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© The Rockefeller University Press,
0021-9525/1997//1209 $5.00
The Journal of Cell Biology, Volume 139, Number 5,
, 1997 1209-1217
Article |
Role for a Glycan Phosphoinositol Anchor in Fc
Receptor Synergy
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
While many cell types express receptors for the Fc domain of IgG (Fc
R), only primate polymorphonuclear neutrophils (PMN) express an FcR linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked FcR (FcRIIIB) cooperates with the transmembrane FcR (FcRIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fc receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fc receptors. Jurkat T cells were stably transfected with cDNA encoding FcRIIA and/or FcRIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca2+]i) to levels not reached by stimulation of either FcRIIA or FcRIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca2+. Cocrosslinking FcRIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca2+]i rise, as did crosslinking CD59 with FcRIIA on PMN, suggesting that interactions between the extracellular domains of the two Fc receptors are not required for synergy. Replacement of the GPI anchor of FcRIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the FcRIIA cytoplasmic tail abolished synergy. While the ITAM of FcRIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked FcRIIA was diminished when cocrosslinked with FcRIIIB. These data demonstrate that FcRIIA association with GPI-linked proteins facilitates FcR signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored FcR of human PMN.
Abbreviations used in this paper: [Ca2+]i, intracytoplasmic Ca2+ concentration; GPI, glycan phosphoinositol; ITAM, immunoreceptor tyrosine-based activation motif; PLC, phospholipase C; PMN, polymorphonuclear neutrophils.
THE binding of immune complexes by polymorphonuclear neutrophils (PMN)1 receptors for the Fc domain of IgG (Fc receptors) induces essential host defense and inflammatory responses such as adhesion, phagocytosis of antibody-coated microorganisms, degranulation, and the respiratory burst (33, 38). PMN activation by immune complexes is important in the pathology of serum sickness, the Arthus reaction, acute glomerulonephritis, rheumatoid arthritis, and other idiopathic inflammatory disorders as well as in host defense against infection. The Fc receptors are a family of hematopoietic cell receptors that share structurally related ligand-binding domains for the Fc portion of immunoglobulins, but which differ in their transmembrane and intracellular domains (for review see 16, 33). These varying cytoplasmic tails presumably give rise to distinct intracellular signals to provide diversity of function.
Primate PMN are unique, because in addition to the transmembrane FcR, FcRIIA, they express the only known eukaryotic nontransmembrane FcR, the glycan phosphoinositol (GPI)-linked FcRIIIB. Ligand binding by transmembrane FcRIIA initiates a tyrosine kinase cascade dependent upon the cytoplasmic tail of this receptor, which contains one copy of an immunoreceptor tyrosine-based activation motif (ITAM) (11, 27), a substrate for phosphorylation by members of the src tyrosine kinase family. The phosphorylated ITAM of FcRIIA can bind to and activate syk tyrosine kinase, which subsequently activates a number of effector pathways (16). In contrast, little is known about the signaling mechanisms of FcRIIIB, the most abundant PMN Fc receptor. Some studies have suggested an inability of FcRIIIB to transduce signals independently. These studies, taken together with this receptor's lack of a cytoplasmic domain, have led to the concept that FcRIIIB is primarily an Fc-binding molecule that aids in immune complex presentation to FcRIIA (1, 13). However, evidence now suggests that FcRIIIB is able to mediate intracellular signaling events, such as the activation of the src family member hck and induction of intracellular calcium fluxes (14, 19, 39, 49). Moreover, FcRIIIB cooperates with FcRIIA in PMN activation. When ligated together, as would occur when PMN bind immune complexes, FcRIIA and FcRIIIB synergize to activate the respiratory burst and to increase intracytoplasmic calcium (44, 47).
