Download Influenza Virus Infections Significant Contribution to Clearance of Fc

Fc Receptor-Mediated Phagocytosis Makes a
Significant Contribution to Clearance of
Influenza Virus Infections
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of June 17, 2017.
Victor C. Huber, Joyce M. Lynch, Doris J. Bucher, Jianhua
Le and Dennis W. Metzger
J Immunol 2001; 166:7381-7388; ;
doi: 10.4049/jimmunol.166.12.7381
http://www.jimmunol.org/content/166/12/7381
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References
Fc Receptor-Mediated Phagocytosis Makes a Significant
Contribution to Clearance of Influenza Virus Infections1
Victor C. Huber,* Joyce M. Lynch,* Doris J. Bucher,† Jianhua Le,† and Dennis W. Metzger2*
I
t is believed that humoral immunity is required for the prevention of influenza infection through neutralization of free
infective particles (1, 2), whereas cell-mediated immunity involving cytotoxic T cells is primarily responsible for lysis of virally infected cells and recovery from infection (3–7). However,
recent studies have indicated that both T helper cells (8) and B
cells (9 –11) have important roles in recovery from influenza infection. Previously, we showed that intranasal (i.n.)3 immunization
of mice in the presence of IL-12 induced large amounts of respiratory IgG2a Ab and led to significant protection from lethal influenza virus challenge (12). The production of IgG2a appears to
be pivotal in anti-viral immunity, as evidenced by the fact that
monoclonal IgG2a Abs protect mice from both influenza (13) and
Ebola (14) infections. Although neutralization of viral particles is
believed to be the primary function of Abs in anti-viral immunity
(15–20), it is also known that IgG2a is the most efficient isotype at
fixing complement (21) and binding to Fc receptors on macrophages (22, 23) and NK cells (24).
The development of mice with genetic disruptions in Fc receptor
expression has allowed detailed study of the importance of these
receptors in the clearance of infections (25). One Fc receptor
knockout mouse that has been developed lacks the common
␥-chain signaling molecule (26) shared by two Fc receptors that
interact with IgG (Fc␥RI and Fc␥RIII), as well as the high affinity
Fc receptor for IgE (Fc⑀RI) (27). Previous studies have shown that
FCR ␥⫺/⫺ mice lack opsonophagocytosis and Ab-dependent cell*Center for Immunology and Microbial Disease, Albany Medical College, Albany,
NY 12208; and †Department of Microbiology and Immunology, New York Medical
College, Valhalla, NY 10595
mediated cytotoxicity (ADCC) (26), and have increased susceptibility to fungal (28) and bacterial (25) infections. However, the
role of Fc receptors in the clearance of viral infections has not yet
been characterized.
In this study, we describe a novel role for Fc receptors in protection against influenza virus challenge. Upon i.n. immunization
with influenza vaccine, FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice produced
equivalent levels of cytokines and specific Abs, yet FcR ␥⫺/⫺ mice
were significantly more susceptible to influenza infection than FcR
␥⫹/⫹ mice. The role of FcR-bearing cells in protection was investigated using mice that are transgenic for human CD3⑀ and thus
lack functional NK cells (29), and by using an in vitro opsonophagocytosis assay with Ab-coated influenza virus and the J774A.1
macrophage cell line. The results are discussed in relation to the
role of Fc receptors and macrophages in mucosal immunity to
influenza virus.
Materials and Methods
Mice
Adult (4 – 8 wk old) BALB/c mice with a genetic disruption in expression
of the FcR ␥-chain (26) were obtained from Taconic Farms (Germantown,
NY). Age-matched FcR ␥⫹/⫹ BALB/c controls were purchased from
Charles River Breeding Laboratories (Raleigh, NC) through the National
Cancer Institute (Bethesda, MD). Adult (C57BL/6J ⫻ CBA/J)F1 mice
transgenic for the human CD3⑀ signaling subunit (29) and nontransgenic
controls were obtained from The Jackson Laboratory (Bar Harbor, ME).
All experiments were performed in accordance with guidelines established
by the Institutional Animal Care and Use Committee at Albany Medical
College (Albany, NY).
