Role for a Glycan Phosphoinositol Anchor in Fcgamma Receptor Synergy

Jennifer M. Green,* Alan D. Schreiber,Dagger and Eric J. Brown*

* Division of Infectious Diseases, Washington University, School of Medicine, St. Louis, Missouri 63110; and Dagger  Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Abstract
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
Abbreviations used in this paper
References


Abstract

While many cell types express receptors for the Fc domain of IgG (Fcgamma R), only primate polymorphonuclear neutrophils (PMN) express an Fcgamma R linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked Fcgamma R (Fcgamma RIIIB) cooperates with the transmembrane Fcgamma R (Fcgamma RIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fcgamma receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fcgamma receptors. Jurkat T cells were stably transfected with cDNA encoding Fcgamma RIIA and/or Fcgamma RIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca2+]i) to levels not reached by stimulation of either Fcgamma RIIA or Fcgamma RIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca2+. Cocrosslinking Fcgamma RIIA 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 Fcgamma RIIA on PMN, suggesting that interactions between the extracellular domains of the two Fcgamma receptors are not required for synergy. Replacement of the GPI anchor of Fcgamma RIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the Fcgamma RIIA cytoplasmic tail abolished synergy. While the ITAM of Fcgamma RIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked Fcgamma RIIA was diminished when cocrosslinked with Fcgamma RIIIB. These data demonstrate that Fcgamma RIIA association with GPI-linked proteins facilitates Fcgamma R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fcgamma R of human PMN.


THE binding of immune complexes by polymorphonuclear neutrophils (PMN)1 receptors for the Fc domain of IgG (Fcgamma 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 Fcgamma 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 Fcgamma R, Fcgamma RIIA, they express the only known eukaryotic nontransmembrane Fcgamma R, the glycan phosphoinositol (GPI)-linked Fcgamma RIIIB. Ligand binding by transmembrane Fcgamma RIIA 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 Fcgamma RIIA 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 Fcgamma RIIIB, the most abundant PMN Fcgamma receptor. Some studies have suggested an inability of Fcgamma RIIIB to transduce signals independently. These studies, taken together with this receptor's lack of a cytoplasmic domain, have led to the concept that Fcgamma RIIIB is primarily an Fc-binding molecule that aids in immune complex presentation to Fcgamma RIIA (1, 13). However, evidence now suggests that Fcgamma RIIIB 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, Fcgamma RIIIB cooperates with Fcgamma RIIA in PMN activation. When ligated together, as would occur when PMN bind immune complexes, Fcgamma RIIA and Fcgamma RIIIB synergize to activate the respiratory burst and to increase intracytoplasmic calcium (44, 47).

Despite the importance of the cooperation between Fcgamma RIIA and Fcgamma RIIIB 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 Fcgamma RIIA and Fcgamma RIIIB in Jurkat T cells, which lack endogenous Fcgamma receptors but are fully competent for tyrosine kinase signaling. In transfected Jurkat T cells, the PMN Fcgamma 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 Fcgamma RIIIB, since a mutant in which the GPI anchor was replaced by the transmembrane domain of CD7 was unable to synergize with Fcgamma RIIA. Moreover, crosslinking other GPI-linked proteins on Jurkat T cells with Fcgamma RIIA also led to a synergistic increase in [Ca2+]i. The increase in [Ca2+]i also required the tyrosines of the Fcgamma RIIA 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-gamma 1 phosphorylation was not altered by the replacement of the GPI anchor of Fcgamma RIIIB with the transmembrane domain of CD7. Thus, synergy between Fcgamma R requires the GPI anchor of Fcgamma RIIIB, but not for an increase in Fcgamma RIIA-dependent tyrosine kinase signaling. We hypothesize instead that the role for the GPI anchor of Fcgamma RIIIB is to sequester Fcgamma RIIA 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 Fcgamma R of primate PMN. Moreover, this could be a general mechanism by which GPI anchored proteins affect signal transduction from transmembrane receptors.


Materials and Methods

Cells and Antibodies

The human Jurkat T cells (American Type Culture Collection, Rockville, MD) were maintained in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) containing 10% heat-inactivated FCS (Hyclone, Logan, UT), 2 mM L-glutamine, 0.1 mM NEAA, 50 mM 2-mercaptoethanol, and 100 µg/ml penicillin and streptomycin under a 5% CO2 atmosphere. The bulk population was cloned before transfection to minimize heterogeneity of the population. Human PMN were freshly purified from the peripheral blood of healthy donors as described (5). The following mAbs were used in this study: IV.3 (anti-CD32, anti-Fcgamma RII; 26), 3G8 (anti-CD16, anti-Fcgamma RIII; 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-Fcgamma RII; 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 gamma -1 (PLC-gamma 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).

