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RIIIB-to-CR3 Proximity on Neutrophils1
*
Department of Biological Sciences, Wayne State University, Detroit, MI 48202; and
Howard Hughes Medical Institute and Department of Internal Medicine and Biological Chemistry, and
Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIIB (CD16b) and inhibits L-selectin
shedding. In this study, we test the hypothesis that sGP interferes
with the physical linkage between CR3 and FcRIIIB. Neutrophils were
stained with rhodamine-conjugated anti-CD16b mAb (which does not
inhibit sGP binding) and fluorescein-conjugated anti-CR3 mAb
reagents and then incubated in media with or without sGP. Physical
proximity between fluorochrome-labeled CR3 and FcRIIIB on individual
cells was measured by resonance energy transfer (RET) imaging,
quantitative RET microfluorometry, and single-cell imaging
spectrophotometry. Cells incubated with control supernatants displayed
a significant RET signal, indicative of physical proximity (<7 nm)
between CR3 and FcRIIIB. In contrast, cells exposed to sGP showed a
significant reduction in the CR3-FcRIIIB RET signal using these
methods. Interestingly, colocalization and cocapping of CR3 and
FcRIIIB were not affected, suggesting that the proximity of these
two receptors is reduced without triggering dissociation. Thus, sGP
alters the physical linkage between FcRIIIB and
CR3. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIIB (CD16b) and
that it acts in vitro to inhibit L-selectin down-regulation
(4), which may affect host defense mechanisms. Because
FcRIIIB is a GPI-linked membrane protein, it is unclear how
sGP-FcRIIIB complexes affect neutrophil activation. One mechanism of
FcRIIIB signaling is via lateral membrane interactions with CR3
(5, 6, 7, 8, 9, 10). Early studies showed that FcRIIIB cocaps with
CR3 and that immune complexes trigger CR3 interactions with
microfilaments (5, 11). Moreover, FcRIIIB has been
observed in proximity to CR3 on neutrophil membranes in the absence of
a capping stimulus using resonance energy transfer (RET) (7, 8). Further studies have shown that certain saccharides and
polysaccharides can affect FcRIIIB-dependent cell activation and
calcium signaling by competing with FcRIIIB for the lectin-like
binding site of CR3 (9). Stockl et al. (10)
have mapped the FcRIIIB binding site to the membrane proximal region
of CD11b. Thus, the interactions of FcRIIIB and CR3 contribute to
inflammatory signaling. Therefore, we hypothesized that the inhibitory
effect of sGP on neutrophil functions may be due to an sGP-mediated
disturbance of the FcRIIIB interactions with CR3. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N-acetyl-D-glucosamine (NADG) was obtained from Sigma (St. Louis, MO). FITC and tetramethylrhodamine isothiocyanate (TRITC) were obtained from Molecular Probes (Eugene, OR).
Secretory glycoprotein
sGP was prepared as described previously (12). Briefly, an expression vector for sGP and a plasmid control were transfected into human 293 cells. Culture supernatants containing sGP or controls were used in the experiments described below.
Preparation of neutrophils
Neutrophils were isolated from peripheral blood obtained from
healthy adults using heparinized tubes, by Ficoll-Hypaque (Sigma)
step-density gradient centrifugation. The cell preparation was 
95%
neutrophils. The purified cells were typically 95% viable, as assessed
by trypan blue exclusion.
Monoclonal Abs
Mouse mAbs to CR3 (anti-Mo1, clone 44) were obtained as
described previously (5). F(ab')2
fragments were prepared as described (5).
F(ab')2 fragments directed against FcRIII
(clone 3G8) were obtained from Medarex (West Lebanon, NH). Intact mAbs
directed against FcRIII clones DJ130c (IgG1) and 1D3 (IgM) were
obtained from Dako (Carpinteria, CA) and Colter/Immunotech (Hialeah,
FL), respectively.
Preparation of FITC- and TRITC-conjugated Abs
mAb or their F(ab')2 fragments were dialyzed against 0.15 M carbonate-bicarbonate buffer at pH 9.3 for 4 h at 4°C (5). Samples were incubated with dyes at a F/P ratio of 40 µg TRITC or 30 µg FITC per mg Abs at room temperature for 4 h. The fluorescent conjugates were separated from unreacted fluorochromes by Sephadex G-25 (Sigma) column chromatography. Purified conjugates were dialyzed against PBS at pH 7.4 overnight at 4°C.
Fluorescence labeling
Neutrophils in suspension were labeled with
fluorochrome-conjugated IgG (DJ130c), IgM (1D3),
F(ab')2 fragments (3G8) of anti-FcRIIIB,
or F(ab')2 fragments of anti-CR3 (clone 44)
mAbs for 30 min at 4°C. The cells were washed twice and then labeled
with a second mAb to create FITC-TRITC pairs.