Despite the importance of the cooperation between FcRIIA and FcRIIIB for PMN function, its mechanism is not understood. As primary, terminally differentiated, nondividing cells, PMN are exceedingly resistant to genetic and cell biological manipulations which have aided characterization of receptor function in other systems. We developed a model system to dissect the functional roles and domains of FcRIIA and FcRIIIB in Jurkat T cells, which lack endogenous Fc receptors but are fully competent for tyrosine kinase signaling. In transfected Jurkat T cells, the PMN Fc receptors synergized to induce a rise in intracytoplasmic Ca2+ concentration ([Ca2+]i) that was greater and more prolonged than from ligation of either receptor individually. This was identical to the effect of coligation of these receptors in PMN (44). The synergistic calcium rise required the influx of extracellular calcium and depended upon the GPI anchor of FcRIIIB, since a mutant in which the GPI anchor was replaced by the transmembrane domain of CD7 was unable to synergize with FcRIIA. Moreover, crosslinking other GPI-linked proteins on Jurkat T cells with FcRIIA also led to a synergistic increase in [Ca2+]i. The increase in [Ca2+]i also required the tyrosines of the FcRIIA ITAM. Surprisingly, we found that phosphorylation of the ITAM was diminished under conditions that led to the synergistic calcium flux and that the kinetics of PLC-1 phosphorylation was not altered by the replacement of the GPI anchor of FcRIIIB with the transmembrane domain of CD7. Thus, synergy between FcR requires the GPI anchor of FcRIIIB, but not for an increase in FcRIIA-dependent tyrosine kinase signaling. We hypothesize instead that the role for the GPI anchor of FcRIIIB is to sequester FcRIIA into specialized membrane domains where signal transduction by the ITAM is altered. This could provide a further level of modulation of activation signals from immune complex binding and may explain many of the functions of the unusual GPI-linked FcR of primate PMN. Moreover, this could be a general mechanism by which GPI anchored proteins affect signal transduction from transmembrane receptors.
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RII; 26), 3G8 (anti-CD16, anti-FcRIII; 9), IH4 (anti-CD55, anti-DAF; 8), MEM-43 (anti-CD59, anti-Protectin), 10G10 (anti-CD59; kindly provided by Dr. Marilyn Telen, Duke University, Durham, NC), MEM-102 (anti-CD48; Harlan Bioproducts, Indianapolis, IN), II1A5 (anti-FcRII; kindly provided by Dr. Jurgen Frey, Universität Bielefeld), and mouse IgG2b isotype control (Sigma Chemical Co., St. Louis, MO). To crosslink primary antibodies, goat F(ab')2 fragments specific for mouse F(ab') or goat F(ab')2 fragments specific for mouse IgG1 or mouse IgG2b (Sigma Chemical Co) were used. Antibody fragments of IV.3, 3G8, or 10G10 were made by standard methods or purchased (Medarex, Annandale, NJ). For FACS® analysis, bound mAbs were detected using FITC-conjugated goat F(ab')2 fragments specific for mouse F(ab') (Sigma Chemical Co.). Anti-phospholipase C -1 (PLC-1) was purchased from Upstate Biotechnology (Lake Placid, NY) or Transduction Laboratories (Lexington, KY). Anti-phosphotyrosine (Upstate Biotechnology) was detected with HRP-conjugated goat antibodies specific for mouse IgG2b (Caltag Laboratories, So. San Francisco, CA).
FcRIIA and FcRIIIB Expression Constructs and Transfection into Jurkat T Cells
The oligos 5'-CCTGAATTCCTCCGGATATCTTTGGTGAC-3' and 5'-AGAGGATCCGCTGCCACTGCTCTTATTAC-3' were used to amplify the human FcRIIIB (CD16) cDNA by RT-PCR of human PMN mRNA (24). The resulting product was digested with EcoRI and HindIII and ligated into similarly digested vectors, pBluescript II SK+/–, pRcCMV, and pCEP4 (Invitrogen, San Diego CA). The intactness of the cDNA was verified by DNA sequencing (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit; Perkin Elmer, Foster City, CA). The FcRIIIB/CD7 construct was made by ligating a HindIII/MluI fragment of the CD16/CD7/syk construct (kindly provided by Dr. Brian Seed, Harvard Medical School, Boston, MA; (20) and a MluI/NotI adaptor (annealed oligonuclotides 5'-CGCGTTAATAGATCGATGC-3' and 5'-GGCCGCATCGATCTATTAA-3' [stop codons underlined]) into HindIII/NotI-digested pRcCMV. This construct encodes the FcRIIIB extracellular domain joined with a CD7 transmembrane domain. The cDNA was verified by DNA sequencing. The cDNAs encoding FcRIIA and FcRIIA with both ITAM tyrosines in the cytoplasmic tail mutated to phenylalanine were prepared as described (7, 27) and cloned into pRcCMV and pCEP4.