Fcgamma RIIA and Fcgamma RIIIB Expression Constructs and Transfection into Jurkat T Cells

The oligos 5'-CCTGAATTCCTCCGGATATCTTTGGTGAC-3' and 5'-AGAGGATCCGCTGCCACTGCTCTTATTAC-3' were used to amplify the human Fcgamma RIIIB (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 Fcgamma RIIIB/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 Fcgamma RIIIB extracellular domain joined with a CD7 transmembrane domain. The cDNA was verified by DNA sequencing. The cDNAs encoding Fcgamma RIIA and Fcgamma RIIA 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 × 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 × 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 2× 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 gamma -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-Fcgamma RII (II1A5), or anti-PLC gamma -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 Fcgamma RIIA or PLC-gamma 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.


Results

Cocrosslinking Fcgamma RIIA and Fcgamma RIIIB Results in a Synergistic [Ca2+]i Rise

Jurkat T cells, which do not express endogenous Fcgamma receptors, were stably transfected with the cDNAs encoding Fcgamma RIIA and Fcgamma RIIIB (J2/3; Fig. 1, top). In addition, stable transfectants were made which express Fcgamma RIIA along with a chimeric receptor consisting of the extracellular portion of Fcgamma RIIIB coupled to the transmembrane domain of CD7 (J2/3-CD7; Fig. 1, middle). A third transfectant was made that expresses Fcgamma RIIIB and an Fcgamma RIIA receptor in which the tyrosines (Y282 and Y298) of the ITAM have been mutated to phenylalanines (27; J2Yright-arrow 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).


Fig. 1. Fluorescent flow cytometric analysis of Fcgamma R expression. Jurkat T cells (106) expressing various Fcgamma receptors were resuspended in 50 µl PBS/5% FCS with 1 µg of the mAb IV.3 (2), specific for Fcgamma RIIA, mAb 3G8 (3), specific for Fcgamma RIIIB, or the mAb MEM-43 (4), specific for CD59. Cells were also stained with a negative control antibody (1). Cells were washed and then stained with F(ab')2 fragments of FITC-conjugated goat anti-mouse antibodies and then analyzed by FACS®. Cells expressing wild-type Fcgamma RIIA and Fcgamma RIIIB (J2/3; top), wild-type Fcgamma RIIA and the chimeric Fcgamma RIIIB/CD7 (J2/3-CD7; middle), or wild-type Fcgamma RIIIB and the mutant Fcgamma RIIA where the tyrosines within the ITAM (Y282 and Y298) are changed to phenylalanine (J2Yright-arrow F/3; bottom) are shown.
[View Larger Version of this Image (14K GIF file)]

Previous studies in PMN have shown that Fcgamma RIIA and Fcgamma RIIIB 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 Fcgamma RIIA are able to flux calcium after receptor ligation (15), suggesting the signaling machinery used by Fcgamma receptors is functional in these cells. Therefore we compared [Ca2+]i in J2/3 cells after crosslinking Fcgamma RIIA and Fcgamma RIIIB individually or after crosslinking both receptors together, using a F(ab')2 crosslinking antibody. Crosslinking Fcgamma RIIA resulted in a significant, short lived rise in [Ca2+]i (Fig. 2, top). In contrast, crosslinking Fcgamma RIIIB alone resulted in a slow rise in [Ca2+]i with a magnitude lower than for Fcgamma RIIA (Fig. 2, top). When both Fcgamma R 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-Fcgamma RII or -Fcgamma RIII 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 Fcgamma 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 Fcgamma RIIIB 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 Fcgamma RIIIB expression. In PMN, Fcgamma RIIIB 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 Fcgamma RIIIB from the cell surface, abolishes the rise in [Ca2+]i after Fcgamma RIIIB crosslinking (35, and data not shown). Nonetheless, the expression level of Fcgamma RIIIB in the transfected Jurkat cells was sufficient to produce a synergistic rise in [Ca2+]i.


Fig. 2. Changes in the [Ca2+]i after crosslinking Fcgamma R. Fura 2-AM pre-loaded J2/3 cells were incubated 30 min with the mAb IV.3 (anti-Fcgamma RII, IgG2b), the mAb 3G8 (anti-Fcgamma RIIIB, IgG1), or both these mAbs (top and middle). J2/3 cells also were incubated with mAb IV.3 and the mAb IB4, specific for beta 2 integrins (bottom). F(ab')2 fragments of goat anti-mouse antibodies (top and bottom), F(ab')2 fragments of goat anti-mouse IgG1 (middle), or F(ab')2 fragments of goat anti-mouse IgG2b (middle) were added to crosslink Fcgamma receptors at 20 s. Each curve is representative of at least three independent experiments. When Fcgamma RIIA was crosslinked with mAb IV.3/anti-IgG1 or Fcgamma RIIIB was crosslinked with mAb 3G8/anti-IgG2b, no rise in [Ca2+]i resulted, demonstrating specificity of the secondary antibodies (data not shown). No rise in [Ca2+]i resulted from the addition of secondary antibodies alone (data not shown).
[View Larger Version of this Image (19K GIF file)]

To determine if the synergistic calcium response required bridging of Fcgamma RIIA and Fcgamma RIIIB together or whether the augmentation in [Ca2+]i could be achieved by simultaneously crosslinking each Fcgamma receptor individually, isotype-specific secondary crosslinking antibodies were used (Fig. 2, middle). Fcgamma RIIA was crosslinked with IV.3, an IgG2b mAb, and goat F(ab')2 fragments specific for mouse IgG2b and Fcgamma RIIIB was crosslinked with 3G8, an IgG1 mAb, and goat F(ab')2 fragments specific for mouse IgG1. When both Fcgamma 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 Fcgamma receptors individually (Fig. 2, middle).

