* Division of Infectious Diseases, Washington University, School of Medicine, St. Louis, Missouri 63110; and Department of
Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
While many cell types express receptors for
the Fc domain of IgG (FcR), only primate polymorphonuclear neutrophils (PMN) express an Fc
R linked
to the membrane via a glycan phosphoinositol (GPI)
anchor. Previous studies have demonstrated that this
GPI-linked Fc
R (Fc
RIIIB) cooperates with the
transmembrane Fc
R (Fc
RIIA) 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 Fc
RIIA and/or Fc
RIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca2+]i) to levels not reached by
stimulation of either Fc
RIIA or Fc
RIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca2+. Cocrosslinking Fc
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 Fc
RIIA 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 Fc
RIIIB with a transmembrane anchor
abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the Fc
RIIA cytoplasmic tail abolished synergy. While the ITAM of
Fc
RIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked Fc
RIIA was diminished when cocrosslinked with Fc
RIIIB. These
data demonstrate that Fc
RIIA association with
GPI-linked proteins facilitates Fc
R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fc
R of human PMN.
THE binding of immune complexes by polymorphonuclear neutrophils (PMN)1 receptors for the Fc domain of IgG (Fc Primate PMN are unique, because in addition to the transmembrane Fc Despite the importance of the cooperation between
Fc 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-Fc Fc The oligos 5 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 [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 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 Cocrosslinking Fc Jurkat T cells, which do not express endogenous Fc
Previous studies in PMN have shown that Fc
To determine if the synergistic calcium response required bridging of Fc To show specificity of the synergy, cells were incubated
with anti-Fc The GPI Anchor Is Necessary and Sufficient for the
Contribution of Fc Primate PMN are the only cells that express a GPI-anchored
Fc
To determine whether any aspect of the extracellular Ig
domains of Fc These experiments demonstrate that the GPI anchor of
Fc Synergy in PMN between Fc The ITAM of Fc Activation of tyrosine phosphorylation and propagation of
a tyrosine kinase cascade by receptor associated ITAMs is
thought to be essential for Fc
The Synergistic Signal Does Not Result in Increased
Tyrosine Phosphorylation of Fc 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 Fc
The Synergistic Calcium Rise Does Not Result from the
Prolonged Tyrosine Phosphorylation of PLC- PLC-
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
Fc
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, Fc The interaction of Fc Our data support the hypothesis that association of
Fc Our data further extend the observations made with
several receptors, including Fc In summary, transfection of human PMN 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.
R, Fc
RIIA, they express the only known
eukaryotic nontransmembrane Fc
R, the glycan phosphoinositol (GPI)-linked Fc
RIIIB. Ligand binding by transmembrane Fc
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 Fc
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 Fc
RIIIB, the most abundant PMN Fc
receptor. Some studies have suggested
an inability of Fc
RIIIB to transduce signals independently. These studies, taken together with this receptor's
lack of a cytoplasmic domain, have led to the concept that
Fc
RIIIB is primarily an Fc-binding molecule that aids in
immune complex presentation to Fc
RIIA (1, 13). However, evidence now suggests that Fc
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, Fc
RIIIB cooperates with Fc
RIIA in PMN activation. When ligated together, as would occur when PMN bind immune complexes,
Fc
RIIA and Fc
RIIIB synergize to activate the respiratory
burst and to increase intracytoplasmic calcium (44, 47).
RIIA and Fc
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 Fc
RIIA and Fc
RIIIB 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 Fc
RIIIB, since a mutant in which the GPI anchor was replaced by the transmembrane
domain of CD7 was unable to synergize with Fc
RIIA.
Moreover, crosslinking other GPI-linked proteins on Jurkat T cells with Fc
RIIA also led to a synergistic increase
in [Ca2+]i. The increase in [Ca2+]i also required the tyrosines of the Fc
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-
1 phosphorylation was not altered by
the replacement of the GPI anchor of Fc
RIIIB with the
transmembrane domain of CD7. Thus, synergy between
Fc
R requires the GPI anchor of Fc
RIIIB, but not for an
increase in Fc
RIIA-dependent tyrosine kinase signaling. We hypothesize instead that the role for the GPI anchor of
Fc
RIIIB is to sequester Fc
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
Fc
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
RII; 26), 3G8 (anti-CD16, anti-Fc
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-Fc
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
-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).
