Effective Targeting of Pathogens to
Neutrophils via Chimeric Surfactant Protein
D/Anti-CD89 Protein
This information is current as
of June 18, 2017.
Paul J. Tacken, Kevan L. Hartshorn, Mitchell R. White, Cees
van Kooten, Jan G. J. van de Winkel, Ken B. M. Reid and
Joseph J. Batenburg
J Immunol 2004; 172:4934-4940; ;
doi: 10.4049/jimmunol.172.8.4934
http://www.jimmunol.org/content/172/8/4934
Subscription
Permissions
Email Alerts
This article cites 38 articles, 18 of which you can access for free at:
http://www.jimmunol.org/content/172/8/4934.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
References
The Journal of Immunology
Effective Targeting of Pathogens to Neutrophils via Chimeric
Surfactant Protein D/Anti-CD89 Protein1
Paul J. Tacken,* Kevan L. Hartshorn,† Mitchell R. White,† Cees van Kooten,‡
Jan G. J. van de Winkel,§ Ken B. M. Reid,¶ and Joseph J. Batenburg2*
T
he ability of bacteria, viruses, and fungi to become resistant to therapeutic drugs together with the continuous
emergence of new pathogens call for new approaches to
the treatment of infectious disease. Recently, the myeloid Fc␣RI
(CD89) has been identified as an effective target molecule for
bispecific Ab-mediated immunotherapy (1). CD89 is expressed exclusively in myeloid cells, including neutrophils, eosinophils, and
monocytes/macrophages (2, 3). The expression of CD89 on neutrophils is induced by chemoattractants (4) and the inflammatory
mediator TNF-␣ (5) and is increased on infiltrated lung neutrophils
isolated from bronchoalveolar lavage fluid of cystic fibrosis patients with chronic lung infection (4). Upon stimulation, CD89
mediates both phagocytosis and induction of a respiratory burst (2,
3). Opsonization of pathogens with bispecific Abs consisting of an
Ab against a specific pathogen coupled to an Ab against CD89
induces phagocytosis and killing of the pathogen by CD89-expressing neutrophils (6, 7). In addition, CD89 targeting results in
enhanced clearance of Bordetella pertussis in the lungs of human
CD89 transgenic mice (6). The anti-CD89 Ab used in the targeting
studies, mouse mAb A77, binds CD89 at an epitope distinct from
the ligand binding domain. Therefore, A77 binding to CD89 will
*Department of Biochemistry and Cell Biology, Graduate School of Animal Health,
Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; †Department of Medicine, Boston University School of Medicine, Boston, MA 02118;
‡
Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands; §Department of Immunology, University Medical Center Utrecht, Utrecht,
The Netherlands; §Genmab, Utrecht, The Netherlands; and ¶Medical Research Council, Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom
Received for publication October 7, 2003. Accepted for publication February
10, 2004.
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.
1
This work was supported by European Commission Contract QLK2-CT-200000325 (to J.J.B.).
2
Address correspondence and reprint requests to Dr. Joseph J. Batenburg, Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht
University, P.O. Box 80176, 3508 TD Utrecht, The Netherlands. E-mail address:
[email protected]
Copyright © 2004 by The American Association of Immunologists, Inc.
not be hampered by high concentrations of Abs already present in
serum/tissues. However, a major disadvantage of bispecific Abs is
the fact that they only bind a narrow range of pathogens.
The present study evaluates the effectiveness of a surfactant protein D (SP-D)3/anti-CD89 chimeric protein as an approach to targeting a broad spectrum of pathogens to CD89. SP-D is secreted
into the airspaces of the lung by the respiratory epithelium and
plays a role in pulmonary host defense. It is a member of the
collectin family, which includes mannan-binding lectin, conglutinin, collectins 43 and 46, and SP-A. SP-D consists of an N-terminal domain, a collagenous domain, an ␣-helical coiled coil neck
domain, and a C-terminal globular carbohydrate recognition domain (CRD), which binds carbohydrates in a calcium-dependent
manner (for review, see Refs. 8 –10). Via its CRD, SP-D binds to
carbohydrate moieties on the surface of a great variety of pathogens. SP-D isolated from the lung consists of dodecamers or higher
order oligomeric complexes, which are formed by association of
trimers of the basic polypeptide chain at their N termini (8, 9).
Binding of multimeric SP-D to microorganisms can stimulate or
inhibit their uptake by phagocytes depending on the specific pathogen involved. Furthermore, binding often leads to aggregation of
the microorganisms, which may help in their removal via mucociliary clearance (reviewed in Refs. 8, 9, and 11).
In theory, a chimeric protein consisting of a trimeric fragment of
human SP-D and an mAb against CD89 should bind a broad range
of pathogens and direct them to CD89 on phagocytic cells. In the
present study we chemically cross-linked a recombinant trimeric
fragment of human SP-D (rfSP-D) to the Fab⬘ of mAb A77. The
resulting rfSP-D/anti-CD89 fusion protein markedly enhanced the
uptake of influenza A virus (IAV), Candida albicans, and Escherichia coli by human neutrophils in vitro. Furthermore, rfSP-D/
3
Abbreviations used in this paper: SP-D, surfactant protein D; CRD, carbohydrate
recognition domain; D-PBS, Dulbecco’s PBS with 1 mM calcium and 0.5 mM magnesium; IAV, influenza A virus; hSP-D, human SP-D; rfSP-D, recombinant fragment
of hSP-D; rhSP-D, recombinant hSP-D; sSMCC, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic 3-sulfo-n-hydroxysuccinimide.