To show specificity of the synergy, cells were incubated with anti-Fcgamma RII mAb IV.3 and the mAb IB4, specific for beta 2 (CD18) integrins (Fig. 2, bottom). The beta 2 integrin LFA-1 is expressed at a level similar to the transfected Fcgamma RIIIB (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 Fcgamma RIIA for [Ca2+]i rise. This result indicates that signaling through Fcgamma RIIA is augmented when cocrosslinked to Fcgamma RIIIB, as would occur under physiological conditions where both Fcgamma receptors are ligated by immune complexes.

The GPI Anchor Is Necessary and Sufficient for the Contribution of Fcgamma RIIIB to Synergy

Primate PMN are the only cells that express a GPI-anchored Fcgamma receptor (32). To determine whether the GPI anchor was necessary for Fcgamma RIIIB contribution to the synergistic increase in [Ca2+]i, stable transfectants were made expressing Fcgamma RIIA and a chimeric Fcgamma RIIIB with the GPI anchor replaced by the transmembrane domain of CD7 (J2/3-CD7; Fig. 1, middle). When Fcgamma RIIA and Fcgamma RIIIB/ CD7 were crosslinked together in these cells, the [Ca2+]i rise was similar to the rise generated when Fcgamma RIIA was crosslinked alone without any synergy from Fcgamma RIIIB (Fig. 3, middle). The inability of the chimeric Fcgamma RIIIB/ CD7 molecule to contribute to the synergistic [Ca2+]i rise was not due to inadequate expression of this protein, since the Fcgamma RIIIB/CD7 molecule was expressed at a greater level than the wild-type Fcgamma RIIIB (Fig. 1, top and middle). This experiment demonstrates that the GPI anchor is necessary for the synergistic [Ca2+]i rise.


Fig. 3. [Ca2+]i in cells expressing the chimeric Fcgamma RIIIB/CD7. J2/3 cells (top), J2/3-CD7 cells (middle), or PMN (bottom) were preloaded with Fura 2-AM. J2/3 and J2/3-CD7 cells were then incubated for 30 min with the mAb IV.3 (anti-Fcgamma RII), mAb 3G8 (anti-Fcgamma RIII), mAb MEM-43 (anti-CD59), or combinations of these mAbs. PMN were incubated with mAb IV.3 F(ab), mAb 10G10 F(ab')2 (anti-CD59), or combinations of these mAbs. Experiments were performed as described in Fig. 2. Each curve is representative of at least three independent experiments. For PMN, the change in [Ca2+]i at 140 s after the addition of crosslinking antibody was calculated and results are shown as the mean ± SEM for three independent experiments (bottom).
[View Larger Version of this Image (19K GIF file)]

To determine whether any aspect of the extracellular Ig domains of Fcgamma RIIIB rise were required for the synergistic [Ca2+]i rise, other GPI-linked proteins expressed by Jurkat cells were cocrosslinked with Fcgamma RIIA. 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 Fcgamma RIIIB. When these GPI-linked proteins, CD59 (Fig. 3, top) and CD48 (not shown), were cocrosslinked with Fcgamma RIIA, a synergistic rise in [Ca2+]i also occurred in Jurkat cells transfected with Fcgamma RIIA 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 Fcgamma RIIIB alone (Fig. 3, top, and data not shown).

These experiments demonstrate that the GPI anchor of Fcgamma RIIIB is required for Fcgamma R cooperation but that other extracellular domains will substitute for Fcgamma RIIIB when cocrosslinked with Fcgamma RIIA. 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 Fcgamma RIIA and Fcgamma RIIIB interaction with the beta 2 integrin CR3 (for review see 30). Moreover, since these cells do not express CR3, this experiment shows that Fcgamma R synergy can occur without this PMN integrin.

Synergy in PMN between Fcgamma RIIA and Fcgamma RIIIB 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, Fcgamma RIIA and CD59 were cocrosslinked and a prolongation in the rise [Ca2+]i was found (Fig. 3, bottom). The synergistic rise in [Ca2+]i with Fcgamma RIIA and CD59 was not as pronounced as with Fcgamma RIIIB and Fcgamma RIIA. No significant synergy between Fcgamma RIIA 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 Fcgamma RIIIB (CD59 has ~13% of the expression of Fcgamma RIIIB, 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 Fcgamma RIIIB on the cell surface by 80% (35 and data not shown).