RIIA and Fc
RIIIB Expression Constructs and
Transfection into Jurkat T Cells
-CCTGAATTCCTCCGGATATCTTTGGTGAC-3
and
5
-AGAGGATCCGCTGCCACTGCTCTTATTAC-3
were used to amplify the human Fc
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 Fc
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 Fc
RIIIB extracellular domain
joined with a CD7 transmembrane domain. The cDNA was verified by
DNA sequencing. The cDNAs encoding Fc
RIIA and Fc
RIIA with both
ITAM tyrosines in the cytoplasmic tail mutated to phenylalanine were prepared as described (7, 27) and cloned into pRcCMV and pCEP4.
)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.
) specific goat F(ab
)2 fragments were added. Intracellular calcium concentrations were calculated as
described (36).
) 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
-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-Fc
RII
(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 Fc
RIIA 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.
Results
RIIA and Fc
RIIIB Results in a
Synergistic [Ca2+]i Rise
receptors, were stably transfected with the cDNAs encoding
Fc
RIIA and Fc
RIIIB (J2/3; Fig. 1, top). In addition, stable transfectants were made which express Fc
RIIA along
with a chimeric receptor consisting of the extracellular
portion of Fc
RIIIB coupled to the transmembrane domain of CD7 (J2/3-CD7; Fig. 1, middle). A third transfectant was made that expresses Fc
RIIIB and an Fc
RIIA 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).
Fig. 1.
Fluorescent flow cytometric analysis of FcR expression. Jurkat T cells (106)
expressing various Fc
receptors were resuspended in
50 µl PBS/5% FCS with 1 µg
of the mAb IV.3 (2), specific
for Fc
RIIA, mAb 3G8 (3),
specific for Fc
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 Fc
RIIA and Fc
RIIIB (J2/3; top), wild-type
Fc
RIIA and the chimeric
Fc
RIIIB/CD7 (J2/3-CD7;
middle), or wild-type Fc
RIIIB and the mutant Fc
RIIA where the tyrosines within the ITAM (Y282 and
Y298) are changed to phenylalanine (J2Y
F/3; bottom)
are shown.
[View Larger Version of this Image (14K GIF file)]
RIIA and
Fc
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 Fc
RIIA 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
Fc
RIIA and Fc
RIIIB individually or after crosslinking
both receptors together, using a F(ab
)2 crosslinking antibody. Crosslinking Fc
RIIA resulted in a significant, short
lived rise in [Ca2+]i (Fig. 2, top). In contrast, crosslinking
Fc
RIIIB alone resulted in a slow rise in [Ca2+]i with a
magnitude lower than for Fc
RIIA (Fig. 2, top). When both Fc
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-Fc
RII or -Fc
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 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 Fc
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 Fc
RIIIB expression. In
PMN, Fc
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 Fc
RIIIB
from the cell surface, abolishes the rise in [Ca2+]i after
Fc
RIIIB crosslinking (35, and data not shown). Nonetheless, the expression level of Fc
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 FcR. Fura 2-AM
pre-loaded J2/3 cells were incubated 30 min with the mAb IV.3
(anti-Fc
RII, IgG2b), the mAb 3G8 (anti-Fc
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
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 Fc
receptors at 20 s. Each curve is representative of
at least three independent experiments. When Fc
RIIA was
crosslinked with mAb IV.3/anti-IgG1 or Fc
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)]
RIIA and Fc
RIIIB 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). Fc
RIIA was crosslinked with IV.3, an IgG2b mAb, and goat F(ab
)2 fragments specific for
mouse IgG2b and Fc
RIIIB 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).
RII mAb IV.3 and the mAb IB4, specific for
2 (CD18) integrins (Fig. 2, bottom). The
2 integrin
LFA-1 is expressed at a level similar to the transfected
Fc
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 Fc
RIIA for [Ca2+]i rise.
This result indicates that signaling through Fc
RIIA is
augmented when cocrosslinked to Fc
RIIIB, as would occur under physiological conditions where both Fc
receptors are ligated by immune complexes.