Cocapping
Cocapping experiments were performed as described previously (5). CR3 was capped using F(ab')2 fragments of clone 44. Cells were capped using goat F(ab')2 fragments of a goat anti-murine F(ab')2 IgG (5). To avoid interference with sGP binding, capped cells were probed for cocapping using clone DJ130c.
Fluorescence microscopy
An axiovert inverted fluorescence microscope with HBO-100 mercury illumination (Carl Zeiss, New York, NY) interfaced to a Dell 410 workstation (Round Rock, TX) via Scion SG-7 video card (Vay Tek, Fairfield, IA) was employed. The fluorescence images were collected by an intensified charge-coupled device camera, model XC-77 (Hamamatsu, Hamamatsu City, Japan) and processed with ScionImage software (Vay Tek). A narrow bandpass-discriminating filter set was used with excitation at 485DF22 nm and emission at 530DF30 nm for FITC and an excitation of 540DF20 nm and emission of 590DF30 nm for rhodamine (Omega Optical, Brattleboro, VT). Long pass dichroic mirrors of 510 nm and 560 nm were used for FITC and TRITC, respectively. For energy transfer imaging, the 485DF22, 5101p, and 590DF30 filter combination was used (13). In some experiments the RET intensity was quantitated using a photomultiplier tube (Hamamatsu) held in a Products for Research (Danvers, MA) housing. The signal was processed using a discriminator/amplifier (model 1762, Photochemical Research Associates, London, Ontario, Canada) and quantitated using a photon counter PRA model 1770. An average of three readings per target cell were used for each datum point. At least 100–150 cells were studied in each trial.
Single-cell imaging spectrophotometry
RET was also assessed using a microscope/imaging spectrophotometer system (e.g., see Ref. 14). To minimize light losses, a Zeiss IM-135 axiovert microscope with a bottom port was employed. The bottom port was fiber-optically coupled with an efficiency near 1.0 to the input side of an Acton-150 (Acton, MA) imaging spectrophotometer. The exit side was connected to a liquid nitrogen-cooled intensifier which was, in turn, attached to a Peltier-cooled I-MAX-512 camera (Princeton Instruments, Trenton, NJ). The collection of spectra or images was controlled by a high-speed Princeton ST-133 interface and a Stanford Research Systems (Sunnyvale, CA) DG-535 delay-gate generator. This high-efficiency, high-sensitivity system allowed medium-resolution emission spectra to be collected from individual cells in less than 1 s. These systems were interfaced to a Dell 410 workstation running Winspec software (Princeton Instruments) to manage and analyze data. Cells were illuminated using an optical filter at 485DF22 nm and a 510lp dichroic mirror. By employing a 520lp emission filter we were able to simultaneously collect spectra at both donor and acceptor emission wavelengths. This enabled us to simultaneously monitor the RET-mediated acceptor emission and the donor’s fluorescence-intensity quenching.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the hypothesis that Ebola virus sGP interferes with
interactions between CR3 and FcRIIIB, we performed RET experiments
with labels attached to CR3 and FcRIIIB on neutrophils after
incubation with sGP or control culture supernatants. For RET imaging,
neutrophils were incubated in the presence of cell culture supernatants
from sGP or control-transfected cells. Samples were first stained for
30 min at 4°C with rhodamine-conjugated reagents directed against
FcRIIIB (CD16) and then with a fluorescein-conjugated reagent
directed against CR3 (CD11b; clone 44). Three reagents directed against
FcRIIIB were employed: F(ab')2 fragments of
clone 3G8, which compete with sGP for FcRIIIB binding; an intact IgM
of clone 1D3; and intact IgG of clone DJ130c, which do not interfere
with the binding of sGP to cells (Ref. 4 and our
unpublished observations). Samples were incubated for 20 min. at 4°C
with various dilutions of control or sGP supernatants and then they
were placed on a 37°C microscope stage for further analyses.
Images of fluorescein, rhodamine and the RET emission channel were
collected with an intensified charge-coupled device camera. A
significant level of RET emission was observed using all
anti-FcRIIIB reagents (clones 3G8, DJ130c, and 1D3) in the
presence of control supernatants (Fig. 1
,
A–I). In contrast, exposure of neutrophils to
sGP supernatants dramatically decreased RET intensity, as assessed by
RET imaging, between CR3 and FcRIIIB (noncompeting clones DJ130c and
1D3) (Fig. 1, L and O). However, the RET level of
sGP-treated cells labeled with the sGP-competing anti-FcRIIIB
(clone 3G8) reagent was not affected (the clone 3G8-labeled receptors
cannot interact with sGP) (Fig. 1, P–R). Thus,
clone 3G8, which blocks sGP binding, served as a negative control.