Received for publication January 23, 2001. Accepted for publication April 10, 2001.
i.n. immunization
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Immunizations were performed as described previously (12). Briefly, mice
were anesthetized by i.p. injection of 80 mg kg⫺1 Ketamine HCl (Fort
Dodge Laboratories, Fort Dodge, IA) and 16 mg kg⫺1 Xylazine (Phoenix
Pharmaceuticals, St. Joseph, MO) diluted in PBS to a final volume of 200
␮l per mouse. The anesthetized mice were inoculated i.n. with 5 ␮g of an
influenza A/PR/8/34 protein preparation containing hemagglutinin subtype
1 (H1) and neuraminidase subtype 1 (N1). In addition, mice were inoculated i.n. with 1 ␮g recombinant murine IL-12 using 1% (v/v) normal
mouse serum in PBS (1% NMS-PBS) as a vehicle. The total volume used
for i.n. immunization was 50 ␮l per mouse. Recombinant murine IL-12
was provided by V. H. Van Cleave (Genetics Institute, Cambridge, MA).
1
This research was supported by National Institutes of Health Grants AI41715 and
HL62120.
2
Address correspondence and reprint requests to Dr. Dennis W. Metzger, Center for
Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208. E-mail address: [email protected]
3
Abbreviations used in this paper: i.n., intranasal; ADCC, Ab-dependent cell-mediated cytotoxicity; H1, hemagglutinin subtype 1; N1, neuraminidase subtype 1; 1%
NMS-PBS, 1% (v/v) normal mouse serum in PBS; BAL, bronchoalveolar lavage.
Copyright © 2001 by The American Association of Immunologists
0022-1767/01/$02.00
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Fc receptors for IgG expressed on macrophages and NK cells are important mediators of opsonophagocytosis and Ab-dependent
cell-mediated cytotoxicity. Phagocyte-mediated opsonophagocytosis is pivotal for protection against bacteria, but its importance
in recovery from infection with intracellular pathogens is unclear. We have now investigated the role of opsonophagocytosis in
protection against lethal influenza virus infection by using FcR ␥ⴚ/ⴚ mice. Absence of the FcR ␥-chain did not affect the expression
of IFN-␥ and IL-10 in the lungs and spleens after intranasal immunization with an influenza subunit vaccine. Titers of serum and
respiratory Abs of the IgM, IgG1, IgG2a, and IgA isotypes in FcR ␥ⴚ/ⴚ mice were similar to levels seen in FcR ␥ⴙ/ⴙ mice.
Nevertheless, FcR ␥ⴚ/ⴚ mice were highly susceptible to influenza infection, even in the presence of anti-influenza Abs from
immune FcR ␥ⴙ/ⴙ mice. NK cells were not necessary for the observed Ab-mediated viral clearance, but macrophages were found
to be capable of actively ingesting opsonized virus particles. We conclude that Fc receptor-mediated phagocytosis plays a pivotal
role in clearance of respiratory virus infections. The Journal of Immunology, 2001, 166: 7381–7388.
7382
PHAGOCYTOSIS CONTRIBUTES TO THE CLEARANCE OF INFLUENZA
Cytokine measurements
Cell culture conditions
Mice were sacrificed by Halothane (Halocarbon Laboratories, River Edge,
NJ) inhalation 24 h after immunization, and RNA from spleens and lungs
was prepared using the Ambion Total RNA Isolation Kit (Ambion, Austin,
TX). Two microliters cDNA prepared using the Life Technologies (Grand
Island, NY) reverse transcription kit were analyzed for IFN-␥ and IL-10 by
real-time PCR with a Perkin-Elmer (Branchburg, NJ) ABI Prism 7700
Sequence Detection System and the TaqMan PCR Reagent Kit. Amplification was performed using the primers, probes, and conditions described
previously (30). Primer (300 nM) and 200 nM probe were used. Primers
and probes were mixed with 3.5 mM MgCl2, 200 ␮M dATP, 200 ␮M
dCTP, 200 ␮M dGTP, 400 ␮M dUTP, 0.025 U ␮l⫺1 AmpliTaq Gold Taq
polymerase, and 0.01 U ␮l⫺1 AmpErase Uracil N-glycosylase in buffer to
a final volume of 25 ␮l. Before amplification, samples were heated to 50°C
for 2 min followed by 95°C for 10 min. The samples were then subjected
to 45 cycles at 95°C for 15 s and 60°C for 1 min. The samples were
quantitated using known concentrations of plasmid DNA encoding murine
IFN-␥ and IL-10 (provided by R. M. Locksley, University of California at
San Francisco) (31). Differences in cytokine expression between groups of
mice were analyzed using Student’s t test with statistical significance reported as p ⬍ 0.05.