The ITAM of Fcgamma RIIA 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 Fcgamma receptor signaling (16, 43). To determine whether this cascade had a role in Fcgamma receptor synergy, Jurkat cells were transfected with Fcgamma RIIIB and a mutant Fcgamma RIIA in which tyrosines Y282 and Y298 contained within the ITAM were mutated to phenylalanines (J2Yright-arrow F/3; Fig. 1, bottom). It has been shown in model systems that these tyrosines are required for [Ca2+]i flux when Fcgamma RIIA is ligated alone (27, 28). No synergistic [Ca2+]i flux occurred in J2Yright-arrow F/3 cells when Fcgamma RIIA was ligated either alone or together with Fcgamma RIIIB, 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 Fcgamma RIIA are required for the synergistic [Ca2+]i rise. Thus both the GPI anchor of Fcgamma RIIIB and the ITAM motif of Fcgamma RIIA are required for synergy in calcium signaling.


Fig. 4. [Ca2+]i flux in cells expressing Fcgamma RIIA containing the ITAM mutation. Fura 2-AM preloaded J2Yright-arrow F/3 cells were incubated with the mAbs IV.3 (anti-Fcgamma RII) and 3G8 (anti-Fcgamma RIII), then analyzed by fluorimetry as described in Fig 2. The mAb C305, specific for the TCR/CD3 complex, was added at 300 sec to demonstrate that these cells are competent to flux [Ca2+]i.
[View Larger Version of this Image (12K GIF file)]

The Synergistic Signal Does Not Result in Increased Tyrosine Phosphorylation of Fcgamma RIIA

Because of the requirement for the ITAM in synergy and the association of GPI-linked proteins with src family kinases (4, 43), we hypothesized that an early step in this synergistic interaction might be an increased tyrosine phosphorylation of the ITAM of Fcgamma RIIA. When Fcgamma RIIA was immunoprecipitated from J2/3 cells after crosslinking Fcgamma RIIA alone, its tyrosine phosphorylation peaked at 1 min and was diminished by 5 min (Fig. 5 A, top). Surprisingly, crosslinking Fcgamma RIIA and Fcgamma RIIIB together did not enhance tyrosine phosphorylation of Fcgamma RIIA as expected but actually diminished detection of the tyrosine phosphorylation of Fcgamma RIIA (Fig. 5 A, top). Averages from three experiments after normalization for the amount of receptor immunoprecipitated showed that Fcgamma RIIA was phosphorylated ~10-fold less under synergistic conditions as compared to ligation of Fcgamma RIIA alone. We also analyzed the tyrosine phosphorylation of Fcgamma RIIA in J2/3-CD7 cells. Ligation of Fcgamma RIIA without Fcgamma RIIIB induced tyrosine phosphorylation of itself to a similar extent and with similar kinetics as in cells expressing both wild-type Fcgamma receptors (Fig. 5 B, bottom). In striking contrast to the results obtained in J2/3 cells by crosslinking both wild-type Fc receptors, cocrosslinking Fcgamma RIIA and Fcgamma RIIIB/CD7 did not significantly diminish the extent or alter the kinetics of Fcgamma RIIA phosphorylation (Fig. 5 A, bottom). To determine if the marked diminution of Fcgamma RIIA tyrosine phosphorylation also occurred when it was crosslinked with other GPI-anchored proteins, Fcgamma RIIA was crosslinked with CD48 or CD59 (Fig. 5 B). Cocrosslinking any GPI-anchored protein with Fcgamma RIIA markedly diminished its tyrosine phosphorylation. In addition, we analyzed the extent of tyrosine phosphorylation of Fcgamma RIIA in PMN after ligating Fcgamma RIIA, individually or together with Fcgamma RIIIB, by using the F(ab) fragment of mAb IV.3 and the F(ab')2 of mAb 3G8. Crosslinking both Fcgamma receptors resulted in ~2-3-fold diminished tyrosine phosphorylation of Fcgamma RIIA when compared to ligating Fcgamma RIIA alone (data not shown).


Fig. 5. Tyrosine phosphorylation of Fcgamma RIIA after crosslinking Fcgamma R. (A) J2/3 (top) or J2/3-CD7 (bottom) cells were incubated with mAb IV.3 (anti-Fcgamma RII) or with mAbs IV.3 and 3G8 (anti-Fcgamma RIII) for 30 min on ice and then warmed 10 min to 37°C. (B) J2/3 cells were incubated with various combinations of mAbs specific for Fcgamma RII, Fcgamma RIII, CD48, or CD59. In both panels, crosslinking F(ab')2 fragments of goat anti-mouse antibodies were added for various amounts of time. At each time point, an aliquot was removed, lysed, and Fcgamma RIIA immunoprecipitated. Proteins were separated by SDS-PAGE, and blots were probed with anti-phosphotyrosine. Cocrosslinking of GPI- but not transmembrane-anchored Fcgamma RIIIB diminishes tyrosine phosphorylation of Fcgamma RIIA. Blots shown are representative of at least five experiments.
[View Larger Version of this Image (28K GIF file)]