Although the RET level was dramatically decreased between CR3 and
noncompeting FcRIIIB reagents in the presence of sGP (Fig. 1, L and O), the anti-CR3 and
anti-FcRIIIB labels remained colocalized at the cell surface
(Fig. 1, J, K, M, and N).
Thus, sGP appeared to block CR3 and FcRIIIB proximity (<7 nm)
without affecting colocalization.
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RIIIB
(clone DJ103c) as described above followed by exposure to control or
sGP supernatants (Table I). After 15 min
at 37°C, there was no statistically significant change in the
staining intensity of either CR3 or FcRIIIB when treated with
control or sGP supernatants. Therefore, incubation with sGP did not
cause the shedding (or displacement) of the labels or quench the
fluorescence of either fluorescein or rhodamine. Quantitative
dose-response studies were then conducted at the RET emission
wavelength. Cells incubated with control supernatants demonstrated
significant RET levels (Fig. 2,
a–c), as qualitatively seen in the images of
Fig. 1
, C, F, and I. Incubation of
neutrophils with sGP dramatically decreased RET intensity between CR3
and FcRIIIB for cells labeled with mAb 1D3 and DJ130c (Fig. 2, a and b). Moreover, the RET intensity decreased
in an sGP dose-dependent fashion. There was no reduction of the RET
intensity from CR3 to FcRIIIB in cells incubated with sGP and
labeled with an anti-FcRIIIB mAb (clone 3G8) that blocks sGP
binding to FcRIIIB (Fig. 1C). This finding is consistent
with the fact that mAb (clone 3G8) blocks sGP binding to cells. Another
GPI-linked protein, urokinase-type plasminogen activator receptor
(uPAR; CD87), is also known to interact with CR3 (15, 16).
To verify the specificity of sGP-FcRIIIB-CR3 interactions, we
performed quantitative RET measurements between CR3 and uPAR in the
presence of sGP. No change in RET intensity was observed (Fig. 2d) suggesting that sGP is specific for the FcRIIIB
interaction with CR3.
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The RET imaging and quantitative microfluorometry experiments
described above measure energy transfer by examining one spectral
region near the acceptor’s emission. Another physical means of
studying RET is the assessment of the donor chromophore’s emission
spectrum in the presence and absence of the acceptor. In contrast to
the above studies, single-cell spectrophotometry simultaneously
measures cell emission properties at many wavelengths across the
visible spectrum. We first examined the emission spectra of cells
individually labeled with either fluorescein anti-CR3 or rhodamine
anti-FcRIIIB reagents alone (Fig. 3A). The fluorescence emission
spectra and intensities of fluorescein and rhodamine labels were
not affected by sGP (Fig. 3B). For RET studies, emission
spectra (>520 nm) were collected from individual cells. For cells
treated with control supernatants, robust acceptor emission at 590
nm was observed in the presence of both the donor and acceptor but not
in the presence of the donor alone (Fig. 3, C, E,
G, and I, bold lines vs thin lines). However,
emission spectra of sGP-treated neutrophils labeled with CR3 and 1D3 or
DJ130c reagents were characterized by minimal emission beyond 570 nm
(Fig. 3, D and F). In contrast, RET emission was
observed between CR3 and FcRIIIB (clone 3G8) or uPAR in both the
presence and absence of sGP (Fig. 3, G–J). A
careful inspection of Fig. 3 reveals a reduction (or quenching) in the
FITC emission intensity for all samples demonstrating RET (Fig. 3, C, E, and G–J). This
reduction in FITC emission is due to energy transfer to TRITC. Thus,
these data provide broad spectral information and confirm RET using
FITC quenching, a second physical parameter.
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RIIIB (clone 1D3; acceptor) at
subsaturating doses with ratios of 5:1, 1:1, and 1:5. Fig. 4 shows representative emission spectra
illustrating the relative amounts of donor-quenching and RET acceptor
emission at three labeling ratios (Fig. 4, A, C,
and E) for control supernatant-treated neutrophils. We next
added a saturating dose of sGP to the same cells of Fig. 4, A, C, and E and collected emission
spectra of the donor (>520 nm) and acceptor (>570 nm) chromophores
(Fig. 4, B, D, and F). Although RET is
not observed in the presence of sGP (e.g., Fig. 4B, thin
line), the presence of sGP allows the direct confirmation of amount of
donor and acceptor chromophores associated with the cells without the
RET effects of donor quenching or acceptor emission. The experiments of
Fig. 4, B, D, and F confirm the
approximate levels of labeling anticipated by the mAb additions to the
samples. When the density of the donor exceeded that of the acceptor
(Fig. 4B), donor-quenching and the longer-wavelength RET
emission were observed in the absence of sGP (Fig. 4A).