The BALB/c macrophage cell line J774A.1 was obtained from the American Type Culture Collection (Manassas, VA). Cells were propagated in
DMEM with 4500 mg L⫺1 glucose, 110 mg L⫺1 sodium pyruvate HCl, and
NaHCO3 (Sigma). In addition, the medium was supplemented with 10%
(v/v) FBS, 4 mM L-glutamine (Life Technologies), 1 mM sodium pyruvate
(Life Technologies), and 10 ␮g ml⫺1 gentamicin (Sigma).
Bronchoalveolar lavage (BAL) fluid Ab analysis
Serum Ab analysis
Mice were immunized i.n. with H1N1 and boosted on days 14 and 28 as
described above. On day 35, serum obtained by bleeding mice from the
orbital plexus was analyzed by the same ELISA used to measure BAL Ab
levels. Titer values were compared for statistical significance using Student’s t test, with significant differences reported as p ⬍ 0.05.
Influenza virus challenge
Anesthetized mice were immunized i.n. with 5 ␮g H1N1 on day 0 and
treated with either 1 ␮g IL-12 in 1% NMS-PBS or 1% NMS-PBS alone on
days 0, 1, 2, and 3. Approximately 30 days later, these mice were challenged i.n. with 1 ⫻ 103 PFU A/PR/8/34 influenza virus in a volume of 40
␮l. Mice were then monitored daily for survival and weight loss. A loss of
33% of initial body weight was considered lethal, and mice that reached
this point were sacrificed by i.p. injection of 100 mg kg⫺1 Pentobarbital.
Passive transfer of serum
Sera obtained from mice 35 days after immunization were pooled and
adjusted to a standard total Ab titer of 7.4 ⫻ 104 ml⫺1 in PBS. This serum
pool was then injected i.p. in a 200-␮l volume into naive mice. Four hours
later, the mice were anesthetized and challenged i.n. with 2.7 ⫻ 102 PFU
A/PR/8/34 virus as described above. Mice were monitored daily for survival and weight loss.
A/PR/8/34 influenza virus was labeled with FITC (Sigma) as described
previously (32). Briefly, 1 ml of concentrated virus (⬃1 ⫻ 109 PFU) was
mixed with 100 ␮l of a 1 mg ml⫺1 solution of FITC in 1 M sodium
carbonate (pH 9.6) for 1 h at 37°C. This mixture was then dialyzed against
PBS for 18 h at 4°C. Opsonophagocytosis of FITC-labeled influenza was
analyzed by a modification of a previously described technique (33). Serum
samples containing an Ab titer of 1.4 ⫻ 103 in 20 ␮l were mixed with 10
␮l FITC-labeled virus at 37°C for 30 min. J774A.1 cells (1 ⫻ 106) were
then incubated with the opsonized A/PR/8/34 virus for 30 min at 37°C.
Extracellular fluorescence was quenched with 20 ␮l of a 0.2 mg ml⫺1
solution of trypan blue, and fluorescence was measured using a BD Biosciences (San Diego, CA) FACSCalibur flow cytometer. In some instances,
cells were photographed using an Olympus (Melville, NY) fluorescence
microscope with an Optronics (Goleta, CA) digital camera and software.
Depletion of total Ig was performed using Sepharose beads (Sigma)
coated with goat anti-mouse total Ig (Southern Biotechnology Associates).
Goat anti-mouse Ig was bound to Sepharose beads as described (34).