0022-1767/04/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Targeting of specific pathogens to FcRs on immune effector cells by using bispecific Abs was reported to result in effective killing
of the pathogens, both in vitro and in vivo. Instead of targeting a specific pathogen to an FcR, we assessed whether a broad
spectrum of pathogens can be targeted to an FcR using surfactant protein D (SP-D). SP-D is a collectin that binds a great variety
of pathogens via its carbohydrate recognition domain. A recombinant trimeric fragment of SP-D (rfSP-D), consisting of the
carbohydrate recognition domain and neck domain of human SP-D, was chemically cross-linked to the Fabⴕ of an Ab directed
against the human Fc␣RI (CD89). In vitro, the chimeric rfSP-D/anti-CD89 protein enhanced uptake of Escherichia coli, Candida
albicans, and influenza A virus by human neutrophils. Blocking of the interaction between rfSP-D/anti-CD89 and either the
pathogen or CD89 abolished its stimulatory effect on pathogen uptake by neutrophils. In addition, rfSP-D/anti-CD89 stimulated
killing of E. coli and C. albicans by neutrophils and enhanced neutrophil activation by influenza A virus. In conclusion, rfSP-D/
anti-CD89 effectively targeted three structurally unrelated pathogens to neutrophils. (Col)lectin-based chimeric proteins may thus
offer promise for therapy of infectious disease. The Journal of Immunology, 2004, 172: 4934 – 4940.
The Journal of Immunology
4935
anti-CD89 enhanced killing of C. albicans and E. coli by neutrophils in in vitro killing assays and enhanced neutrophil activation
by IAV.
Materials and Methods
Materials
The previously described hybridoma that produces the A77 mAb (mIgG1)
directed against human CD89 (12) and the hybridoma that produces mAb
22 (mIgG1) directed against human Fc␥RI (CD64) (13) were provided by
Medarex (Annandale, NJ). Production of rCD89 and Abs 2D11 and 7D7
has been described previously (14). Human SP-D (hSP-D) was isolated
from bronchoalveolar lavage fluid of alveolar proteinosis patients and
checked for purity by SDS-PAGE and subsequent silver staining and Western blotting as described previously (15). Recombinant human SP-D (rhSPD), produced in CHO-K1 cells as described previously (16), was a gift
from Dr. E. Crouch (Washington University School of Medicine, St. Louis,
MO). Rabbit anti-hSP-D antiserum was detailed previously (15). Goat anti(mouse IgG) peroxidase conjugate was purchased from Nordic (Tilburg,
The Netherlands).
Recombinant fSP-D production
Production of rfSP-D/anti-CD89 and rfSP-D/anti-CD64
The rfSP-D was dialyzed against PE buffer (0.1 M sodium phosphate and
2.5 mM EDTA buffer, pH 7.4). A 20-fold molar excess of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic 3-sulfo-n-hydroxysuccinimide (sSMCC; Pierce,
Rockford, IL) was added to the rfSP-D. After incubation for 2 h in the dark
(at room temperature), rfSP-D/sSMCC was dialyzed against PE buffer.
mAb A77 was digested with pepsin (Sigma-Aldrich, St. Louis, MO) to
generate F(ab⬘)2, followed by reduction with 10 mM ME amine (SigmaAldrich) to generate Fab⬘. Fab⬘ were added to rfSP-D/sSMCC in a molar
ratio of 1.2:1. The solution was incubated in the dark at room temperature
for 3 h, followed by overnight incubation at 4°C. Subsequently, unbound
sites were alkylated by adding iodoacetamide (Sigma-Aldrich) to a final
concentration of 25 mM, followed by 30-min incubation at room temperature. The protein mixture was loaded onto a Superdex 200 column (50-ml
bed volume; Pharmacia Biotech, Uppsala, Sweden), and fractions were
collected and analyzed by SDS-PAGE and silver staining. Fractions containing unbound rfSP-D or Fab⬘ were discarded. Fractions containing coupled proteins were pooled and subjected to maltose-agarose affinity purification (Sigma-Aldrich). Endotoxins were removed by polymyxin B
treatment, as described by Wright and coworkers (18). Endotoxin levels of
polymyxin B-treated protein samples were determined by Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) and did not exceed 18
pg/␮g protein. The chimeric rfSP-D/anti-CD89 protein was analyzed by
SDS-PAGE, silver staining, and Western blotting. Recombinant fSP-D/
anti-CD64 was produced by cross-linking Fab⬘ of mAb 22 to rfSP-D according to the same procedure used for rfSP-D/anti-CD89. Fig. 1 shows a
diagram of the chimeric proteins, Ab fragments, and SP-D preparations
used in these studies.
Isolation of human neutrophils
Heparinized venous blood was drawn from healthy volunteers. Neutrophils
were separated by Ficoll-Histopaque (Sigma-Aldrich) density gradient centrifugation. The neutrophil layer was collected, and remaining erythrocytes
were removed by hypotonic lysis. Neutrophils were suspended in Dulbecco’s PBS with 1 mM calcium and 0.5 mM magnesium (D-PBS; Life Technologies, Grand Island, NY). Cell viability was ⬎98% as determined by
trypan blue exclusion. All studies involving human neutrophils were approved by the ethics committees of Utrecht University and Boston University, where these studies were conducted.