Equal amounts of the donor and acceptor chromophores were used for the
spectra of Fig. 4, C and D. A bimodal spectrum
showing fluorescence emission from the donor and RET was noted in the
absence of sGP. When the number of the acceptor molecules exceeded the
donors (Fig. 4F), the relative RET emission intensity
increased dramatically (Fig. 4, F vs C). These
findings further confirm the results described above by demonstrating
the dependence on donor:acceptor ratio and the ability of sGP to block
RET at these ratios. Moreover, to our knowledge, this is the first
demonstration of the label density-dependence of RET in cells.
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These experiments demonstrated that energy transfer between CR3
and FcRIIIB was reduced by sGP. These data reflect the molecular
proximity (7 nm) of receptors, not their global distribution. The
colocalization experiments mentioned above (Fig. 1, J,
K, M, and N) suggest that CR3 and
FcRIIIB remain associated, although the labeled receptors no longer
display RET. To directly test this possibility, we performed cocapping
experiments. As previously reported (5), significant
levels of CR3 capping and CR3-FcRIIIB cocapping were observed (Fig. 5, A–C; Table II); however, sGP had no influence on
cocapping (Fig. 5, D and E; Table II). Fig. 5
shows micrographs of the same cell recorded before
(A–C) and after (D–F)
exposure to sGP. In this experiment, sGP was added directly to cells on
a tissue culture plate at 37°C on a heating stage. Thus, sGP appears
to intercalate into preformed caps to reduce RET. To ensure the
specificity of this effect, we also treated cells with the saccharide
NADG, which has been previously shown to reduce CR3-FcRIIIB
cocapping (5). NADG treatment substantially decreased
CR3-FcRIIIB cocapping without affecting the ability of CR3 to cap on
cells or the amount of tagged mAb on cells (data not shown). Thus, sGP
cannot dislodge FcRIIIB from CR3 caps, although it reduces receptor
proximity. However, NADG can dislodge FcRIIIB from CR3 caps in both
the presence and absence of sGP (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIIB, a GPI-linked
membrane protein, could mediate transmembrane signaling and
physiological functions (18, 19). This paradoxical ability
of FcRIIIB can be explained, at least in part, by its ability to
physically interact with CR3 (5, 6, 7, 8), which apparently is
linked to the functional activity of FcRIIIB (9).
Indeed, several additional neutrophil membrane proteins including the
urokinase receptor (CD87), CD14, and FcRII (CD32) apparently
interact with CR3 (15, 20, 21, 22, 23). These interactions have
been observed by a variety of techniques including cocapping, RET,
lateral diffusion, immunoprecipitation, gene transfection, and gene
knock-out experiments (5, 6, 7, 8, 9, 15, 16, 20, 21, 22, 23). Clinical
aberrations in these interactions have also been observed in
neutrophils from certain pyoderma gangrenosum patients (24, 25). These studies suggest a potential mechanism that accounts
for transmembrane signaling by certain GPI-anchored receptors. To our
knowledge, the present studies provide the first report of a product of
an infectious agent that affects receptor interactions on plasma
membranes, presumably to blunt host responses.
Exposure of cells to buffer alone or to supernatants from control
cultures had no effect on RET between FcRIIIB and CR3. The presence
of energy transfer under control conditions was confirmed by optical
imaging, quantitative microfluorometry, and single-cell imaging
spectrophotometry. Both the acquisition of acceptor fluorescence
emission and quenching of donor fluorescence emission were noted (thus
demonstrating that the donor lost energy while the acceptor gained
energy). Because RET is strongly distance-dependent, the labels
attached to FcRIIIB and CR3 must be within 7 nm of one another
(approximately molecular proximity). The intensity of RET is also
dependent upon the relative numbers of donor and acceptor chromophores.
Using an imaging spectrophotometry system, we have confirmed the
dependence of RET on the relative number of donor and acceptor labels
in this system. Therefore, as we have previously reported
(5, 6, 7), FcRIIIB and CR3 are in close physical proximity
on human neutrophils.
We have found that the sGP of Ebola virus dramatically reduces
FcRIIIB-to-CR3 RET on living human neutrophils. This reduction was
inferred by studies employing RET imaging, quantitative measurements,
and spectrophotometry of single cells. The decrease in RET was found to
be dependent upon the concentration of sGP, as illustrated by Fig. 2.