The resulting plasmids were introduced into clones of Jurkat T cells by electroporation. Cells (107) in 400 µl HEBS (25 mM Hepes, pH 7.05, 140 mM NaCl, 750 mM Na2HPO4) and plasmid (30 µg in 100 µl HEBS) were added to a 0.4-mm-gap width cuvette and electroporated at 1,000 µF, 330 v (Electroporator II; Invitrogen). After electroporation, cells were cultured for 36 to 48 h in normal propagation media. Cells were transferred to selective media (propagation media plus 1.4 mg/ml geneticin/G418 [Gibco Laboratories] and/or 600 µg/ml hygromycin B [Boehringer Mannheim, Indianapolis, IN]) and cultured for 2 to 3 wk. High protein-expressing cell populations were selected by fluorescence-activated cell sorting using mAb IV.3 or mAb 3G8. Briefly, cells (106) were resuspended in 50 µl PBS/5% FCS with 1 µg antibody and incubated on ice for 45 min. Cells were washed and then incubated an additional 30 min with F(ab')2 fragments of goat anti–mouse IgG-FITC (Sigma Chemical Co.). Cells were analyzed on a flow cytometer (Coulter Electronics, Hialeah, FL) or sorted using a fluorescence-activated cell sorter (Becton Dickenson, Palo Alto, CA). All cDNAs were introduced into at least two different Jurkat clones and all experiments yielded equivalent results in all clones.
[Ca2+]i Measurements
Jurkat transfectants were loaded with 3 µM Fura 2-AM (Molecular Probes, Eugene, OR) in RPMI 1640/10% FCS for 40 min in the dark at 37°C. PMN were loaded with 5 µM Fura-2 AM in Hanks Balanced Salt Solution (HBSS; Gibco Laboratories), 1 mM MgCl2, 1 mM CaCl2, and 1% vol/vol human serum albumin (HBSS++) for 25 min in the dark at 37°C. Cells (6 x 106) were washed once, resuspended in RPMI 1640/10% FCS or HBSS++ containing the appropriate mAbs, and incubated 30 min on ice. Cells were washed three times and resuspended in 2 ml calcium buffer (25 mM Hepes, pH 7.4, 125 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mg/ml D-glucose, 1 mg/ml BSA, 1 mM CaCl2, 0.5 mM MgCl2). Changes in fluorescence, using excitation wavelengths of 340 and 380 nm and the emission wavelength of 510 nm, were measured with a spectrofluorimeter (F-2000; Hitachi Instruments, Danbury, CT) equipped with a thermostatic cuvette holder maintained at 37°C. Cells were warmed to 37°C for 5 min and added to the cuvette; then 10 µl mouse F(ab') specific goat F(ab')2 fragments were added. Intracellular calcium concentrations were calculated as described (36).
Receptor Crosslinking, Immunoprecipitation, and Western Blots
Cells (1–2 x 107) were incubated in RPMI 1640/10% FCS containing the mAb IV.3 (15 µg/ml) or the mAbs IV.3 and 3G8 (15 µg/ml each) for 30 min on ice. Cells were washed three times, resuspended in 0.5 ml RPMI 1690 with 10% FCS, and then warmed to 37°C for 10 min. Crosslinking mouse F(ab') specific goat F(ab')2 fragments (20 µl) were added for various times. Cells were lysed with an equal volume of 2x lysis buffer (100 mM Tris-HCl, pH 7.4, 2% NP-40, 0.5% deoxycholate, 300 mM NaCl, 2 mM EDTA, 2 mM NaF, 250 µM Na3VO4, 1 mM Na2MoO4, 1 mM Na2H2P2O7, 10 ng/ml calyculin, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 15 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride) at 4°C. Samples were centrifuged 5 min at 14,000 g. Resulting supernatants were rotated overnight with 75 µl of a 1:1 slurry of Gamma Bind plus Sepharose (Pharmacia Biotech, Piscataway, NJ). For PLC -1 immunoprecipitations, 10 µl of polyclonal antibodies were added to each sample. Beads were washed extensively and resuspended in reducing cocktail (50% vol/vol glycerol, 250 mM Tris-HCl, pH 6.8, 5% wt/vol SDS, 570 mM 2-mercaptoethanol, bromphenol blue). Samples were boiled for 5 min and then subjected to SDS-PAGE and electrotransfer onto Immobilon-P (Milipore, Bedford, MA) membranes. Blots were probed with anti-phosphotyrosine, anti-FcRII (II1A5), or anti-PLC -1. Bound antibodies were detected with HRP-conjugated mouse specific goat antibodies. Antibody reactive protein was visualized using enhanced chemiluminescence (ECL; Amersham Intl., Arlington Heights, IL). Tyrosine phosphorylation of FcRIIA or PLC-1 under different conditions was compared by normalizing the amount of phosphorylation, determined by densitometry of the anti-phosphotyrosine blots, to the amount of protein precipitated, as determined by reprobing the same blots with antibodies to the relevant protein. Multiple experiments were combined for analysis by comparing all experimental conditions to the ratio obtained for wild-type receptors in the same experiment.