Measurement of pathogen uptake by human neutrophils
FITC-labeled IAV Phillipines/82 (H3N2; Phil82) and Puerto Rico/8/34
(H1N1; PR8) strains and FITC-labeled C. albicans strain 448585 (American Type Culture Collection, Manassas, VA) were prepared as described
previously (7, 19). Unless indicated otherwise, the Phil82 strain was used
in the IAV uptake experiments. Heat-killed, FITC-labeled E. coli K12 was
obtained from Molecular Probes (Leiden, The Netherlands). Experiments
on the uptake of IAV, E. coli K12, and C. albicans by human neutrophils
were performed essentially as described previously (7, 20, 21). Pathogens
were incubated for 30 min at 4°C (E. coli and C. albicans) or 37°C (IAV)
in D-PBS, or in D-PBS containing various concentrations of rfSP-D,
(r)hSP-D, and/or rfSP-D/anti-CD89. In experiments in which pathogens
were incubated with both rfSP-D/anti-CD89 and (r)hSP-D, both proteins
were added to the pathogens simultaneously. Where indicated, 7D7, 2D11,
rCD89, 100 mM maltose, or 5 mM EDTA was added to the experimental
samples and their controls. Subsequently, aliquots of samples were incubated with neutrophils at 37°C for 30 min (IAV), 20 min (E. coli), or 1 h
(C. albicans). The pathogen:neutrophil ratios were 10:1 for IAV, 12:1 for
E. coli K12, and 4:1 for C. albicans. Trypan blue (0.2 mg/ml) was added
to the samples to quench extracellular fluorescence. After washing with
PBS (Life Technologies), neutrophils were fixed with 2% (w/v) paraformaldehyde in PBS, and neutrophil-associated fluorescence was measured
using flow cytometry. The mean neutrophil fluorescence (⬎1000 cells
counted/sample) and the percentage of control neutrophil fluorescence
were calculated. For C. albicans, the number of fluorescent cells was determined (i.e., cells that displayed fluorescence ⬎99% that of untreated
neutrophils).
Measurement of neutrophil activation
IAV-Phil82 was incubated in D-PBS or in D-PBS containing 34 ␮g/ml
rfSP-D/anti-CD89 or rfSP-D in a volume of 50 ␮l for 30 min at 37°C.
Subsequently, samples were incubated with 4 ⫻ 106 neutrophils in a total
volume of 2 ml. H2O2 production was measured by the oxidation of scopoletin as described previously (22).
C. albicans and E. coli killing experiments
C. albicans strain 448585 (American Type Culture Collection) was cultured overnight at 37°C in Sabouraud dextrose broth (Sigma-Aldrich). E.
coli strain 25922 (American Type Culture Collection) was cultured overnight at 37°C in Luria-Bertoni broth (Sigma-Aldrich). Pathogens were
washed three times with PBS and were incubated in quadruplicate in 96well plates (U-bottom; Greiner, Alphen aan den Rijn, The Netherlands) for
10 min at room temperature with D-PBS, D-PBS containing rfSP-D, or
D-PBS containing rfSP-D/anti-CD89. Subsequently, either D-PBS alone or
5 ⫻ 105 neutrophils suspended in D-PBS (pathogen:neutrophil ratio, 4:1)
were added to the samples to a total volume of 135 ␮l. Plates were shaken
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Production of rfSP-D, consisting of eight Gly-X-Y repeats of the collagen
region, ␣-helical coiled coil neck region, and CRD, has been described
previously (17). In brief, rfSP-D was expressed in E. coli, in which it was
present in inclusion bodies, and was purified by a procedure involving
denaturation and renaturation of inclusion body material, followed by gel
and affinity chromatography. The purity of the rfSP-D was checked by
SDS-PAGE and subsequent Coomassie Blue staining and Western
blotting.
FIGURE 1. Diagram of wild-type, recombinant, and chimeric proteins.
Proteins used in this study include hSP-D isolated from bronchoalveolar
lavage fluid of alveolar proteinosis patients (hSP-D), full-length rhSP-D,
rfSP-D, an F(ab⬘)2 of the A77 mouse anti-human CD89 mAb (anti-CD89
F(ab⬘)2), an Fab⬘ of the A77 mAb (anti-CD89 Fab⬘), an Fab⬘ of the mAb
22 mouse anti-human CD64 (anti-CD64 Fab⬘), a chimeric protein consisting of rfSP-D and anti-CD89 (rfSP-D/anti-CD89) and a chimeric protein
consisting of rfSP-D and anti-CD64 (rfSP-D/anti-CD64). Note that this is
a schematic diagram, and that chemical cross-linking between rfSP-D and
the Fab⬘ resulted in a variety of chimeric products consisting of rfSP-D
coupled to one or more Fab⬘. Furthermore, cross-linking will not be restricted to the N terminus of rfSP-D.
4936
rfSP-D/ANTI-CD89 PROTEIN TARGETS PATHOGENS TO NEUTROPHILS
at 37°C at 400 rpm for 1 h (E. coli) or 2 h (C. albicans). Water was added
to lyse neutrophils by hypotonic shock. This did not affect pathogen viability, as determined in control experiments with known numbers of pathogens. Serial dilutions of the samples were prepared in PBS. E. coli samples
were plated on Luria-Bertoni agar (Sigma-Aldrich), whereas C. albicans
samples were plated on Sabouraud dextrose agar plates (Sigma-Aldrich).
CFUs were determined after incubation at 37°C for 24 h. Killing was
expressed as: percent killing ⫽ 100 ⫻ [CFU from control wells (without
neutrophils) ⫺ CFU from experimental wells]/[CFU from control wells
(without neutrophils)].
Statistics
Unless indicated otherwise, statistical analysis of the data was conducted
by ANOVA, whereupon, provided that the F-test indicated a significant
difference ( p ⬍ 0.05) among groups, comparisons with one particular condition were made with Dunnett’s test.