One potential mechanism for the reduction in FcRIIIB-to-CR3
proximity is the dissociation of FcRIIIB-CR3 complexes, as we have
previously noted in studies of saccharides and polysaccharides (e.g.,
Ref. 9). However, this is apparently not the case for sGP.
Surprisingly, the fluorescence staining patterns demonstrated
colocalization and cocapping of FcRIIIB and CR3 in the presence of
sGP (Figs. 1 and 5; Table II). A simple explanation consistent with
these observations is that sGP may intercalate between FcRIIIB and
CR3, thereby separating the labels by a greater distance and reducing
RET, without causing dissociation of FcRIIIB from CR3.
Alternatively, sGP could induce an extraordinary conformational change
in the receptors causing the orientation and/or distance between the
chromophores to be altered, or sGP could bind to FcRIIIB in such a
fashion that the line of sight between the labels is interrupted,
thereby blocking RET. Thus, several lines of evidence support the
hypothesis that sGP induces a physical change in the nature of
FcRIIIB-to-CR3 interactions in cell membranes.
Although a simple sGP intercalation model may be a considerable advance
in our understanding of sGP’s effect on neutrophils, it is only a
partial explanation. Although previous studies have shown that
anti-FcRIIIB clone 3G8 blocks sGP binding to cells
(4), FcRIIIB apparently is a necessary but insufficient
condition because transfectants expressing CR3 and FcRIIIB were
unable to bind sGP (our unpublished observations). We speculate that
another unidentified component of these CR3-containing membrane
complexes participates in sGP-to-cell binding. Nonetheless, sGP binding
diminishes cell function. Immune complexes, which activate neutrophil
metabolic flux (26), are unable to do so in the presence
of sGP, but can in the presence of control supernatants (our
unpublished observations). Similarly, the percentage of neutrophils
binding or internalizing IgG-opsonized SRBCs is reduced from 51 ±
15% in the presence of Fab fragments of anti-FcRII (clone IV.3) to
8 ± 8% in the presence of both anti-FcRII and sGP (data not
shown). Thus, whatever the sufficient conditions are for sGP binding to
cells might be, FcRIIIB-mediated functions and its normal physical
interactions with CR3 are disrupted.
We suggest that a step in the pathogenic effects of Ebola virus
infections involves the disruption of normal FcRIIIB-to-CR3
interactions by sGP. However, this disruption may not be limited to
FcRIIIB-mediated cell activation. Our findings raise the interesting
possibility that rationally designed compounds directed at exodomain
receptor-integrin interactions may be useful as anti-inflammatory
drugs.
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Footnotes |
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2 Current address: Vaccine Research Center, National Institutes of Health, Building 9, Room 1N124D, Bethesda, MD 20892.
3 Address correspondence and reprint requests to Dr. Howard R. Petty, Department of Biological Sciences, Wayne State University, Detroit, MI 48202. E-mail address:
4 Abbreviations used in this paper: sGP, secretory glycoprotein; RET, resonance energy transfer; NADG, N-acetyl-D-glucosamine; TRITC, tetramethylrhodamine isothiocyanate; uPAR, urokinase-type plasminogen activator receptor.
Received for publication July 16, 1999. Accepted for publication October 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor III on human neutrophils. J. Immunol. 150:3030.[Abstract]
receptor IIIB and the complement receptor type 3. J. Immunol. 153:1769.[Abstract]
receptor type IIIB with complement receptor type 3 in fibroblast transfectants: evidence from lateral diffusion and resonance energy transfer experiments. J. Mol. Biol. 247:597.[Medline]
-chain (CD11b) potentially involved in intramembrane complex formation with glycosylphosphatidylinositol-anchored FcRIIIB (CD16) molecules. J. Immunol. 154:5452.[Abstract]
1 and 3 integrins of fibrosarcoma cells: dependence on extracellular matrix components. Cancer Res. 57:1682.RIIIpmn mediates transmembrane signaling events distinct from FcRII. J. Exp. Med. 171:1239.2-integrins, and src-kinases within a single receptor complex of human monocytes. J. Exp. Med. 181:1381.RIIA (CD32) tail-minus mutant. J. Immunol. 157:5660.[Abstract]
M2, CD11b/CD18) and FcRIII cooperate in generation of a neutrophil respiratory burst: requirement for FcRII and tyrosine phosphorylation. J. Cell Biol. 125:1407.x2; CD11c/CD18) oscillations on neutrophils in a mild form of pyoderma gangrenosum. J. Invest. Dermatol. 111:154.[Medline]
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