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RIIA and FcRIIIB Results in a Synergistic [Ca2+]i Rise receptors, were stably transfected with the cDNAs encoding FcRIIA and FcRIIIB (J2/3; Fig. 1, top). In addition, stable transfectants were made which express FcRIIA along with a chimeric receptor consisting of the extracellular portion of FcRIIIB coupled to the transmembrane domain of CD7 (J2/3-CD7; Fig. 1, middle). A third transfectant was made that expresses FcRIIIB and an FcRIIA receptor in which the tyrosines (Y282 and Y298) of the ITAM have been mutated to phenylalanines (27; J2Y
F/3, Fig. 1, bottom). FACS® analysis indicated that each mutant receptor is expressed at a level at least comparable to that of the corresponding wild-type receptor (Fig. 1).
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RIIA and FcRIIIB in PMN cooperate to generate a calcium flux that is greater than the sum of the calcium fluxes generated by crosslinking either receptor individually (44). In addition, it has been shown that Jurkat cells that were stably transfected with FcRIIA are able to flux calcium after receptor ligation (15), suggesting the signaling machinery used by Fc receptors is functional in these cells. Therefore we compared [Ca2+]i in J2/3 cells after crosslinking FcRIIA and FcRIIIB individually or after crosslinking both receptors together, using a F(ab')2 crosslinking antibody. Crosslinking FcRIIA resulted in a significant, short lived rise in [Ca2+]i (Fig. 2, top). In contrast, crosslinking FcRIIIB alone resulted in a slow rise in [Ca2+]i with a magnitude lower than for FcRIIA (Fig. 2, top). When both FcR were crosslinked together, there was an increase in the maximum [Ca2+]i rise and a prolongation of the increase (Fig. 2, top). Synergy did not require the Fc fragment of either anti-FcRII or -FcRIII mAb, since similar results were obtained by using the F(ab) fragment of the mAb IV.3 and the F(ab')2 fragment of the mAb 3G8 (data not shown). Neither the addition of antibodies specific for Fc receptors alone nor the crosslinking goat F(ab')2 fragments alone induced a rise in [Ca2+]i (Fig. 2, top and data not shown). In PMN, crosslinking FcRIIIB is able to mediate a rise in intracellular calcium by itself. This difference between the Jurkat transfectants and PMN is most likely due to the level of FcRIIIB expression. In PMN, FcRIIIB is extremely abundant on the cell surface (12, 13). Phosphatidylinositol-specific phospholipase C (PLC) treatment of PMN, an enzyme that cleaves GPI-linked proteins and that removes 80% of the FcRIIIB from the cell surface, abolishes the rise in [Ca2+]i after FcRIIIB crosslinking (35, and data not shown). Nonetheless, the expression level of FcRIIIB in the transfected Jurkat cells was sufficient to produce a synergistic rise in [Ca2+]i.
