ca2/ entry, Ca2/ influx following intracellular stores depletion (33), is responsible for the bulk of fMLF as well as agonist-stimulated Ca2/ influx. We have demonstrated that the thapsigargin-sensitive intracellular Ca2/ stores are empty for many minutes in antigenstimulated RBL-2H3 cells, creating conditions that favor Ca2/ influx. In contrast, our observation that fMLF causes only partial depletion of thapsigargin-sensitive Ca2/ stores provides a reasonable explanation for the smaller magnitude of the fMLF-stimulated Ca2/ influx response. Studies of Ca2/ responses to receptor antagonists revealed another difference between the FceR1 and FPR signaling pathways. [Ca2/]i levels in fMLF-treated cells decrease almost immediately to baseline upon the addition of the FPR antagonist t-boc that prevents further occupation of the FPR. These results indicate that FPRmediated Ca2/ entry requires continuous signaling. In contrast, antagonizing the cross-linking of FceR1 with 10 mM DNP-lysine did not return the antigen-stimulated elevation in [Ca2/]i to basal levels during the 6 minutes of our assay, although excess DNP-lysine did immediately inhibit secretion. These results suggest that FceR1 cross-linking activates Ca2/ influx via a pathway that does not require continuous receptor crosslinking. The evidence (above) linking sustained antigen-induced Ca2/ influx to the empty state of the Ca2/ stores provides an obvious mechanism for continued Ca2/ influx without continued receptor signalling. Consistent with our hypothesis, Zhang and McCloskey (27) showed that DNP-lysine addition to antigen-stimulated cells causes a relatively slow reduction in the antigen-stimulated Ca2/ current, thought to be the capacitative Ca2/ entry current, measured using the perforated patch clamp method. On the other hand, our results appear to contradict work by Fewtrell and Sherman (34) showing an immediate inhibition of DNPBSA-induced 45Ca influx by the addition of DNP-lysine to quin2-buffered RBL-2H3 cells; by Maeyama et al. (35) showing that DNP-lysine causes a rapid drop in fluorescence of quin2-labelled cells; and by Weetall et al. (36) showing that adding monovalent hapten to cells activated with a bivalent antigen causes a rapid decrease in [Ca2/]i indicated by indo-1 fluorescence. The two fluorimetric studies were performed under conditions of suboptimal stimulation (less than 10% receptor occupancy in one case; a weak antigen in the other). Continued receptor crosslinking may be more important for maintaining stimulated Ca2/ influx under these conditions than under our conditions of maximal stimulation. Our time-resolved microscopic assays of
[Ca2/]i levels in single, adherent fura-2-labeled cells are not easily comparable with Fewtrell and Sherman’s radio-isotopic study of 45Ca2/ influx in suspensions of quin2-loaded cells. Importantly, simultaneous FPR and FceR1 activation produces Ca2/ influx responses that begin sooner and are more sustained than those in cells activated by antigen alone. These data support the hypothesis that antigen and fMLF activate receptor-specific Ca2/ mobilization responses in the RBLFPR transfectants. We speculate that the more rapid and sustained Ca2/ responses of co-stimulated cells may in turn potentiate Ca2/-dependent signaling responses that include the activation of PLC isoforms responsible for Ins(1,4,5)IP3 synthesis (37) and secretion. Rather than potentiating antigen-induced responses, the RBLFPR cell line generated independently by Ali et al. (13) responded to fMLF with Ca2/ mobilization, Ins(1,4,5)P3 production and secretion. When we measured fMLF-stimulated secretion in cytochalasin Btreated cells, the conditions used by Ali and colleagues, net secretion was still less than 6% (data not shown). Our RBLFPR transfectants expressed 1-3 1 105 FPR/ cell, while Ali et al. report approximately 9 1 104 FPR/ cell. We are unable to explain why fMLF addition to our RBLFPR cells prepares these cells for enhanced antigenmediated functional responses whereas it directly elicits responses in another line. We note, however, that occupying the G protein-coupled adenosine receptors of RBL-2H3 cells also causes no secretion but enhances antigen-induced secretion (38). In summary, we have demonstrated that mast cell responses to activating the tyrosine kinase-coupled FceR1 can be enhanced through the simultaneous activation of the G protein-coupled FPR. Our results implicate receptor-specific mechanisms that regulate Ca2/ release from stores, Ca2/ re-uptake into stores, and Ca2/ influx in the FPR-mediated potentiation of FceR1coupled responses. ACKNOWLEDGMENTS We thank A. Marina Martinez for technical assistance. The ratio imaging microscope and flow cytometer used in this study are shared instruments maintained in the Cytometry and Microscopy shared facilities of the UNM Cancer Research and Treatment Center. This work was supported in part by NIH Grants GM50562 (BSW), HL56384 and GM49814 (JMO), AI19032 and RR01315 (LAS), and AI35997 (R. Posner, Northern Arizona University, for DEL).
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