The Synergistic Calcium Rise Does Not Result from the Prolonged Tyrosine Phosphorylation of PLC-gamma 1

PLC-gamma 1 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 Fcgamma RIIA induces the tyrosine phosphorylation of PLC-gamma 1, which leads to its activation (25, 42). To determine whether prolonged activation of PLC-gamma 1 could account for the synergistic increase in [Ca2+]i, its tyrosine phosphorylation was examined. In agreement with previous studies, crosslinking Fcgamma RIIA in the transfected Jurkat cells resulted in tyrosine phosphorylation of PLC-gamma 1 that was visible by 1 min (data not shown, and 42). Crosslinking Fcgamma RIIIB and Fcgamma RIIA in J2/3 cells resulted in tyrosine phosphorylation of PLC-gamma 1, which was not different from cocrosslinking Fcgamma RIIA and the chimeric Fcgamma RIIIB/CD7 in J2/3-CD7 cells (Fig. 6). Thus, Fcgamma receptor synergy is independent of the tyrosine phosphorylation of PLC-gamma 1.


Fig. 6. The tyrosine phosphorylation of PLC-gamma 1 after crosslinking various Fcgamma R. J2/3 (squares) or J2/3-CD7 (triangles) cells were incubated with mAbs IV.3 (anti-Fcgamma RII) and 3G8 (anti- Fcgamma RIII), warmed to 37°C, and crosslinking initiated by addition of F(ab')2 fragments of goat anti-mouse antibodies. At each time point, an aliquot was removed, PLC-gamma 1 was immunoprecipitated, and proteins were separated by SDS-PAGE. Blots were probed with anti-phosphotyrosine and subsequently with anti-PLC-gamma 1 antibodies to determine the relative phosphorylation of the immunoprecipitated enzyme, as described in Materials and Methods. Three independent experiments from both cell types were analyzed by densitometry, and the mean and SEM of the three experiments are shown.
[View Larger Version of this Image (13K GIF file)]

The Synergistic Rise in [Ca2+]i Requires the Influx of Extracellular Calcium

To determine the source of Ca2+ for the synergistic [Ca2+]i rise in the J2/3 cells, changes in Fura-2 fluorescence were measured in the presence of extracellular EGTA to prevent calcium influx from the medium. The synergistic [Ca2+]i rise was inhibited almost immediately after addition of EGTA, indicating that calcium influx through plasma membrane channels is largely responsible for the prolonged [Ca2+]i rise (Fig. 7 A, left) as found in PMN (44). Similarly, the synergistic [Ca2+]i rise induced by cocrosslinking Fcgamma 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 Fcgamma 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 Fcgamma RIIA with Fcgamma RIIIB 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 Fcgamma RIIIB 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).


Fig. 7. The synergistic rise in [Ca2+]i requires the influx of extracellular calcium. Changes in Fura 2-AM fluorescence after receptor crosslinking in J2/3 cells was measured as in Fig. 2 in the absence or presence of 2 mM EGTA to prevent calcium influx from the medium. (A) 2 mM EGTA was added 280 s after crosslinking. (B) 2 mM EGTA was added immediately before receptor crosslinking. Also shown is no added EGTA. (C) 2 mM EGTA was added at 0 or 300 s.
[View Larger Version of this Image (24K GIF file)]


Discussion

Since the discovery that GPI-linked proteins can transduce proliferative signals, attention has focused on the mechanism by which these proteins, anchored into the outer leaflet of the plasma membrane by their fatty acyl chains, can signal to the cell cytoplasm. Two distinct but not mutually exclusive paradigms have developed. One model suggests that GPI-linked proteins can sequester into specialized membrane domains, especially after clustering (for review see 29, 34). These domains, which are defined by their insolubility in Triton X-100, contain characteristic lipid components, such as glycosphingolipids and cholesterol, but may be depleted in certain phospholipids. GPI-linked proteins are enriched ~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, Fcgamma RIIIB 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, alpha vbeta 3 (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 Fcgamma RIIA and Fcgamma RIIIB on human PMN presents an opportunity to test these hypotheses concerning signal transduction by GPI-linked proteins. When immune complexes bind to PMN, Fcgamma RIIA and Fcgamma RIIIB 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 Fcgamma receptor synergy on PMN. In Jurkat cells without Mac-1, Fcgamma RIIA and Fcgamma RIIIB 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 Fcgamma RIIIB, also can synergize with Fcgamma RIIA 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 Fcgamma 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 Fcgamma RIIA with glycolipid domains enriched in GPI-linked proteins fundamentally alters subsequent signaling. Cocrosslinking Fcgamma RIIA with any of the GPI-linked proteins induced the synergistic increase in [Ca2+]i and, surprisingly, decreased the extent of Fcgamma RIIA tyrosine phosphorylation. When Fcgamma RIIIB was expressed with a transmembrane domain, its synergy with Fcgamma RIIA was abolished, as was its effect on Fcgamma RIIA tyrosine phosphorylation. These data support the hypothesis that the membrane environment of Fcgamma RIIA is altered by crosslinking it with GPI- anchored proteins. This altered environment modulates the Fcgamma RIIA-generated signal in fundamental ways. We initially expected that the synergistic [Ca2+]i rise would be associated with increased phosphorylation of the ITAM of Fcgamma RIIA, 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 Fcgamma RIIA has diminished tyrosine phosphorylation after cocrosslinking with Fcgamma RIIIB, because ligation with GPI-linked proteins causes Fcgamma RIIA 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-gamma 1. Calcium mobilization after crosslinking Fcepsilon RI activates a sphingosine kinase that produces sphingosine-1-phosphate as a second messenger for intracellular calcium mobilization (6). Alternatively, localization of the Fcgamma 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 Fcgamma 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 Fcgamma receptor, Fcgamma RIIB, inhibits phagocytosis mediated by Fcgamma RIIA. Decreased tyrosine phosphorylation induced by Fcgamma RIIB after interaction with IgG ligand may be responsible for this inhibition of Fcgamma RIIA-mediated phagocytosis (Hunter, S., and A.D. Schreiber, unpublished results).