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RIIA and FcRIIIB together or whether the augmentation in [Ca2+]i could be achieved by simultaneously crosslinking each Fc receptor individually, isotype-specific secondary crosslinking antibodies were used (Fig. 2, middle). FcRIIA was crosslinked with IV.3, an IgG2b mAb, and goat F(ab')2 fragments specific for mouse IgG2b and FcRIIIB was crosslinked with 3G8, an IgG1 mAb, and goat F(ab')2 fragments specific for mouse IgG1. When both Fc receptors were individually and simultaneously crosslinked, no synergistic rise in [Ca2+]i was found (Fig. 2, middle), paralleling results found in PMN (44). In fact, the resulting rise in [Ca2+]i appeared to be additive of the rises obtained by crosslinking both Fc receptors individually (Fig. 2, middle).
To show specificity of the synergy, cells were incubated with anti-FcRII mAb IV.3 and the mAb IB4, specific for β2 (CD18) integrins (Fig. 2, bottom). The β2 integrin LFA-1 is expressed at a level similar to the transfected FcRIIIB (data not shown). Moreover, LFA-1 synergizes with the ITAM-containing T cell antigen receptor to prolong an increase in [Ca2+]i (45). However, there was no synergy between LFA-1 and FcRIIA for [Ca2+]i rise. This result indicates that signaling through FcRIIA is augmented when cocrosslinked to FcRIIIB, as would occur under physiological conditions where both Fc receptors are ligated by immune complexes.
The GPI Anchor Is Necessary and Sufficient for the Contribution of FcRIIIB to Synergy
Primate PMN are the only cells that express a GPI-anchored Fc receptor (32). To determine whether the GPI anchor was necessary for FcRIIIB contribution to the synergistic increase in [Ca2+]i, stable transfectants were made expressing FcRIIA and a chimeric FcRIIIB with the GPI anchor replaced by the transmembrane domain of CD7 (J2/3-CD7; Fig. 1, middle). When FcRIIA and FcRIIIB/ CD7 were crosslinked together in these cells, the [Ca2+]i rise was similar to the rise generated when FcRIIA was crosslinked alone without any synergy from FcRIIIB (Fig. 3, middle). The inability of the chimeric FcRIIIB/ CD7 molecule to contribute to the synergistic [Ca2+]i rise was not due to inadequate expression of this protein, since the FcRIIIB/CD7 molecule was expressed at a greater level than the wild-type FcRIIIB (Fig. 1, top and middle). This experiment demonstrates that the GPI anchor is necessary for the synergistic [Ca2+]i rise.
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RIIIB rise were required for the synergistic [Ca2+]i rise, other GPI-linked proteins expressed by Jurkat cells were cocrosslinked with FcRIIA. CD48 (not shown) and CD59 (protectin) (Fig. 1) are both expressed by parental Jurkat cells and by each of the transfectants at levels equal to or greater than FcRIIIB. When these GPI-linked proteins, CD59 (Fig. 3, top) and CD48 (not shown), were cocrosslinked with FcRIIA, a synergistic rise in [Ca2+]i also occurred in Jurkat cells transfected with FcRIIA alone (data not shown), in J2/3 cells (Fig. 3, top), and in J2/3-CD7 cells (Fig. 3, middle). In all of these cells, ligation of CD59 alone produced a [Ca2+]i rise similar to that elicited by crosslinking FcRIIIB alone (Fig. 3, top, and data not shown).
These experiments demonstrate that the GPI anchor of FcRIIIB is required for FcR cooperation but that other extracellular domains will substitute for FcRIIIB when cocrosslinked with FcRIIA. This is strong evidence against the hypothesis that interaction between the extracellular domains of the receptors is required for synergy, as has been proposed for FcRIIA and FcRIIIB interaction with the β2 integrin CR3 (for review see 30). Moreover, since these cells do not express CR3, this experiment shows that FcR synergy can occur without this PMN integrin.
Synergy in PMN between FcRIIA and FcRIIIB was found for the rise in [Ca2+]i (data not shown and 44), the respiratory burst (data not shown and 44, 47, 49), and degranulation (data not shown). To determine if the synergistic rise in [Ca2+]i could also be obtained in PMN with other GPI-anchored proteins, FcRIIA and CD59 were cocrosslinked and a prolongation in the rise [Ca2+]i was found (Fig. 3, bottom). The synergistic rise in [Ca2+]i with FcRIIA and CD59 was not as pronounced as with FcRIIIB and FcRIIA. No significant synergy between FcRIIA and CD59 was found in assays of degranulation or respiratory burst. This was true for CD48, CD55, and CD66b, other GPI-linked proteins on PMN, as well (data not shown). This is most likely due to a lower level of expression of these GPI-anchored proteins on PMN as compared to FcRIIIB (CD59 has 
13% of the expression of FcRIIIB, CD48 has 1%, CD55 has 6%, and CD66b has 9%; data not shown). This is consistent with the lack of a synergistic rise in [Ca2+]i obtained in PMN treated with phosphatidylinositol-specific PLC, which reduces the amount of FcRIIIB on the cell surface by 80% (35 and data not shown).