Briefly, 1 mg goat anti-mouse total Ig was mixed with cyanogen bromideactivated Sepharose beads at pH 3. After blocking unbound sites with 1 M
ethanolamine (Sigma), serum samples were mixed with the coated beads
overnight at 4°C and supernatants were collected. Depletion of total and
specific Ig from serum was confirmed by ELISA.
Confocal microscopy
Following incubation with FITC-labeled virus particles and quenching of
extracellular fluorescence with trypan blue as described above, J774A.1
cells were washed with PBS and placed onto a poly-L-lysine-coated coverslip. Images of optical sections, taken at 0.3-␮m intervals in the z-direction, were collected on a Nikon Diaphot inverted microscope (Melville,
NY) attached to a Noran-Oz laser scanning confocal microscope system
(Noran Instruments, Middleton, WI). Maximum intensity projection fluorescence images, fluorescence images of a single optical slice, and transmitted light images were generated using the Noran Intervision 3D and 2D
software packages, respectively.
Results
Cytokine expression in the lungs and spleens of immunized FcR
␥⫺/⫺ and FcR ␥⫹/⫹ mice
Cytokine mRNA levels were quantitated in the lungs and spleens
of mice 24 h after i.n. immunization with H1N1, either alone or
with IL-12 as an adjuvant. After immunization with the vaccine
alone, low levels of IFN-␥ and IL-10 were detected in the lungs
and the spleens (Table I), with no significant differences between
FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice. As previously described (12), IL-12
codelivery with the vaccine led to significant increases ( p ⬍ 0.05)
in both IFN-␥ and IL-10 levels in the lungs and the spleens of FcR
␥⫹/⫹ mice. Similar increases in cytokine expression were observed in FcR ␥⫺/⫺ mice after vaccine and IL-12 coadministration.
Systemic and respiratory anti-influenza Ab responses in FcR
␥⫺/⫺ and FcR ␥⫹/⫹ mice
After immunization with the H1N1 vaccine in the presence or
absence of IL-12, sera, and BAL fluids were analyzed for IgM,
IgG1, IgG2a, IgA, and total influenza-specific Ab. After i.n. inoculation of vaccine only, mice showed dominant expression of IgA
in the BAL fluid (Fig. 1), whereas IgM, IgG1, and IgA dominated
in the serum (Fig. 2). After codelivery of IL-12, IgM, and IgG2a
expression was increased in mucosal secretions, whereas IgG2a
was the only Ab isotype showing increased expression in serum.
With the exception of serum IgM levels, no significant differences
between FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice were observed. Influenza-
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Anesthetized mice were inoculated i.n. with 5 ␮g of H1N1 on day 0 and
with either 1 ␮g recombinant murine IL-12 in 1% NMS-PBS or 1% NMSPBS alone on days 0, 1, 2, and 3. The total volume used for each daily i.n.
inoculation was 50 ␮l per mouse. Mice were then boosted i.n. with 3 ␮g
H1N1 on days 14 and 28. Mice initially receiving IL-12 were given another
1-␮g dose of IL-12 i.n. on day 28. On day 35, mice were sacrificed by
inhalation of Halothane, and their lungs were immediately washed with 2
ml PBS containing 5 mM EDTA. Blood contamination was tested in the
BAL fluid using Albustix (sensitivity of 150 ␮g ml⫺1) (Bayer, Elkhart, IN),
and those BAL fluid samples with measurable levels of albumin were discarded. BAL fluid was stored at ⫺80°C after centrifugation at 12,000 ⫻ g
for 5 min to remove cellular debris.
Anti-H1N1 Abs were analyzed using isotype-specific ELISAs (12).
Briefly, 96-well microtiter plates (Nalge Nunc, Rochester, NY) were
coated by incubation with 1 ␮g ml⫺1 A/PR/8/34 virus (Charles River,
North Frankin, CT) in PBS overnight at 4°C. The plates were washed with
PBS containing 0.3% (v/v) Brij-35 (Sigma, St. Louis, MO) and then
blocked with PBS containing 5% (v/v) FBS (HyClone, Logan, UT) and
0.3% (v/v) Brij-35 for 1 h at room temperature. Two-fold serial dilutions
of BAL fluids were added to the plates and incubated overnight at 4°C.