In summary, transfection of human PMN Fcgamma 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 Fcgamma RIIIB and the ITAM of Fcgamma RIIA. 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 Fcgamma RIIA molecules are expressed on the cell surface together with 10 to 20 times more Fcgamma RIIIB (12, 13). Thus it is highly likely that whenever Fcgamma RIIA is ligated by an immune complex, it is in association with several GPI-linked Fcgamma RIIIB and that the modulated signal which occurs because of association with GPI domains is the major mechanism of immune complex-mediated PMN activation.


Footnotes

Received for publication 29 April 1997 and in revised form 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

We thank Dr. Ming-jie Zhou (Molecular Probes, Inc.) for the PCR clone of CD16, Dr. Brian Seed for the CD16/CD7/zeta 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.


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.


References

1. Anderson, C.L., L. Shen, D.M. Eicher, M.D. Wewers, and J.K. Gill. 1990. Phagocytosis mediated by three distinct Fc gamma  receptor classes on human leukocytes. J. Exp. Med. 171: 1333-1345 [Abstract].
2. Bengtsson, T., M.E. Jaconi, M. Gustafson, K.-E. Magnusson, J.-M. Theler, D.P. Lew, and O. Stendahl. 1993. Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium. Eur. J. Cell Biol. 62: 49-58
3. Berger, M., D.L. Birx, E.M. Wetzler, J.J. O'Shea, E.J. Brown, and A.S. Cross. 1985. Calcium requirements for increased complement receptor expression during neutrophil activation. J. Immunol. 135: 1342-1348 [Abstract/Free Full Text].
4. Brown, D.. 1993. The tyrosine kinase connection: how GPI anchored proteins activate T cells. Curr. Opin. Immunol. 5: 349-354
5. Brown, E.J.. 1994. In vitro assays of phagocytic function of human peripheral blood leukocytes: receptor modulation and signal transduction. Methods Cell Biol. 45: 147-164
6. Choi, H.U., J. Kim, and J. Kinet. 1996. Calcium mobilization via sphingosine kinase in signaling by the Fcepsilon RI antigen receptor. Nature. 380: 634-636
7. Clark, M.R., S.G. Stuart, R.P. Kimberly, P.A. Ory, and I.M. Goldstein. 1991. A single amino acid distinguishes the high-responder from the low-responder form of Fc receptor II on human monocytes. Eur. J. Immunol. 21: 1911-1916
8. Coyne, K.E., S.E. Hall, S. Thompson, M.A. Arce, T. Kinoshita, T. Fujita, D.J. Anstee, W. Rosse, and D.M. Lublin. 1992. Mapping of epitopes, glycosylation sites, and complement regulatory domains in human decay accelerating factor. J. Immunol. 149: 2906-2913 [Abstract/Free Full Text].
9. Fleit, H.B., S.D. Wright, and J.C. Unkeless. 1982. Human neutrophil Fc gamma  receptor distribution and structure. Proc. Natl. Acad. Sci. USA. 79: 3275-3279 [Abstract].
10. Fujimoto, T.. 1993. Calcium pump of the plasma membrane is localized in caveolae. J. Cell Biol. 120: 1147-1157 [Abstract].
11. Huang, M.-M., Z. Indik, L.F. Brass, J.A. Hoxie, A.D. Schreiber, and J.S. Brugge. 1992. Activation of Fcgamma RII induces tyrosine phosphorylation of multiple proteins including Fcgamma RII. J. Biol. Chem. 267: 5467-5473 [Abstract/Free Full Text].
12. Huizinga, T.W.J., C.E. Van der Schoot, C. Jost, R. Klaassen, M. Kleijer, A.E.G.K. van dem Borne, D. Roos, and P.A.T. Tetteroo. 1988. The PI-linked receptor Fcgamma RIII is released on stimulation of neutrophils. Nature. 333:667-669.
13. Huizinga, T.W., F. van Kemenade, L. Koenderman, K.M. Dolman, A.E. von dem Borne, P. A. Tetteroo, and D. Roos. 1989. The 40-kDa Fc gamma  receptor (Fcgamma RII) on human neutrophils is essential for the IgG-induced respiratory burst and IgG-induced phagocytosis. J. Immunol. 142: 2365-2369 [Abstract/Free Full Text].