Results
Generation of rfSP-D/anti-CD89 and rfSP-D/anti-CD64
Effect of rfSP-D/anti-CD89 on pathogen uptake by neutrophils
The uptake of three different pathogens at various concentrations
of rfSP-D/anti-CD89 or rfSP-D is shown in Fig. 3. The rfSP-D/
anti-CD89 significantly enhanced uptake of IAV by neutrophils
(Fig. 3A), C. albicans (Fig. 3B), and E. coli K12 (Fig. 3C) compared with control samples, whereas no significant enhancement
was seen with rfSP-D. Uptake of unopsonized C. albicans by neutrophils was relatively low compared with uptake of unopsonized
IAV and E. coli K12; only 4% of neutrophils had ingested C.
FIGURE 3. Recombinant fSP-D/anti-CD89 enhances pathogen uptake
by neutrophils. Freshly isolated human neutrophils were incubated with
FITC-labeled IAV (A), C. albicans (B), and E. coli K12 (C), as described
in Materials and Methods. Pathogens were either unopsonized (control) or
opsonized with various concentrations of rfSP-D (E) or rfSP-D/antiCD89
(F). After incubation at 37°C, the mean neutrophil fluorescence (IAV and
E. coli K12) or the number of fluorescent neutrophils (C. albicans) was
determined by FACS analysis, and the percentages of the control value
were calculated. Data are the mean ⫾ SEM of at least four experiments.
Significant difference from unopsonized control, as determined by repeated
measures ANOVA and Dunnett’s test: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.
albicans at the end of the incubation (data not shown). For this
reason, uptake of C. albicans by neutrophils was assessed by determining the number of fluorescent neutrophils, rather than mean
neutrophil fluorescence. Stimulation of uptake of pathogens by
neutrophils in the presence of rfSP-D/anti-CD89 was confirmed by
fluorescence microscopy (data not shown).
FIGURE 2. SDS-PAGE and Western blot analysis of rfSP-D/antiCD89. The rfSP-D and the Fab⬘ of A77 were chemically cross-linked, as
described in Materials and Methods. The resulting protein mixture was
analyzed by SDS-PAGE and silver staining (A). Subsequently, the protein
mixture was subjected to gel filtration to separate coupled proteins from
free rfSP-D and Fab⬘. Fractions containing coupled protein were pooled
and subjected to maltose-agarose affinity purification. The affinity-purified proteins were subjected to SDS-PAGE, followed by silver staining (B). In addition, purified proteins were subjected to Western blot analysis with goat anti(mouse IgG) antiserum (C) and with rabbit anti-hSP-D antiserum (D).
Effects of anti-CD89 Fab⬘, anti-CD89 F(ab⬘)2, and rfSP-D/antiCD64 on uptake of E. coli by neutrophils
To determine whether the rfSP-D/anti-CD89-mediated increase in
pathogen uptake was not due solely to binding of its Fab⬘ to CD89,
experiments were conducted to determine the effect of Fab⬘ or
F(ab⬘)2 from mAb A77 (anti-CD89 Fab⬘ and anti-CD89 F(ab⬘)2,
respectively) on uptake of E. coli K12 by neutrophils. In contrast
to rfSP-D/anti-CD89, neither anti-CD89 Fab⬘ nor anti-CD89
F(ab⬘)2 stimulated uptake (Fig. 4).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Chemical cross-linking of the rfSP-D to the Fab⬘ of A77 resulted
in a mixture of proteins (Fig. 2A). Size separation by gel filtration,
followed by affinity purification to isolate proteins containing a
functional rfSP-D CRD domain, resulted in the isolation of crosslinked proteins with an apparent molecular mass of 100 kDa or
more (Fig. 2, B–D). The cross-linked proteins stained positively
for mouse IgG (Fig. 2C) and human SP-D (Fig. 2D). No bands
were visible in control experiments in which sera from nonimmunized goat and rabbit were used or in experiments testing for crossreactivity between the rabbit anti-hSP-D antiserum and anti-CD89
Fab⬘ and between the goat anti-(mouse IgG) peroxidase conjugate
and rfSP-D (data not shown). Similar patterns were obtained upon
generation of rfSP-D/anti-CD64 (data not shown).
The Journal of Immunology
4937
Table I. Blocking rfSP-D/anti-CD89 binding to either pathogens or
CD89 inhibits its effect on pathogen uptake by neutrophilsa
Pathogen Uptake (% of unopsonized control)
To exclude the possibility that chimeric proteins of rfSP-D and
Fab⬘ would nonspecifically stimulate neutrophils, the effect of
rfSP-D/anti-CD64 on E. coli K12 uptake by neutrophils was studied. The rfSP-D/anti-CD64 protein should not target pathogens to
neutrophils, as CD64 is an FcR that is not expressed on freshly
isolated neutrophils (23). Fig. 4 shows that, in contrast to rfSP-D/
anti-CD89, rfSP-D/anti-CD64 did not enhance E. coli K12 uptake.
Effect of blocking CD89 and CRD on rfSP-D/antiCD89-mediated pathogen uptake
SP-D binds carbohydrates on microorganisms via its CRD in a
calcium-dependent manner (9). This binding can be abolished by
chelation of calcium with EDTA or addition of sugars (24). To
determine whether the rfSP-D/anti-CD89-mediated increase in
pathogen uptake by neutrophils required binding of the CRD of the
rfSP-D moiety to the pathogen, CRD-pathogen interaction was
blocked by performing uptake experiments in the presence of either 100 mM maltose or 5 mM EDTA. The rfSP-D/anti-CD89mediated increase in pathogen uptake by neutrophils was significantly reduced by both maltose and EDTA (Table I). In addition,
uptake of the IAV strain PR8 by neutrophils was studied in the
presence and the absence of rfSP-D/anti-CD89. PR8 lacks the high
mannose carbohydrate moieties on hemagglutinin, a surface glycoprotein of IAV, that are required for SP-D binding (20). As
expected, the uptake of rfSP-D/anti-CD89-opsonized PR8 by neutrophils was not significantly enhanced compared with that of unopsonized PR8 (Table I). These observations suggest that CRDpathogen interaction is essential for rfSP-D/anti-CD89-mediated
pathogen uptake.