After washing, alkaline phosphatase-conjugated goat anti-mouse isotypespecific Abs (Southern Biotechnology Associates, Birmingham, AL) were
added to the plates and incubated for 1 h at room temperature. p-nitrophenyl phosphate substrate (Sigma) was added to the plates, and OD at 405 nm
was measured using a Bio-Tek Microplate Autoreader (Bio-Tek Instruments, Winooski, VT). The reciprocal BAL dilution corresponding to 50%
maximal binding was reported as the titer. Titer values for each group were
compared for statistical significance using Student’s t test. Significant differences are reported as p ⬍ 0.05.
Opsonophagocytosis assay
The Journal of Immunology
7383
Table I. Cytokine responses in lungs and spleens of FcR ␥⫺/⫺ and FcR ␥⫹/⫹ micea
IFN-␥ Copy No. (⫻10⫺5)
Mouse
Genotype
FcR
FcR
FcR
FcR
Immunogen
⫹/⫹
␥
␥⫺/⫺
␥⫹/⫹
␥⫺/⫺
H1N1
H1N1
H1N1 ⫹ IL-12
H1N1 ⫹ IL-12
IL-10 Copy No. (⫻10⫺3)
Lung
Spleen
Lung
Spleen
7.3 ⫾ 1.2
8.9 ⫾ 1.8
39.3 ⫾ 3.8*
65.7 ⫾ 17.4*
8.1 ⫾ 1.4
6.7 ⫾ 0.8
39.0 ⫾ 18.9*
23.6 ⫾ 1.7*
⬍0.3 ⫾ 0
⬍0.3 ⫾ 0
5.9 ⫾ 3.1*
2.9 ⫾ 1.4*
1.4 ⫾ 1.6
5.0 ⫾ 5.8
32.5 ⫾ 14.2*
30.0 ⫾ 16.1
a
Mean copy numbers are shown ⫾ SD of three mice per group. *, p ⬍ 0.05 compared to mice receiving H1N1 alone using Student’s t test. There were no significant
differences between FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice. Hypoxanthine phosphoribosyl transferase expression was examined by RT-PCR, and was equal in all samples analyzed (data
not shown).
specific IgG2b and IgG3 levels were also measured, and no significant differences were seen between the two groups (data not
shown).
Susceptibility of FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice to influenza
infection
⫹/⫹
Protective effects of passively transferred immune serum in FcR
␥ ⫺/⫺ and FcR ␥⫹/⫹ mice
FcR ␥
and FcR ␥
mice were infected with 1 ⫻ 10 PFU
A/PR/8/34 influenza virus either after no pretreatment or 30 days
after exposure to a single dose of the influenza subunit vaccine ⫾
IL-12. In the group of mice that received no vaccine, FcR ␥⫺/⫺
mice were somewhat more susceptible to infection than FcR ␥⫹/⫹
mice (9.9 ⫾ 1.5 days mean survival for FcR ␥⫺/⫺ mice compared
with 11.6 ⫾ 0.5 days mean survival for FcR ␥⫹/⫹ mice) (Fig. 3A).
However, the difference in susceptibility was significantly greater
after i.n. immunization with the H1N1 vaccine, with 13.3 ⫾ 5.0
days and 25% survival among FcR ␥⫺/⫺ mice compared with
17.0 ⫾ 5.6 days with 63% survival among FcR ␥⫹/⫹ mice (Fig.
