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Science 30 October 1998:
Vol. 282. no. 5390, p. 843
DOI: 10.1126/science.282.5390.843a
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Technical Comments
Ebola Virus, Neutrophils, and Antibody Specificity
Zhi-yong Yang et al. (1)
report that the secreted form of the Ebola virus glycoprotein (sGP)
specifically interacts with neutrophils through the immunoglobulin G
(IgG) Fc receptor IIIb (Fc RIIIb, CD16), which is exclusively
expressed on neutrophils. Yang et al. conclude that sGP
"inhibits early neutrophil activation, which likely affects the host
response to [Ebola] infection." The study raised considerable
interest because it provided at least a partial explanation for the
high pathogenicity of Ebola virus (2, 3). The study,
however, did not exclude the possibility that the rabbit antibody used
to detect the binding of Ebola sGP mediated the binding of rabbit
IgG-sGP immune complexes to neutrophil FcRIIIb through its Fc
portion. To clarify this issue, we performed flow cytometry to
study the putative interaction of sGP with neutrophils with the use of
three human monoclonal antibody Fab fragments specific for sGP (Fab KS14, K518, and LS4) (4, 5) and rabbit IgG F(ab')2 fragments to sGP (6).
First, we confirmed that we could detect the binding of sGP-rabbit
antiserum immune complexes with neutrophils in flow cytometry with the
use of a fluorescein isothiocyanate (FITC)-labeled goat antibody to rabbit IgG, as previously shown by Yang et al.
(7). In contrast with this result, however, we could
not detect binding of sGP to neutrophils with the use of three human
monoclonal Fab fragments under similar conditions (Fab LS4 shown in
Fig. 1A; Fab KS14 and K518 yielded
similar results). It is unlikely that this result is due to overlapping
binding sites of the Fab fragments and the putative receptor binding
site on sGP, because strong binding of all three human Fabs to
neutrophil-bound sGP was found in the presence of the rabbit antibody
to sGP (Fig. 1B). These data suggest that the binding of Ebola sGP to
neutrophils may be mediated by the rabbit IgG used to detect the sGP,
rather than by a specific sGP-neutrophil interaction.
Fig. 1.
Flow cytometry assays.
Incubation with primary antibody, black solid lines; control
FITC-conjugated antibodies alone, red dotted lines. (A)
Binding of sGP and Fab fragment (LS4) to neutrophils. No binding was
detected when Fab fragments (LS4, KS14, and K518) (20 µg/mL) were
premixed with sGP and incubated with neutrophils (left panel: without
sGP, right panel: with sGP). Detection was performed with fluorescein
isothiocyanate (FITC)-conjugated goat IgG F(ab')2
fragments to human IgG F(ab')2 (1:100) (Pierce,
Rockford, Illinois) (data of KS14 and K518, not shown). (B)
Binding of Fab fragment LS4 and sGP to neutrophils in the presence of
rabbit anti-sGP serum. Fab LS4 (20 µg/ml) and rabbit anti-sGP serum
(1:250) were mixed in the absence (left panel) or the
presence (right panel) of sGP and incubated with neutrophils (1.25 × 105 cells). Binding of Fab LS4 to neutrophils was
detected only in the presence of both sGP and rabbit anti-sGP serum.
Detection was performed with FITC-conjugated goat IgG
F(ab')2 fragments to human IgG
F(ab')2. (C) Binding of sGP
(1:10) and purified rabbit IgG against sGP (5 µg/ml) to
neutrophils without (left panel) and with (right panel) sGP. Detection
was done by FITC-conjugated goat IgG F(ab')2 fragments to
rabbit IgG F(ab')2 (1:100) (Jackson, West Grove,
Pennsylvania). (D) Binding of sGP and rabbit IgG
F(ab')2 fragments against sGP (5 µg/mL) to neutrophils.
No binding of rabbit IgG F(ab')2 fragments against sGP to
neutrophils was observed in the absence (left panel) or presence (right
panel) of sGP. Detection was done with the use of FITC-conjugated goat
IgG F(ab')2 fragments to rabbit IgG F(ab')2.
Incubations were performed in a 50-µL volume at 4°C for 60 min with
sGP and the primary antibody, followed by a 20 min incubation with the
FITC-conjugated antibodies. Dilution of sGP, antibodies, serum, and
neutrophils and the washing of cells were done with phosphate-buffered
saline (PBS) containing 1% fetal calf serum (FCS) and 0.01%
NaN3.
[View Larger Version of this Image (31K GIF file)]
To investigate whether the rabbit antibody -dependent binding of
Ebola sGP was mediated by the Fc moiety of the rabbit IgG, we prepared
F(ab')2 fragments. Rabbit F(ab')2 retained the
same reactivity against sGP as did the whole rabbit IgG to sGP, as measured by enzyme-linked immunosorbent assay (ELISA) (Fig.
2). The rabbit IgG tested alone gave a
weak signal in flow cytometry, which was increased significantly in the
presence of sGP (Fig. 1C). In contrast, rabbit IgG F(ab')2
fragments to sGP did not bind to neutrophils either in the absence or
presence of sGP (Fig. 1D).
Fig. 2.