To determine whether rfSP-D/anti-CD89 binding to CD89 on
the neutrophil was required for enhancement of pathogen uptake,
experiments were performed in the presence of either soluble
rCD89 or mAbs directed against human CD89. The mAb 7D7 has
been described to bind CD89 at a similar epitope as A77 (14), the
mAb used for generation of rfSP-D/anti-CD89. In contrast, mAb
2D11 recognizes a different site in CD89 (14). Unfortunately,
rCD89 dramatically affected basal pathogen uptake levels by neutrophils at concentrations of 10 ␮g/ml and higher (data not shown).
Therefore, we chose to determine the effect of rCD89 on rfSP-D/
anti-CD89-mediated uptake at the relatively low rCD89 concentration of 4 ␮g/ml. Although the blocking effect of 4 ␮g/ml rCD89
on rfSP-D/anti-CD89-mediated E. coli uptake by neutrophils did
IAV-Phil82
IAV-PR8
C. albicans
E. coli K12
None
Maltose
EDTA
rCD89
7D7
2D11
196 ⫾ 23
122 ⫾ 4b
102 ⫾ 5c
ND
ND
ND
104 ⫾ 2
ND
ND
ND
ND
ND
309 ⫾ 31
129 ⫾ 14c
108 ⫾ 5c
156 ⫾ 24c
ND
ND
293 ⫾ 28
143 ⫾ 20c
106 ⫾ 2c
180 ⫾ 15
103 ⫾ 6c
274 ⫾ 56
a
Freshly isolated human neutrophils were incubated with FITC-labeled IAV, C.
albicans, and E. coli K12, as described in Materials and Methods. Experiments were
performed in D-PBS (no addition) or in D-PBS containing 100 mM maltose, 5 mM
EDTA, 4 ␮g/ml rCD89, 50 ␮g/ml 7D7, or 50 ␮g/ml 2D11. Pathogens were either
unopsonized or opsonized with 7.2 ␮g/ml (in the case of IAV-Phil82 and IAV-PR8),
16 ␮g/ml (in the case of C. albicans), or 0.5 ␮g/ml (in the case of E. coli K12)
rfSP-D/anti-CD89. After incubation at 37°C, the mean neutrophil fluorescence or the
number of fluorescent neutrophils was determined by FACS analysis. Data represent
the mean neutrophil fluorescence (IAV-Phil82, IAV-PR8, and E. coli K12) or the
number of fluorescent neutrophils (C. albicans) as a percentages of the unopsonized
control value ⫾ SEM of at least four experiments (except for IAV-PR8, which was
tested in three experiments). The significant difference from D-PBS (no addition) was
determined by ANOVA and Dunnett’s test.
b
p ⬍ 0.05.
c
p ⬍ 0.01.
not reach statistical significance, rCD89 significantly reduced
rfSP-D/anti-CD89-mediated C. albicans uptake (Table I). Furthermore, 50 ␮g/ml mAb 7D7 blocked the effect of rfSP-D/anti-CD89
on E. coli uptake by neutrophils, whereas 50 ␮g/ml 2D11 had no
such effect (Table I). Thus, blocking the interaction between rfSPD/anti-CD89 and CD89 on the neutrophils blocked rfSP-D/antiCD89-mediated pathogen uptake.
Effect of rfSP-D/anti-CD89 in combination with SP-D
In the presence of SP-D, uptake of IAV (20) and E. coli K12 (21)
by human neutrophils has been reported to be increased, whereas
uptake of C. albicans (25) by rat alveolar macrophages was reported to be decreased. Recombinant hSP-D optimally enhanced
uptake of E. coli by human neutrophils at concentrations of 0.5–1
␮g/ml (20), which is on the same order as SP-D levels found in
human serum (26). We chose to determine the effect of rfSP-D/
anti-CD89 on E. coli and C. albicans uptake by neutrophils in the
presence of SP-D at a concentration of 0.88 ␮g/ml hSP-D. For
experiments on IAV uptake by neutrophils, the effect of rfSP-D/
anti-CD89 was tested at different rhSP-D concentrations.
Uptake of IAV by neutrophils was increased to maximally
164 ⫾ 20% by rhSP-D compared with unopsonized control samples. Simultaneous addition of 7.2 ␮g/ml rfSP-D/anti-CD89 significantly increased IAV uptake by neutrophils at all rhSP-D concentrations tested (Fig. 5A), whereas no significant effect was seen
with 7.2 ␮g rfSP-D. In the presence of 0.88 ␮g/ml hSP-D, the
uptake of C. albicans was 252 ⫾ 20% of that without opsonization. Opsonization with 0.88 ␮g/ml hSP-D in combination with 8
␮g/ml rfSP-D/anti-CD89 significantly increased uptake to 309 ⫾
26%, whereas opsonization with 8.88 ␮g/ml hSP-D stimulated uptake to a significantly lower extent than opsonization with 0.88
␮g/ml hSP-D or with 0.88 ␮g/ml hSP-D plus 8 ␮g/ml rfSP-D/
anti-CD89 (Fig. 5B). Microscopic analyses suggested that the decrease in uptake at higher hSP-D levels was due to the formation
of large aggregates of C. albicans, which could not readily be
taken up by the neutrophils (data not shown). Opsonization of E.