3B). Codelivery of IL-12 with vaccine enhanced protection in FcR
␥⫹/⫹ mice (19.9 ⫾ 3.2 days with 88% survival), as seen previously
(12), but failed to have any effect in FcR ␥⫺/⫺ mice (12.4 ⫾ 5.7
days with 25% survival) (Fig. 3C). Weight loss, expressed as a
Sera obtained from FcR ␥⫺/⫺ and FcR ␥⫹/⫹ BALB/c mice after
immunization with H1N1 and IL-12 were pooled, adjusted to a
total Ab titer of 7.4 ⫻ 104 ml⫺1, and transferred i.p. into naive
mice. Four hours later, the recipients were challenged i.n. with
2.7 ⫻ 102 PFU A/PR/8/34 virus. The dose of virus chosen for
infection was an amount that allowed differences in protective efficacy to be optimally detectable (⬃63% survival among naive
FcR ␥⫹/⫹ BALB/c mice after delivery of immune serum). As expected, normal serum from unimmunized mice failed to protect
FcR ␥⫹/⫹ mice regardless of whether the serum was derived from
FcR ␥⫹/⫹ mice (9.1 ⫾ 1.1 days with 0% survival) (Fig. 5A) or FcR
␥ ⫺/⫺ mice (9.1 ⫾ 0.8 days with 0% survival) (Fig. 5B). Immune
serum from FcR ␥⫹/⫹ mice protected FcR ␥⫹/⫹ mice to the expected level (16.9 ⫾ 6.0 days with 63% survival), but had signif-
FIGURE 1. Respiratory Ab responses in FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice.
Titer values are reported as the reciprocal BAL dilution corresponding to
50% maximal binding on the titration curve. Each symbol represents the
titer value for an individual mouse, with the line representing the mean titer
value for the group. Each FcR ␥⫹/⫹ group consisted of four mice, whereas
each FcR ␥⫺/⫺ group contained six mice. ⴱ, p ⬍ 0.05 compared with mice
receiving H1N1 alone.
FIGURE 2. Serum Ab responses in FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice. Titer
values are reported as the reciprocal serum dilution corresponding to 50%
maximal binding on the titration curve. Each symbol represents the titer
value for an individual mouse, with the line representing the mean titer
value for the group. Each FcR ␥⫹/⫹ group consisted of four mice, whereas
each FcR ␥⫺/⫺ group contained six mice. ⴱ, p ⬍ 0.05 compared with mice
receiving H1N1 alone. ⴱⴱ, p ⬍ 0.05 compared with FcR ␥⫹/⫹ mice.
3
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⫺/⫺
percentage of the initial body weight, was measured as a sign of
morbidity (Fig. 4). In all instances, mice lost weight until approximately day 10, at which point the mice that survived recovered to
preinfection levels.
7384
PHAGOCYTOSIS CONTRIBUTES TO THE CLEARANCE OF INFLUENZA
icantly reduced efficacy in FcR ␥⫺/⫺ mice (11.6 ⫾ 4.0 days with
13% survival) (Fig. 5C). Similarly, immune serum from FcR ␥⫺/⫺
mice protected FcR ␥⫹/⫹ mice (18.0 ⫾ 4.2 days with 63% survival), but not FcR ␥⫺/⫺ mice (11.0 ⫾ 1.5 days with 0% survival)
(Fig. 5D). Again, weight loss was monitored (Fig. 6), and mice lost
weight until approximately day 11, at which time the mice that
survived the infection began to regain weight. These results show
that FcR ␥⫺/⫺ mice are fully capable of producing protective Abs,
yet are significantly more susceptible to influenza, likely due to a
failure to effectively clear the infection through the action of Fc
receptor-bearing cells.
Protective effects of passively transferred immune serum in
CD3⑀-transgenic mice
To determine the potential role of ADCC mediated by NK cells in
the observed protective effects, passive transfer experiments were
performed with CD3⑀ mice, which lack both NK and T cells (29).
Serum was obtained from FcR ␥⫹/⫹ BALB/c mice after immunization with H1N1 and IL-12, and passively transferred into naive
CD3⑀ mice that were subsequently challenged as described above.
As expected, normal serum from unimmunized mice failed to protect either wild-type or CD3⑀ mice (11.3 ⫾ 0.5 days with 0%
survival for wild-type mice and 11.3 ⫾ 0.8 with 0% survival for
CD3⑀ mice) (Fig. 7A). In addition, transfer of immune serum protected both wild-type and CD3⑀ mice and resulted in 100% survival of each strain through day 17 of the influenza infection (Fig.