Binding of antibodies and antibody
fragments to sGP by ELISA. Wells were coated with sGP (1:10
in PBS) at 4°C overnight. After washing 5× with PBS/0.05% Tween 20, the wells were blocked with 4% (w/v) nonfat dry milk (Bio-Rad,
Hercules, California)/PBS. Rabbit IgG and IgG F(ab')2
fragments to sGP were serially diluted and added to the wells.
Detection was done with alkaline phosphatase-conjugated goat IgG to
rabbit IgG F(ab')2 (1:500) (Pierce, Rockford, Illinois).
Human Fab LS4 was included as a control and detected by alkaline
phosphatase-conjugated goat IgG to human IgG F(ab')2
(1:500) (Pierce). Incubations were performed at 37°C for
60 min. Rabbit IgG and IgG F(ab')2 fragments showed similar
binding reactivity to sGP.
[View Larger Version of this Image (24K GIF file)]
Finally, in contrast, to Yang et al., we did not detect any
absorption of sGP by purified neutrophils (Fig.
3), which makes it difficult to predict
the presence of any low affinity receptor for sGP on neutrophils.
Fig. 3.
Absorption test of sGP with purified neutrophils. sGP
(1:10) was incubated with purified neutrophils
(1×107) at 4°C for 60 min in a 50-µl volume, after
which the cells were removed by centrifugation. Supernatant containing
unabsorbed sGP was serially diluted in 1% FCS/PBS/0.01% NaN3 and
transferred to ELISA wells coated with rabbit IgG against sGP (4 µg/ml). Plate was washed and incubated with Fab LS4 (4 µg/ml).
Bound Fab was detected with alkaline phosphatase-conjugated goat IgG to
human IgG F(ab')2 (1:500) (Pierce, Rockford,
Illinois). sGP not incubated with neutrophils was also serially diluted
and detected in the same ELISA. ELISA incubations were performed at
37°C for 60 min, and wells were washed 5× with 0.05% Tween 20-PBS
at each step. No absorption of sGP by neutrophils was as observed
compared with the control wells (no incubation with
neutrophils).
[View Larger Version of this Image (17K GIF file)]
Thus, we conclude that Ebola sGP does not bind FcRIIIb (CD16) or any
other receptor on neutrophils and that the rabbit IgG against sGP used
for detection (1) bound to FcRIIIb through its Fc moiety
as an immune complex with sGP.
Toshiaki Maruyama
Department of Immunology,
Scripps Research Institute,
10550 North Torrey Pines Road,
La Jolla, CA 92037, USA
Michael J. Buchmeier
Department of Neuropharmacology,
Scripps Research Institute
Paul W. H. I. Parren
Department of Immunology,
Scripps Research Institute
Dennis R. Burton
Department of Immunology and
Department of Molecular Biology,
Scripps Research Institute
E-mail: burton{at}scripps.edu.
REFERENCES AND NOTES
-
Z.-y. Yang,
et al.,
Science
279,
1034
(1998)
[Abstract/Free Full Text]
.
-
I. Wickelgran, ibid., p. 983.
-
H.-D. Klenk,
V. E. Volchkov,
H. Feldmann,
Nature Med.
4,
388
(1998)
[CrossRef] [ISI] [Medline]
.
-
T. Maruyama et al., J. Infect.
Dis., in press.
-
Human Fab phage display libraries were constructed from bone
marrow RNA from two donors who recovered from infection with Ebola
virus during the 1995 outbreak in Kikwit, Democratic Republic of the
Congo. The three antibody Fab fragments (KS14, K518, and LS4) were
generated by panning the libraries against sGP-containing supernatants
derived from Vero E6 cells infected with the 1995 strain of Ebola Zaire
virus [provided by A. Sanchez at the Centers for Disease Control and
Prevention (CDC), Atlanta, GA].
-
Rabbit serum raised against sGP was provided by A. Sanchez,
CDC, Atlanta.
-
T. Maruyama et al., data not shown.
28 May 1998; accepted 20 August
1998
Response: Maruyama et al. have made an
interesting observation with regard to the binding of sGP of Ebola
virus to the neutrophil. Consistent with our previous report
(1), these investigators find that sGP binds to neutrophils
with the use of antiserum to the viral glycoprotein. They also
unexpectedly have found that a purified F(ab')2 antibody
fragment derived from this serum does not display similar reactivity.
We have confirmed this result and agree with this finding (Fig. 1, A
and B). Although it raises the
possibility that sGP binding could be secondary to antigen-antibody complexes binding to the Fc receptor, this explanation does not account
for all of the available data. For example, when neutrophils are
preincubated with purified rabbit immunoglobulin, sGP binding to these
cells is not inhibited, as would be expected if mediated by Ig binding.
In addition, the transmembrane form of GP, which should form similar
antigen-antibody complexes with this antisera, does not interact with
neutrophils, whereas it does bind to endothelial cells (1).
Finally, sGP can be depleted from cell culture media by absorption with
purified neutrophils (1). This latter result was not
observed by Maruyama et al., possibly because their enriched
sGP supernatant derives from a different source that is significantly
contaminated by other viral proteins, including the highly antigenic
nucleoprotein and VP40, which would not be absorbed, yet would be
detected in their ELISA assay.