RIIIB to Synergy
receptor (32). To determine whether the GPI anchor
was necessary for Fc
RIIIB contribution to the synergistic
increase in [Ca2+]i, stable transfectants were made expressing Fc
RIIA and a chimeric Fc
RIIIB with the GPI
anchor replaced by the transmembrane domain of CD7
(J2/3-CD7; Fig. 1, middle). When Fc
RIIA and Fc
RIIIB/ CD7 were crosslinked together in these cells, the [Ca2+]i
rise was similar to the rise generated when Fc
RIIA was
crosslinked alone without any synergy from Fc
RIIIB
(Fig. 3, middle). The inability of the chimeric Fc
RIIIB/
CD7 molecule to contribute to the synergistic [Ca2+]i rise
was not due to inadequate expression of this protein, since
the Fc
RIIIB/CD7 molecule was expressed at a greater
level than the wild-type Fc
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 FcRIIIB/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-Fc
RII), mAb 3G8
(anti-Fc
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)]
RIIIB rise were required for the synergistic
[Ca2+]i rise, other GPI-linked proteins expressed by Jurkat
cells were cocrosslinked with Fc
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 Fc
RIIIB. When these GPI-linked
proteins, CD59 (Fig. 3, top) and CD48 (not shown), were
cocrosslinked with Fc
RIIA, a synergistic rise in [Ca2+]i
also occurred in Jurkat cells transfected with Fc
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 Fc
RIIIB alone (Fig. 3, top, and data not shown).
RIIIB is required for Fc
R cooperation but that other
extracellular domains will substitute for Fc
RIIIB when
cocrosslinked with Fc
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 Fc
RIIA and Fc
RIIIB interaction with the
2 integrin CR3 (for review see 30). Moreover,
since these cells do not express CR3, this experiment shows
that Fc
R synergy can occur without this PMN integrin.
RIIA and Fc
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, Fc
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
Fc
RIIA and CD59 was not as pronounced as with Fc
RIIIB and Fc
RIIA. No significant synergy between Fc
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
Fc
RIIIB (CD59 has ~13% of the expression of Fc
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 Fc
RIIIB
on the cell surface by 80% (35 and data not shown).
RIIA Is Required for Calcium Flux
receptor signaling (16, 43).
To determine whether this cascade had a role in Fc
receptor synergy, Jurkat cells were transfected with Fc
RIIIB
and a mutant Fc
RIIA 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 Fc
RIIA is ligated alone (27, 28). No synergistic [Ca2+]i flux occurred in J2Y
F/3 cells when Fc
RIIA was
ligated either alone or together with Fc
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 Fc
RIIA are required for the synergistic [Ca2+]i rise. Thus both the GPI
anchor of Fc
RIIIB and the ITAM motif of Fc
RIIA are
required for synergy in calcium signaling.
Fig. 4.
[Ca2+]i flux in cells expressing FcRIIA containing the
ITAM mutation. Fura 2-AM preloaded J2Y
F/3 cells were incubated with the mAbs IV.3 (anti-Fc
RII) and 3G8 (anti-Fc
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)]
RIIA
RIIA. When Fc
RIIA
was immunoprecipitated from J2/3 cells after crosslinking
Fc
RIIA alone, its tyrosine phosphorylation peaked at 1 min and was diminished by 5 min (Fig. 5 A, top). Surprisingly, crosslinking Fc
RIIA and Fc
RIIIB together did
not enhance tyrosine phosphorylation of Fc
RIIA as expected but actually diminished detection of the tyrosine
phosphorylation of Fc
RIIA (Fig. 5 A, top). Averages from
three experiments after normalization for the amount of
receptor immunoprecipitated showed that Fc
RIIA was
phosphorylated ~10-fold less under synergistic conditions as compared to ligation of Fc
RIIA alone. We also analyzed the tyrosine phosphorylation of Fc
RIIA in J2/3-CD7
cells. Ligation of Fc
RIIA without Fc
RIIIB 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 Fc
RIIA and Fc
RIIIB/CD7 did not significantly diminish the extent or alter the kinetics of Fc
RIIA phosphorylation (Fig. 5 A, bottom). To determine if the marked diminution of Fc
RIIA tyrosine phosphorylation also occurred when it was crosslinked with
other GPI-anchored proteins, Fc
RIIA was crosslinked
with CD48 or CD59 (Fig. 5 B). Cocrosslinking any GPI-anchored protein with Fc
RIIA markedly diminished its tyrosine phosphorylation. In addition, we analyzed the extent of tyrosine phosphorylation of Fc
RIIA in PMN after
ligating Fc
RIIA, individually or together with Fc
RIIIB,
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 Fc
RIIA
when compared to ligating Fc
RIIA alone (data not shown).