7B). The ability of CD3⑀ mice to survive an influenza infection
after passive transfer of serum demonstrates that neither NK nor T
FIGURE 4. Weight loss by FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice after influenza virus challenge. Mice were challenged with infectious A/PR/8/34 virus after either no pretreatment (A) or after immunization with H1N1 (B)
or H1N1 ⫹ IL-12 (C). Each group contained eight mice.
cells play an important role in Ab-mediated recovery from
infection.
Opsonophagocytosis of influenza virus by murine macrophages
An opsonophagocytosis assay was next used to measure the ability
of macrophages to ingest opsonized influenza virus particles. Abcoated, FITC-labeled A/PR/8/34 virus particles were mixed with
1 ⫻ 106 J774A.1 BALB/c cells, and the cells were analyzed by
flow cytometry. Use of serum from H1N1 ⫹ IL-12-immunized
mice resulted in an approximate 10-fold shift in the mean fluorescence intensity compared with normal mouse serum (Fig. 8A). Visualization of the cells by fluorescence microscopy (Fig. 8B)
showed more viral uptake by cells after incubation of virus with
serum from H1N1 ⫹ IL-12-immunized mice.
Three independent experiments revealed that serum from
H1N1-immunized mice increased mean fluorescence intensity values compared with nonimmune serum (Fig. 8C), and that the inclusion of IL-12 in the immunization regimen noticeably increased
the resulting efficacy of macrophage opsonophagocytosis. Depletion of total mouse Ig from immune serum reduced the ability of
the preparation to mediate opsonophagocytosis to the level observed with normal mouse serum, showing that Abs were responsible for the observed uptake of influenza virus. Blocking of
Fc␥RII and Fc␥RIII on the J774A.1 cells with 2.4G2 mAb before
exposure to the opsonized virus did not reduce opsonophagocytic
activity (data not shown), suggesting the importance of Fc␥RI in
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FIGURE 3. Survival of FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice after influenza
virus challenge. Mice were challenged with infectious A/PR/8/34 virus
after either no pretreatment (A) or after immunization with H1N1 (B) or
H1N1 ⫹ IL-12 (C). Each group contained eight mice.
The Journal of Immunology
7385
Discussion
the observed phagocytosis of virus. Confocal microscopy (Fig. 9)
was used to confirm that Ab-coated viral particles were internalized by the cells. Additional experiments showed that peritoneal
macrophages from FcR ␥⫺/⫺ mice were not able to phagocytose
virus as efficiently as FcR ␥⫹/⫹ macrophages, as expected (data
not shown).
FIGURE 6. Weight loss by FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice after passive
transfer of serum. Mice were challenged with A/PR/8/34 virus 4 h after i.p.
transfer of serum from either unimmunized mice (A and B) or from mice
immunized with H1N1 ⫹ IL-12 (C and D). Each group contained eight
mice.
FIGURE 7. Survival of CD3⑀-transgenic mice and wild-type controls
after passive transfer of serum. Mice were challenged with A/PR/8/34 virus
4 h after i.p. transfer of serum from either unimmunized mice (A) or from
mice immunized with H1N1 ⫹ IL-12 (B). Each wild-type control group
contained eight mice, whereas each CD3⑀-transgenic group contained
seven mice.
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FIGURE 5. Survival of FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice after passive
transfer of serum. Mice were challenged with A/PR/8/34 virus 4 h after i.p.
transfer of serum from either unimmunized mice (A and B) or from mice
immunized with H1N1 ⫹ IL-12 (C and D). Each group contained eight
mice.
After i.n. immunization with an influenza subunit vaccine, FcR
␥⫺/⫺ and FcR ␥⫹/⫹ mice showed similar cytokine and Ab responses, but FcR ␥⫺/⫺ mice were significantly more susceptible to
influenza infection than FcR ␥⫹/⫹ mice. IL-12 codelivery with the
vaccine enhanced protection in FcR ␥⫹/⫹ mice, but did not affect
survival of FcR ␥⫺/⫺ mice. Passive transfer of immune serum into
naive FcR ␥⫺/⫺ and FcR ␥⫹/⫹ mice directly demonstrated the
crucial role for host Fc receptors in protection of mice from influenza infection. Furthermore, passive transfer of serum into mice
lacking T and NK cells demonstrated a lack of involvement of NK
cell-mediated ADCC reactions in the observed viral clearance. A
viral opsonophagocytosis assay revealed that macrophages ingest
opsonized influenza virus. These findings implicate a pivotal role
for phagocytosis in the clearance of influenza virus.