coli with 0.88 ␮g/ml hSP-D had at best a minor effect on the
uptake of the bacteria (Fig. 5C). The uptake with 0.88 ␮g/ml
hSP-D was significantly increased by simultaneous addition of 2
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 4. Anti-CD89 Fab⬘, anti-CD89 F(ab⬘)2, and rfSP-D/anti-CD64
do not affect uptake of E. coli by neutrophils. FITC-labeled E. coli K12
were incubated in D-PBS (control) or in D-PBS containing 0.5 ␮g/ml
rfSP-D/anti-CD89 (f), rfSP-D/anti-CD64 (䡺), anti-CD89 F(ab⬘)2 (o), or
anti-CD89 Fab⬘ (s). Subsequently, samples were incubated with freshly
isolated neutrophils, as described in Materials and Methods. After incubation at 37°C, the mean neutrophil fluorescence was determined by FACS
analysis, and the percentages of the control value were calculated. Data are
the mean ⫾ SEM of at least four experiments. Significant difference from
control, as determined by ANOVA and Dunnett’s test: ⴱ, p ⬍ 0.01.
Addition
4938
rfSP-D/ANTI-CD89 PROTEIN TARGETS PATHOGENS TO NEUTROPHILS
␮g/ml rfSP-D/anti-CD89, but not by increasing the hSP-D concentration from 0.88 to 2.88 ␮g/ml.
These data indicate that at SP-D concentrations such as those
found in human serum (26), uptake of the investigated pathogens
by neutrophils can be more effectively stimulated by addition of
rfSP-D/anti-CD89 than by addition of a similar amount of SP-D.
Effect of rfSP-D/anti-CD89 on neutrophil activation and killing
of pathogens by neutrophils
The effect of rfSP-D/anti-CD89 on neutrophil activation by IAV
was determined by assessing neutrophil H2O2 production. Al-
though opsonization of IAV with rfSP-D did not significantly affect neutrophil activation, opsonization with rfSP-D/anti-CD89 resulted in a significant increase in H2O2 production compared with
unopsonized IAV (Fig. 6A).
Classical CFU assays were performed with C. albicans and E.
coli to assess whether rfSP-D/anti-CD89 enhances killing of
pathogens by neutrophils. The clinical isolate E. coli ATCC 25922
was used for killing experiments, as the E. coli K12 strain used in
the uptake experiments did not survive the hypotonic shock required to lyse neutrophils (data not shown). After 2 h of incubation, 21 ⫾ 4% of the unopsonized C. albicans were killed. Opsonization with rfSP-D did not significantly enhance killing, whereas
killing was significantly increased by opsonization with rfSP-D/
anti-CD89 (Fig. 6B). One-hour incubation of E. coli with neutrophils resulted in 15 ⫾ 2% killing. Similar to the results obtained
for C. albicans, opsonization of E. coli with rfSP-D did not significantly affect killing, whereas killing was significantly increased
by opsonization with rfSP-D/anti-CD89 (Fig. 6B).
Discussion
The implementation of Abs in clinical therapy has grown rapidly
over the last 2 decades. Abs now represent ⬎30% of biopharmaceuticals in clinical trials and are evaluated for treatment of cancer,
autoimmune diseases, transplant rejection, and infectious diseases
(27). Bispecific Abs are powerful tools for redirection of immune
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 5. Effect of rfSP-D/anti-CD89 in combination with SP-D.
Freshly isolated human neutrophils were incubated with FITC-labeled IAV
(A), C. albicans (B), and E. coli K12 (C). IAV was opsonized with various
concentrations of rhSP-D (E), opsonized with various concentrations of
rhSP-D in combination with 7.2 ␮g/ml rfSP-D/anti-CD89 (F), or opsonized with 0 or 8 ␮g/ml rhSP-D in combination with 7.2 ␮g/ml rfSP-D (Œ).
C. albicans was opsonized with 0.88 ␮g/ml hSP-D (䡺), 0.88 ␮g/ml hSP-D
in combination with 8 ␮g/ml rfSP-D/anti-CD89 (f), or 8.88 ␮g/ml hSP-D
(o). E. coli K12 was opsonized with 0.88 ␮g/ml hSP-D (䡺), 0.88 ␮g/ml
hSP-D in combination with 2 ␮g/ml rfSP-D/anti-CD89 (f), or 2.88 ␮g/ml
hSP-D (o). Data are the mean ⫾ SEM of at least four experiments. A, A
univariant ANOVA was performed on the logarithm of the fluorescence
values, with rhSP-D concentration, additives (rfSP-D/anti-CD89, rfSP-D,
or none), and the interaction between rhSP-D and additives as explanatory
factors and experiment number as a random variable. In those cases where
the F test indicated the significance of a difference (p ⬍ 0.05), multiple
comparisons were made with the Bonferroni correction. This analysis indicated that at each rhSP-D concentration tested, addition of rfSP-D/antiCD89 resulted in a significant additional effect: ⴱ, p ⫽ 0.000. B and C, Bars
indicated by the same letter differ significantly according to ANOVA, followed by the Student-Newman-Keuls test: A and B, p ⬍ 0.05; C, p ⬍
0.001; D and E, p ⬍ 0.01.
FIGURE 6. Recombinant fSP-D/anti-CD89 enhances neutrophil activation by IAV and stimulates killing of C. albicans and E. coli by neutrophils.