14. Hundt, M., and R.E. Schmidt. 1992. The glycosylphosphatidylinositol-linked Fcgamma receptor III represents the dominant receptor structure for immune complex activation of neutrophils. Eur. J. Immunol. 22: 811-816
15. Hunter, S., M. Kamoun, and A.D. Schreiber. 1994. Transfection of an Fcgamma receptor cDNA induces T cells to become phagocytic. Proc. Natl. Acad. Sci. USA. 91: 10232-10236 [Abstract/Free Full Text].
16. Indik, Z.K., J.G. Park, S. Hunter, and A.D. Schreiber. 1995. The molecular dissection of Fcgamma receptor mediated phagocytosis. Blood. 86: 4389-4399 [Abstract/Free Full Text].
17. Jaconi, M.E.E., D.P. Lew, J.-L. Carpentier, K.E. Magnusson, M. Sjogren, and O. Stendahl. 1990. Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils. J. Cell Biol. 110: 1555-1564 [Abstract].
18. Jaconi, M.E., J.M. Theler, W. Schlegel, and P.D. Lew. 1993. Cytosolic free Ca2+ signals in single adherent human neutrophils: generation and functional role. Eur. J. Pediatr. 152: S26-S32
19. Kimberly, R.P., J.W. Ahlstrom, M.E. Click, and J.C. Edberg. 1990. The glycosyl phosphatidylinositol-linked Fcgamma RIII on PMN mediates transmembrane signaling events distinct from Fcgamma RII. J. Exp. Med. 171: 1239-1255 [Abstract/Free Full Text].
20. Kolanus, W., C. Romeo, and B. Seed. 1993. T cell activation by clustered tyrosine kinases. Cell. 74: 171-183
21. Krauss, J.C., H. Poo, W. Xue, L. Mayo-Bond, R.F. Todd, and H.R. Petty. 1994. Reconstitution of antibody-dependent phagocytosis in fibroblasts expressing Fcgamma RIIIB and the complement receptor type 3.  J. Immunol. 153: 1769-1777 [Abstract/Free Full Text].
22. Lanier, L.L., and J.H. Phillips. 1996. Inhibitory MHC class I receptors on NK cells and T cells. Immunol. Today. 17: 86-91
23. Lew, P.D., A. Monod, F.A. Waldvogel, B. Dewald, M. Baggiolini, and T. Pozzan. 1986. Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils. J. Cell Biol. 102: 2197-2204 [Abstract].
24. Lindberg, F.P., H.D. Gresham, E. Schwarz, and E.J. Brown. 1993. Molecular cloning of integrin-associated protein: an immunoglobulin family member with multiple membrane spanning domains implicated in alpha vbeta 3-dependent ligand binding. J. Cell Biol. 123: 485-496 [Abstract].
25. Liscovitch, M., and L.C. Cantley. 1994. Lipid second messengers. Cell. 77: 329-334
26. Looney, R.J., D.H. Ryan, K. Takahashi, H.B. Fleit, H.J. Cohen, G.N. Abraham, and C.L. Anderson. 1986. Identification of a second class of IgG Fc receptors on human neutrophils. A 40-kilodalton molecule also found on eosinophils. J. Exp. Med. 163: 826-836 [Abstract].
27. Mitchell, M.A., M.-M. Huang, P. Chien, Z.K. Indik, X.Q. Pan, and A.D. Schreiber. 1994. Substitutions and deletions in the cytoplasmic domain of the phagocytic receptor Fcgamma RIIA: effect on receptor tyrosine phosphorylation and phagocytosis. Blood. 84: 1753-1759 [Abstract/Free Full Text].
28. Odin, J.A., J.C. Edberg, C.J. Painter, R.P. Kimberly, and J.C. Unkeless. 1991. Regulation of phagocytosis and [Ca2+]i flux by distinct regions of an Fc receptor. Science. 254: 1785-1788
29. Parton, R.G., and K. Simons. 1995. Digging into caveolae. Science. 269: 1398-1399
30. Petty, H.R., and R. Todd III.. 1996. Integrins as promiscuous signal transduction devices. Immunol. Today. 17: 209-212
31. Phillips, J.H., J.E. Gumperz, P. Parham, and L.L. Lanier. 1995. Superantigen-dependent, cell-mediated cytotoxicty inhibited by MHC class I receptors on T lymphocytes. Science. 268: 403-405
32. Ravetch, J.V., and B. Perussia. 1989. Alternative membrane forms of Fcgamma RIII (CD16) on human natural killer cells and neutrophils: cell type-specific expression of two genes that differ in single nucleotide substitutions. J. Exp. Med. 170: 481-497 [Abstract].