Fig. 5.
Tyrosine phosphorylation of FcRIIA after crosslinking Fc
R. (A) J2/3 (top) or J2/3-CD7 (bottom) cells were incubated with mAb IV.3 (anti-Fc
RII) or with mAbs IV.3 and 3G8
(anti-Fc
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 Fc
RII, Fc
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 Fc
RIIA immunoprecipitated.
Proteins were separated by SDS-PAGE, and blots were probed
with anti-phosphotyrosine. Cocrosslinking of GPI- but not transmembrane-anchored Fc
RIIIB diminishes tyrosine phosphorylation of Fc
RIIA. Blots shown are representative of at least five
experiments.
[View Larger Version of this Image (28K GIF file)]
1
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 Fc
RIIA
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 Fc
RIIA in the transfected Jurkat cells resulted in tyrosine phosphorylation of PLC-
1 that was visible by 1 min
(data not shown, and 42). Crosslinking Fc
RIIIB and Fc
RIIA in J2/3 cells resulted in tyrosine phosphorylation of
PLC-
1, which was not different from cocrosslinking Fc
RIIA and the chimeric Fc
RIIIB/CD7 in J2/3-CD7 cells
(Fig. 6). Thus, Fc
receptor synergy is independent of the
tyrosine phosphorylation of PLC-
1.
Fig. 6.
The tyrosine phosphorylation of PLC-1 after crosslinking various Fc
R. J2/3 (squares) or J2/3-CD7 (triangles) cells
were incubated with mAbs IV.3 (anti-Fc
RII) and 3G8 (anti-
Fc
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-
1 was immunoprecipitated,
and proteins were separated by SDS-PAGE. Blots were probed
with anti-phosphotyrosine and subsequently with anti-PLC-
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)]
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 Fc
RIIA with Fc
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 Fc
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
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,
v
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.
RIIA and Fc
RIIIB on human
PMN presents an opportunity to test these hypotheses
concerning signal transduction by GPI-linked proteins.
When immune complexes bind to PMN, Fc
RIIA and
Fc
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 Fc
receptor
synergy on PMN. In Jurkat cells without Mac-1, Fc
RIIA
and Fc
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 Fc
RIIIB, also can synergize
with Fc
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 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).
RIIA with glycolipid domains enriched in GPI-linked
proteins fundamentally alters subsequent signaling. Cocrosslinking Fc
RIIA with any of the GPI-linked proteins
induced the synergistic increase in [Ca2+]i and, surprisingly,
decreased the extent of Fc
RIIA tyrosine phosphorylation. When Fc
RIIIB was expressed with a transmembrane domain, its synergy with Fc
RIIA was abolished, as
was its effect on Fc
RIIA tyrosine phosphorylation. These
data support the hypothesis that the membrane environment of Fc
RIIA is altered by crosslinking it with GPI-
anchored proteins. This altered environment modulates the Fc
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
Fc
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 Fc
RIIA has diminished tyrosine phosphorylation after cocrosslinking with Fc
RIIIB, because ligation with
GPI-linked proteins causes Fc
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-
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).
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, Fc
RIIB, inhibits phagocytosis
mediated by Fc
RIIA. Decreased tyrosine phosphorylation induced by Fc
RIIB after interaction with IgG ligand
may be responsible for this inhibition of Fc
RIIA-mediated phagocytosis (Hunter, S., and A.D. Schreiber, unpublished results).
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 Fc
RIIIB and the ITAM of
Fc
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 Fc
RIIA molecules are expressed on the cell surface together with 10 to 20 times
more Fc
RIIIB (12, 13). Thus it is highly likely that whenever Fc
RIIA is ligated by an immune complex, it is in association with several GPI-linked Fc
RIIIB and that the
modulated signal which occurs because of association with
GPI domains is the major mechanism of immune complex-mediated PMN activation.
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.eduWe thank Dr. Ming-jie Zhou (Molecular Probes, Inc.) for the PCR clone
of CD16, Dr. Brian Seed for the CD16/CD7/ 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.
[Ca2+]i, intracytoplasmic Ca2+ concentration; GPI, glycan phosphoinositol; ITAM, immunoreceptor tyrosine-based activation motif; PLC, phospholipase C; PMN, polymorphonuclear neutrophils.
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