Neutrophil activation by IAV was determined by measuring neutrophil
H2O2 production (A). Unopsonized IAV (control; 䡺), IAV opsonized with
34 ␮g/ml rfSP-D (o), or IAV opsonized with 34 ␮g/ml rfSP-D/anti-CD89
(f) were incubated with neutrophils, and H2O2 production was determined
as described in Materials and Methods. Data represent nanomoles of H2O2
produced in 3 min by 4 ⫻ 106 neutrophils and are the mean ⫾ SEM of four
independent experiments. Killing of C. albicans and E. coli (ATCC 25922)
by neutrophils was assessed, as described in Materials and Methods (B). C.
albicans was either unopsonized (control; 䡺), opsonized with 16 ␮g/ml
rfSP-D (o), or opsonized with 16 ␮g/ml rfSP-D/anti-CD89 (f). E. coli
(ATCC 25922) was either unopsonized (control; 䡺), opsonized with 2
␮g/ml rfSP-D (o) or opsonized with 2 ␮g/ml rfSP-D/anti-CD89 (f). Data
are the mean ⫾ SEM of four experiments. Significant difference from
unopsonized controls, as determined by ANOVA and Dunnett’s test: ⴱ,
p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.
The Journal of Immunology
In its present form, rfSP-D/anti-CD89 might trigger an immunogenic reaction in humans, as it contains a murine Fab⬘. This
should be overcome by cross-linking rfSP-D, which has the human
amino acid sequence, to a fully human anti-CD89 Ab. The rfSP-D
part can be expected to be nonimmunogenic, as SP-D is present in
blood (26).
CD89 is expressed on neutrophils and various types of macrophages (34). This expression pattern makes it a suitable trigger
molecule for cell-mediated killing (1). Most likely, i.v. application
of rfSP-D/anti-CD89 would lead to targeting of pathogens to neutrophils and monocytes in blood, whereas intranasal application
would lead to targeting alveolar macrophages and infiltrated neutrophils. As an alternative for CD89, targeting to CD64 might be
preferable when the objective is to induce antigenic memory.
CD64 is not constitutively expressed on neutrophils, but is expressed on APCs such as monocytes/macrophages and dendritic
cells (23, 35). Furthermore, targeting of weak Ags to CD64 elicits
potent humoral responses and has been reported to induce immunological memory in human CD64 transgenic mice (29, 36). Although CD89 is expressed on monocytes/macrophages, reports on
whether CD89 is expressed on dendritic cells contradict each other
(37, 38). Thus, it remains to be determined whether targeting
CD89 induces humoral responses as potently as targeting CD64.
There are many cell surface molecules that could be used as a
target for immunotherapy. Future studies will have to reveal which
target molecule gives the best results for each individual targeting
strategy.
In conclusion, the present paper describes highly effective targeting of three structurally unrelated pathogens to immune effector
cells using a (col)lectin/anti-FcR chimeric protein. The strategy of
combining the broad pathogen recognition ability of (col)lectins
with the key role that FcRs play in host defense opens up new
ways for treatment and prevention of infectious disease.
Acknowledgments
We thank Dr. Erica Crouch for her gift of rhSP-D, Dr. Henk Haagsman for
his intellectual input, and the people of Eijkman-Winkler Institute at University Medical Center Utrecht for isolation of neutrophils.
References
1. Valerius, T., B. Stockmeyer, A. B. van Spriel, R. F. Graziano,
I. E. van den Herik-Oudijk, R. Repp, Y. M. Deo, J. Lund, J. R. Kalden,
M. Gramatzki, and J. G. J. van de Winkel. 1997. Fc␣RI (CD89) as a novel trigger
molecule for bispecific antibody therapy. Blood 90:4485.
2. Morton, H. C., M. van Egmond, and J. G. J. van de Winkel. 1996. Structure and
function of human IgA Fc receptors (Fc␣R). Crit. Rev. Immunol. 16:423.
3. Shen, L. 1992. Receptors for IgA on phagocytic cells. Immunol. Res. 11:273.
4. Hostoffer, R. W., I. Krukovets, and M. Berger. 1993. Increased Fc␣R expression
and IgA-mediated function on neutrophils induced by chemoattractants. J. Immunol. 150:4532.
5. Hostoffer, R. W., I. Krukovets, and M. Berger. 1994. Enhancement by tumor
necrosis factor-␣ of Fc␣ receptor expression and IgA-mediated superoxide generation and killing of Pseudomonas aeruginosa by polymorphonuclear leukocytes. J. Infect. Dis. 170:82.
6. Hellwig, S. M. M., A. B. van Spriel, J. F. P. Schellekens, F. R. Mooi, and
J. G. J. van de Winkel. 2001. Immunoglobulin A-mediated protection against
Bordetella pertussis infection. Infect. Immun. 69:4846.
7. van Spriel, A. B., I. E. van den Herik-Oudijk, N. M. van Sorge, H. A. Vilé,
J. A. G. van Strijp, and J. G. J. van de Winkel. 1999. Effective phagocytosis and
killing of Candida albicans via targeting Fc␥RI (CD64) or Fc␣RI (CD89) on
neutrophils. J. Infect. Dis. 179:661.
8. Clark, H., and K. B. M. Reid. 2002. Structural requirements for SP-D function in
vitro and in vivo: therapeutic potential of recombinant SP-D. Immunobiology
205:619.
9. Crouch, E. C. 2000. Surfactant protein-D and pulmonary host defense. Respir.
Res. 1:93.
10. Holmskov, U., S. Thiel, and J. C. Jensenius. 2003. Collectins and ficolins: humoral lectins of the innate immune defense. Annu. Rev. Immunol. 21:547.