The ITAM of FcRIIA Is Required for Calcium Flux
Activation of tyrosine phosphorylation and propagation of a tyrosine kinase cascade by receptor associated ITAMs is thought to be essential for Fc receptor signaling (16, 43). To determine whether this cascade had a role in Fc receptor synergy, Jurkat cells were transfected with FcRIIIB and a mutant FcRIIA in which tyrosines Y282 and Y298 contained within the ITAM were mutated to phenylalanines (J2Y F/3; Fig. 1, bottom). It has been shown in model systems that these tyrosines are required for [Ca2+]i flux when FcRIIA is ligated alone (27, 28). No synergistic [Ca2+]i flux occurred in J2Y F/3 cells when FcRIIA was ligated either alone or together with FcRIIIB, although these cells were fully competent to increase [Ca2+]i in response to antigen receptor ligation (Fig. 4). Therefore, these tyrosines in the cytoplasmic tail of FcRIIA are required for the synergistic [Ca2+]i rise. Thus both the GPI anchor of FcRIIIB and the ITAM motif of FcRIIA are required for synergy in calcium signaling.
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RIIARIIA. When FcRIIA was immunoprecipitated from J2/3 cells after crosslinking FcRIIA alone, its tyrosine phosphorylation peaked at 1 min and was diminished by 5 min (Fig. 5 A, top). Surprisingly, crosslinking FcRIIA and FcRIIIB together did not enhance tyrosine phosphorylation of FcRIIA as expected but actually diminished detection of the tyrosine phosphorylation of FcRIIA (Fig. 5 A, top). Averages from three experiments after normalization for the amount of receptor immunoprecipitated showed that FcRIIA was phosphorylated 10-fold less under synergistic conditions as compared to ligation of FcRIIA alone. We also analyzed the tyrosine phosphorylation of FcRIIA in J2/3-CD7 cells. Ligation of FcRIIA without FcRIIIB induced tyrosine phosphorylation of itself to a similar extent and with similar kinetics as in cells expressing both wild-type Fc receptors (Fig. 5 B, bottom). In striking contrast to the results obtained in J2/3 cells by crosslinking both wild-type Fc receptors, cocrosslinking FcRIIA and FcRIIIB/CD7 did not significantly diminish the extent or alter the kinetics of FcRIIA phosphorylation (Fig. 5 A, bottom). To determine if the marked diminution of FcRIIA tyrosine phosphorylation also occurred when it was crosslinked with other GPI-anchored proteins, FcRIIA was crosslinked with CD48 or CD59 (Fig. 5 B). Cocrosslinking any GPI-anchored protein with FcRIIA markedly diminished its tyrosine phosphorylation. In addition, we analyzed the extent of tyrosine phosphorylation of FcRIIA in PMN after ligating FcRIIA, individually or together with FcRIIIB, by using the F(ab) fragment of mAb IV.3 and the F(ab')2 of mAb 3G8. Crosslinking both Fc receptors resulted in 2–3-fold diminished tyrosine phosphorylation of FcRIIA when compared to ligating FcRIIA alone (data not shown).
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11 is one of several PLC isoforms that converts phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-triphosphate leading to the release of intracellular stores of calcium. In several cell types, crosslinking FcRIIA induces the tyrosine phosphorylation of PLC-1, which leads to its activation (25, 42). To determine whether prolonged activation of PLC-1 could account for the synergistic increase in [Ca2+]i, its tyrosine phosphorylation was examined. In agreement with previous studies, crosslinking FcRIIA in the transfected Jurkat cells resulted in tyrosine phosphorylation of PLC-1 that was visible by 1 min (data not shown, and 42). Crosslinking FcRIIIB and FcRIIA in J2/3 cells resulted in tyrosine phosphorylation of PLC-1, which was not different from cocrosslinking FcRIIA and the chimeric FcRIIIB/CD7 in J2/3-CD7 cells (Fig. 6). Thus, Fc receptor synergy is independent of the tyrosine phosphorylation of PLC-1.