Fig. 1.
Potentiation by IgG of sGP binding to human
neutrophils detected by F(ab')2 reagent. (A)
Freshly isolated human neutrophils from healthy donors (2 × 106) were incubated with sGP or control supernatants for 30 min on ice. Cells were spun and washed once with 1 ml of ice-cold PBS.
One hundred microliters of diluted antiserum to GP/sGP
(1:1000) was added and incubated on ice for 30 min. Cells
were spun again and washed with 1 ml of PBS. Finally, 100 µl of
1:75 diluted FITC-conjugated affiniPure (Fab')2
fragment goat anti-rabbit IgG-(Fab')2 (Jackson,
West Grove, Pennsylvania) was added and incubated on ice for 30 min.
Cells were washed once with PBS and resuspended in PBS + 1%
formaldehyde for fluorescence-activated cell sorter (FACS) analysis.
(B) Cells were incubated with sGP and control supernatants
as in (A), and followed by an incubation of 1:100 diluted
purified IgG (Fab')2 fragment rabbit antibody to GP/sGP
(0.4 mg/ml). Finally, the cells were incubated with FITC
(Fab')2 fragment goat antibody to rabbit
IgG(Fab')2 for 30 min on ice. Cells were washed once with 1 ml of ice-cold PBS after each step and resuspended in PBS + 1%
formaldehyde for FACS analysis. (C) Cells were pre-incubated
with 1 µg of rabbit IgG (Sigma, St. Louis, Missouri) in 100 µl on
ice for 30 min, followed by the incubations described in (B). Cells
were washed once with 1 ml of ice-cold PBS after each step.
[View Larger Version of this Image (16K GIF file)]
Another possibility was recently suggested by an experiment in which we
preincubated neutrophils with purified, nonspecific rabbit IgG before
adding sGP (Fig. 1). This preincubation step restored sGP binding to
neutrophils as detected by the F(ab')2 antibody fragment
(compare Fig. 1B with 1C). This finding raises the possibility that
engagement of FcIII (CD16) by immunoglobulin is required for sGP to
bind to the neutrophil. In this case, FcR occupancy would be necessary
for sGP binding to be detected by the antibody to sGP
F(ab')2 fragment and is consistent with the result of
Maruyama et al. A third potential explanation, suggested by
a reviewer, is that engagement of FcIII by immunoglobulin may
trigger neutrophils to express another activation antigen that binds
sGP. This model is also consistent with the available data. Because
neutrophils undergo variable activation during isolation in vitro, this
alternative could also account for differences observed in sGP
depletion by cellular absorption.
In summary, the observation of Maruyama et al. provides
further insight and suggests alternative mechanisms of sGP interaction with neutrophils. We had previously reported that CD16 alone was not
sufficient for binding to sGP. Together, the data suggest that sGP
binding to neutrophils is dependent on CD16, although additional
studies will be required to determine whether CD16 is directly or
indirectly responsible for the interaction. Regardless of the details
of the interaction of sGP with CD16 and binding to neutrophils, our
observation (1) that the secreted and transmembrane forms of
the Ebola glycoproteins have distinct cellular specificities remains
unchanged. It also remains important to determine whether sGP can be
detected on neutrophils from infected patients before its relevance to
disease progression and pathogenesis can be fully understood.
We would like to take this opportunity to correct an error in our
report. A FACS profile from the monocyte cell population was
inadvertently scanned into the far-left graph in figure 1A in our
report (1, p. 1034). The correct figure part is shown here
(as Fig. 2). This change does not alter the conclusion of the report in any way. We would like to thank Brett Lindenbach and
the Virology Journal Club at the Washington University School of
Medicine for bringing this error to our attention and also apologize
for any confusion this error may have caused.
Fig. 2.
Correct image, showing a FACS profile from
lymphocytes, that should have appeared in our report (1, p.
1034) as the far-left graph in figure 1A.
[View Larger Version of this Image (22K GIF file)]
Zhi-yong Yang
Rafael Delgado
Ling Xu
Robert F. Todd
Elizabeth G. Nabel
Anthony Sanchez
Gary J. Nabel
Howard Hughes Medical Institute,
University of Michigan Medical Center,
Ann Arbor, MI 48109-0650, USA
E-mail: gnabel{at}umich.edu
REFERENCES
-
Z-y. Yang
et al.,
Science
279,
1034
(1998)
.
9 July 1998; accepted 20 August 1998
THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
- The Ebola virus VP35 protein functions as a type I IFN antagonist.
- C. F. Basler, X. Wang, E. Mühlberger, V. Volchkov, J. Paragas, H.-D. Klenk, A. García-Sastre, and P. Palese (2000)
PNAS
| Abstract »
| Full Text »
- The Ebola virus VP35 protein functions as a type I IFN antagonist.
- C. F. Basler, X. Wang, E. Muhlberger, V. Volchkov, J. Paragas, H.-D. Klenk, A. Garcia-Sastre, and P. Palese (2000)
PNAS
97, 12289-12294
| Abstract »
| Full Text »
| PDF »
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