33. Ravetch, J.V., and J.P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9: 457-492
34. Rodgers, W., and J.K. Rose. 1996. Exclusion of CD45 inhibits activity of p56 associated with glycolipid-enriched membrane domains. J. Cell Biol. 135: 1515-1523 [Abstract].
35. Rosales, C., and E.J. Brown. 1991. Two mechanisms for IgG Fc-receptor-mediated phagocytosis by human neutrophils. J. Immunol. 146: 3937-3944 [Abstract/Free Full Text].
36. Rosales, C., and E.J. Brown. 1992. Signal transduction by neutrophil immunoglobulin G Fc receptors. Dissociation of [Ca2+] rise from IP3. J. Biol. Chem. 267: 5265-5271 [Abstract/Free Full Text].
37. Rosales, C., and E.J. Brown. 1992. Calcium channel blockers nifedipine and diltiazem inhibit Ca2+ release from intracellular stores in neutrophils. J. Biol. Chem. 267: 1443-1448 [Abstract/Free Full Text].
38. Rosales, C., and E.J. Brown. 1993. Neutrophil receptors and modulation of the immune response. In The Neutrophil. J.S. Abramson and J.G. Wheeler, editors. IRL Press, Oxford. 23-62.
39. Salmon, J.E., N.L. Brogle, J.C. Edberg, and R.P. Kimberly. 1991. Fcgamma receptor III induces actin polymerization in human neutrophils and primes phagocytosis mediated by Fcgamma receptor II. J. Immunol. 146: 997-1004 [Abstract/Free Full Text].
40. Sehgal, G., K. Zhang, R.F. Todd, L.A. Boxer, and H.R. Petty. 1993. Lectin-like inhibition of immune-complex receptor-mediated stimulation of neutrophils: effects on cytosolic calcium release and superoxide production. J. Immunol. 150: 4571-4580 [Abstract/Free Full Text].
41. Sei, Y., M. Takemura, F. Gusovsky, P. Skolnick, and A. Basile. 1995. Distinct mechanisms for Ca2+ entry induced by OKT3 and Ca2+ depletion in Jurkat T cells. Exp. Cell Res. 216: 222-231
42. Shen, Z., C.T. Lin, and J.C. Unkeless. 1994. Correlations among tyrosine phosphorylation of Shc, p72syk, PLC-gamma 1, and [Ca2+]i flux in Fcgamma RIIA signaling. J. Immunol. 152: 3017-3023 [Abstract/Free Full Text].
43. Stefanova, I., V. Horejsi, I.J. Ansotegui, W. Knapp, and H. Stockinger. 1991. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science. 254: 1016-1019
44. Vossebeld, P.J.M., J. Kessler, A.E.G.K. Von dem Borne, D. Roos, and A.J. Verhoeven. 1995. Heterotypic Fcgamma R clusters evoke a synergistic Ca2+ response in human neutrophils. J. Biol. Chem. 270: 10671-10679 [Abstract/Free Full Text].
45. Wacholtz, M.C., S.S. Patel, and P.E. Lipsky. 1989. Leukocyte function- associated antigen 1 is an activation molecule for human T cells. J. Exp. Med. 170: 431-448 [Abstract].
46. Wei, Y., M. Lukashev, D.I. Simon, S.C. Bodary, S. Rosenberg, M.V. Doyle, and H.A. Chapman. 1996. Regulation of integrin function by the urokinase receptor. Science. 273: 1551-1554 [Abstract].
47. Zhou, M.-J., and E.J. Brown. 1994. CR3 (Mac-1, aMb2, CD11b/CD18) and Fcgamma RIII cooperate in generation of a neutrophil respiratory burst: requirement for Fcgamma RII and tyrosine phosphorylation. J. Cell Biol. 125: 1407-1416 [Abstract].
48. Zhou, M., R.F. Todd III, J.G.J. Van de Winkel, and H.R. Petty. 1993. Cocapping of the leukoadhesin molecules complement receptor type 3 and lymphocyte function-associated antigen-1 with Fcgamma receptor III on human neutrophils: possible role of lectin-like interactions. J. Immunol. 150: 3030-3041 [Abstract/Free Full Text].
49. Zhou, M.-J., D.M. Lublin, D.C. Link, and E.J. Brown. 1995. Distinct tyrosine kinase activation and Triton X-100 insolubility upon Fcgamma RII or Fcgamma RIIIB ligation in human polymorphonuclear leukocytes: implications for immune complex activation of the respiratory burst. J. Biol. Chem. 270: 13553-13560 [Abstract/Free Full Text].

Copyright © 1997 by The Rockefeller University Press.