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RIIA and CD59 was abolished by the addition of EGTA (Fig. 7 A, middle). As a control, the changes in intracellular calcium were measured after the T-cell receptor complex (TCR/CD3) was crosslinked with the mAb C305 (Fig. 7 A, right). Previous studies have shown that the rise in intracellular calcium after TCR crosslinking results from an initial rise derived from intracellular stores followed by a secondary sustained calcium influx through plasma membrane channels that can be abolished by the addition of EGTA (41). The addition of EGTA to Jurkat cells treated only with crosslinking secondary antibody does cause a small decrease in the amount of intracellular calcium, but this small depletion does not account for the large loss in the synergistic calcium influx from extracellular stores, as previously shown in PMN (37; Fig. 7, A and C, left). The changes in intracellular calcium also were measured when EGTA was added immediately before Fc receptor crosslinking (Fig. 7 B, left). Crosslinking led to an initial rise in [Ca2+]i, but the synergistic [Ca2+]i rise was substantially diminished after cocrosslinking FcRIIA with FcRIIIB or CD59 (Fig. 7 B, middle and right). The magnitude of the [Ca2+]i rise also was diminished in the presence of EGTA, again demonstrating that a significant contribution to the [Ca2+]i rise is due to the influx of extracellular calcium (Fig. 7 B). The slow rise in [Ca2+]i after crosslinking either FcRIIIB or CD59 alone was abolished in the presence of EGTA (Fig. 7 C, right, and data not shown). EGTA treated cells do not produce a flux in [Ca2+]i after the addition of crosslinking secondary antibodies alone (Fig. 7 C, left).
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200-fold in these domains, and there is evidence for concentration of Src kinases, G protein–coupled receptors, and heterotrimeric G proteins in these membrane domains as well. This has led some investigators to hypothesize that these domains function in signal transduction, and indeed crosslinking of GPI-linked proteins leads to rapid induction of tyrosine phosphorylation (43). On the other hand, some src family kinases sequestered in these domains have low specific activity, suggesting that these glycolipid domains function not in signaling but as a reservoir of signaling molecules that can be recruited to other parts of the membrane (34).
The second model for signal transduction by GPI-linked proteins involves their physical association with transmembrane proteins. For example, FcRIIIB has been shown to associate with the integrin Mac-1, as has the GPI-linked urokinase receptor (uPAR), which also can associate with another integrin, 
vβ3 (21, 46). These physical associations have functional consequences, for example, induction of IgG-mediated phagocytosis in transfected 3T3 cells (21), or cellular adhesion to vitronectin (46). Thus, it is possible that GPI-linked proteins transduce information to the cytoplasm through physical interaction with transmembrane proteins.
The interaction of FcRIIA and FcRIIIB on human PMN presents an opportunity to test these hypotheses concerning signal transduction by GPI-linked proteins. When immune complexes bind to PMN, FcRIIA and FcRIIIB are brought into proximity. While synergy between the receptors in signal transduction in response to immune complexes has been shown, interpretation is complicated by the interaction of both receptors with other membrane proteins such as Mac-1 (40, 48), and by the inability to use molecular genetic techniques to probe receptor function in these primary cells. For these reasons, we have developed a model system to understand Fc receptor synergy on PMN. In Jurkat cells without Mac-1, FcRIIA and FcRIIIB can synergize to increase [Ca2+]i, demonstrating that extracellular domain association with Mac-1 is not required for at least this aspect of synergy. Indeed, since coligation of two other GPI-linked proteins, otherwise structurally unrelated to FcRIIIB, also can synergize with FcRIIA to increase [Ca2+]i, it is unlikely that extracellular domain interactions other than with multivalent ligands are required to induce synergy between the transmembrane and GPI-linked Fc receptors. The synergistic increase in [Ca2+]i may be important in numerous PMN functions, including degranulation (3, 23), actin polymerization (2), and phagocytosis (17, 18).