Although there was variability in IFN-␥ expression in the
spleens of FcR ␥⫹/⫹ mice and the lungs of FcR ␥⫺/⫺ mice after
i.n. vaccination and IL-12 treatment, there were no statistically
significant differences between the groups. There was also no significant difference in IL-10 expression between FcR ␥⫺/⫺ and FcR
␥⫹/⫹ mice regardless of whether they received the vaccine alone
or the vaccine with IL-12. The increase in IL-10 copy number
observed after IL-12 treatment has been previously seen in our
laboratory (12, 35) and others (36 –38), and is believed to be important for down-regulating IFN-␥ levels, thus reducing potential
toxicity (39).
A study by Vora et al. (40) demonstrated that FcR ␥⫺/⫺ mice
respond the same as FcR ␥⫹/⫹ mice with regard to anti(4-hydroxy-3-nitrophenyl)acetyl serum Ab production. In general,
anti-influenza Ab expression in both serum and BAL of FcR ␥⫺/⫺
and FcR ␥⫹/⫹ mice in our experiments was similar, although FcR
␥⫺/⫺ mice had significantly higher serum IgM expression and noticeably higher levels of serum IgG1 and total Ab after vaccination. Significant increases in IgG1 expression in the absence of the
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PHAGOCYTOSIS CONTRIBUTES TO THE CLEARANCE OF INFLUENZA
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FIGURE 8. Analysis of J774A.1 cells after exposure to FITC-labeled A/PR/8/34 influenza virus. Flow cytometric (A) and fluorescence microscopic (B)
analysis of J774A.1 cells exposed to virus incubated with serum either from unimmunized mice or from mice immunized with H1N1 ⫹ IL-12. Additional
flow cytometry results using Ig-depleted sera are shown in C and are representative of three independent experiments. Absorption of serum with Sepharose
beads coated with normal goat Ig did not reduce opsonophagocytic activity (data not shown).
FcR ␥-chain have been reported by Kleinau et al. (41), but the
reason for this increase is unknown. The mechanism behind the
significant increase in serum IgM reported here is also unknown.
Two of four FcR ␥⫹/⫹ mice receiving H1N1 in the absence of
IL-12 displayed IgG2a Ab titers in their mucosal secretions. However, the IgG2a seen in these FcR ␥⫹/⫹ mice did not appear to
mediate the difference in survival rates between FcR ␥⫺/⫺ and FcR
␥⫹/⫹ mice because both types of mice given exogenous IL-12
The Journal of Immunology
7387
FIGURE 9. Analysis of J774A.1 cells by confocal microscopy. Transmitted light (A), fluorescence of an entire z-series (B), and a single optical section
taken at ⬃2.1 ␮m from the bottom of the coverslip (C) are shown for J774A.1 cells exposed to Ab-coated virus.
cells are also likely to be responsible for their protective functions
(45– 49). Such cytokines may in turn cause activation of Fc receptor-bearing macrophages, which then mediate virus clearance (50,
51). In addition to their ability to ingest Ab-coated particles by
opsonophagocytosis, macrophages also could potentially destroy
infected cells by ADCC (52). Interestingly, B cells but not cytotoxic lymphocytes were recently found to be required for heterosubtypic immunity to influenza virus infection (53). Our findings
have important implications for antiviral vaccination strategies and
stress the need for the targeting of Ab responses at mucosal sites
that preferentially stimulate Fc receptor-mediated host
mechanisms.
Acknowledgments
We thank Dr. Victor H. Van Cleave and Dr. Richard M. Locksley for their
generous contributions of recombinant murine IL-12 and cytokine-expressing bacterial plasmids, respectively. In addition, we thank Dr. Joseph Mazurkiewicz and the Albany Medical College Imaging Facility for assistance
with confocal microscopy.
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