Our data support the hypothesis that association of FcRIIA with glycolipid domains enriched in GPI-linked proteins fundamentally alters subsequent signaling. Cocrosslinking FcRIIA with any of the GPI-linked proteins induced the synergistic increase in [Ca2+]i and, surprisingly, decreased the extent of FcRIIA tyrosine phosphorylation. When FcRIIIB was expressed with a transmembrane domain, its synergy with FcRIIA was abolished, as was its effect on FcRIIA tyrosine phosphorylation. These data support the hypothesis that the membrane environment of FcRIIA is altered by crosslinking it with GPI- anchored proteins. This altered environment modulates the FcRIIA-generated signal in fundamental ways. We initially expected that the synergistic [Ca2+]i rise would be associated with increased phosphorylation of the ITAM of FcRIIA, because src family kinases, which phosphorylate ITAMs, have been found to be concentrated in these domains. However, our finding of decreased tyrosine phosphorylation is consistent with the report that CD45, the major transmembrane tyrosine phosphatase present on lymphocytes, is excluded from glycolipid-enriched membrane domains, resulting in lower specific activity of the lymphocyte src kinases in these domains (34). We propose that FcRIIA has diminished tyrosine phosphorylation after cocrosslinking with FcRIIIB, because ligation with GPI-linked proteins causes FcRIIA to be brought into membrane domains with less-active src kinases. It is also possible that an additional signaling pathway is used to mediate synergistic calcium signaling, since the prolonged rise in intracellular calcium is not due to the prolonged tyrosine phosphorylation of PLC-1. Calcium mobilization after crosslinking Fc
RI activates a sphingosine kinase that produces sphingosine-1-phosphate as a second messenger for intracellular calcium mobilization (6). Alternatively, localization of the Fc receptors within specialized membrane domains may activate the synergistic influx of extracellular calcium. Indeed, a plasma membrane calcium pump has been identified in caveolae (10).
Our data further extend the observations made with several receptors, including Fc receptors, that there may be interaction on the cell surface between receptors recognizing the same ligand. For example, T cells express two distinct receptors that interact with MHC class I molecules, one that mediates the positive signal, the T cell receptor, and a second receptor, NKB1, that mediates an inhibitory signal (22, 31). It has been observed in phagocytic cells that the Fc receptor, FcRIIB, inhibits phagocytosis mediated by FcRIIA. Decreased tyrosine phosphorylation induced by FcRIIB after interaction with IgG ligand may be responsible for this inhibition of FcRIIA-mediated phagocytosis (Hunter, S., and A.D. Schreiber, unpublished results).
In summary, transfection of human PMN Fc receptors into the Jurkat cell line has allowed for the further dissection of the mechanism by which these receptors cooperate in immune complex–induced PMN activation. We have defined two essential structural components of the synergistic signal, the GPI-anchor of FcRIIIB and the ITAM of FcRIIA. Moreover, we have shown that synergy can occur in the absence of the phagocyte integrin Mac-1, previously postulated to be an essential component for synergy. In PMN, 10,000 to 20,000 FcRIIA molecules are expressed on the cell surface together with 10 to 20 times more FcRIIIB (12, 13). Thus it is highly likely that whenever FcRIIA is ligated by an immune complex, it is in association with several GPI-linked FcRIIIB and that the modulated signal which occurs because of association with GPI domains is the major mechanism of immune complex-mediated PMN activation.
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Acknowledgments |
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cDNA, Dr. Andrew Chan for the C305 mAb, Dr. Jurgen Frey for the II1A5 mAb, and Drs. Doug Lublin and Scott Blystone (Washington University, St. Louis, MO) for helpful discussions. This work was supported by grants from the National Institutes of Health and the Arthritis Foundation to E.J. Brown. J.M. Green is supported as a Lucille P. Markey Pathway postdoctoral fellow.
Submitted: 29 April 1997
Revised: 13 August 1997
Address all correspondence to Dr. Eric J. Brown, Division of Infectious Diseases, Washington University School of Medicine, 660 S. Euclid Ave., Box 8051, St. Louis, MO 63110. Tel.: (314)362-2125. Fax: (314) 362-9230. E-mail: ebrown{at}id.wustl.edu
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