11. Crouch, E., and J. R. Wright. 2001. Surfactant proteins A and D and pulmonary
host defense. Annu. Rev. Physiol. 63:521.
12. Monteiro, R. C., M. D. Cooper, and H. Kubagawa. 1992. Molecular heterogeneity
of Fc␣ receptors detected by receptor-specific monoclonal antibodies. J. Immunol. 148:1764.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
effector cells to specific targets. FcRs on immune effector cells
have been recognized as efficient trigger molecules for bispecific
Ab-mediated killing of both tumor cells and pathogens (1, 28 –30).
As a novel approach for treatment of infectious disease, we use
the ability of SP-D to recognize a wide variety of microorganisms
as a means to target pathogens to CD89. The rfSP-D/anti-CD89
chimeric protein effectively targets IAV, E. coli, and C. albicans to
neutrophils, leading to pathogen internalization. In contrast to
rfSP-D/anti-CD89, a molecule lacking the rfSP-D moiety (antiCD89 Fab⬘), a molecule containing a different Fab⬘ (rfSP-D/antiCD64), and a molecule lacking the Fab⬘ (rfSP-D) do not stimulate
uptake of E. coli by neutrophils. In addition, pathogen targeting to
neutrophils is blocked when EDTA or maltose hampers the CRDpathogen interaction, or when rfSP-D/anti-CD89 binding to the
neutrophil CD89 is reduced by rCD89 or CD89-blocking Ab.
Moreover, rfSP-D/anti-CD89 does not stimulate uptake of the IAV
PR8 strain, which has been described not to bind SP-D. These
findings indicate that rfSP-D/anti-CD89 functions as it was designed to do, namely, stimulating pathogen uptake by binding to
the pathogen via its rfSP-D CRD domain and by binding CD89 via
its Fab⬘ domain.
With regard to the blocking experiments with EDTA, it is relevant to note that in some of our experiments the level of uptake
in EDTA control samples differed from that in samples without
EDTA. Depending on the pathogen, EDTA increased, decreased,
or had no effect on uptake levels. However, uptake was never
completely blocked (data not shown). Apparently, uptake of pathogens by neutrophils can largely continue in the presence of EDTA.
This resembles the observations by Della Bianca et al. (31), who
observed that phagocytosis of Saccharomyces cerevisiae by neutrophils was not, or was only partly, inhibited by calcium depletion
depending on the receptors involved. Signal transduction upon
triggering of CD89 on neutrophils was found to depend on release
of calcium from an intracellular store that is not affected by extracellular addition of a calcium chelator (32). Therefore, the inhibition by EDTA of the stimulatory effect of rfSP-D/anti-CD89
on pathogen uptake by neutrophils (Table I) is probably not due to
a blockage of CD89-dependent uptake per se, but, rather, to blockage of binding of the rfSP-D moiety to the pathogens.
SP-D by itself has been reported to stimulate neutrophil uptake
of IAV (20) and E. coli (21). However, our data indicate that at
SP-D concentrations found in human serum (26), pathogen uptake
by neutrophils is more effectively stimulated by addition of rfSPD/anti-CD89 than by SP-D. The trimeric rfSP-D protein did not
significantly increase pathogen uptake by neutrophils at any of the
concentrations used in our studies.
In addition to increasing uptake, rfSP-D/anti-CD89 enhances
killing of both E. coli and C. albicans by human neutrophils in
vitro. With regard to IAV, targeting to neutrophils is likely to
incapacitate the virus, as IAV does not replicate in neutrophils
(33), whereas rfSP-D/anti-CD89 augments the activation of neutrophils by IAV (Fig. 6A), thus increasing their capacity to neutralize the virus.
SP-D binds to an overlapping, but distinct, range of pathogens
compared with other collectins and lectins. Together, the (col)lectins cover a broad spectrum of viral, fungal, and bacterial pathogens and could be useful tools in immunotherapy. As SP-D is
highly expressed in the lung, FcR targeting with SP-D-based chimeric proteins might prove useful in the treatment or prevention of
pulmonary infections. In addition, the chimeric proteins could be
applied systemically. A similar approach, using i.v. administration
of bispecific Abs directed against C. albicans and human Fc␥RI
(CD64), has been shown to effectively protect CD64 transgenic
mice from lethal invasive candidiasis (29).
4939
4940
rfSP-D/ANTI-CD89 PROTEIN TARGETS PATHOGENS TO NEUTROPHILS
26. Leth-Larsen, R., C. Nordenbaek, I. Tornoe, V. Moeller, A. Schlosser, C. Koch,
B. Teisner, P. Junker, and U. Holmskov. 2003. Surfactant protein D (SP-D)
serum levels in patients with community-acquired pneumonia. Clin. Immunol.
108:29.
27. Hudson, P. J., and C. Souriau. 2003. Engineered antibodies. Nat. Med. 9:129.
28. Segal, D. M., G. J. Weiner, and L. M. Weiner. 2001. Introduction: bispecific
antibodies. J. Immunol. Methods 248:1.
29. van Spriel, A. B., I. E. van den Herik-Oudijk, and J. G. J. van de Winkel. 2001.
Neutrophil Fc␥RI as target for immunotherapy of invasive candidiasis. J. Immunol. 166:7019.
30. van Spriel, A. B., H. H. van Ojik, and J. G. J. van de Winkel. 2000. Immunotherapeutic perspective for bispecific antibodies. Immunol. Today 21:391.
31. Della Bianca, V., M. Grzeskowiak, and F. Rossi. 1990. Studies on molecular
regulation of phagocytosis and activation of the NADPH oxidase in neutrophils:
IgG- and C3b-mediated ingestion and associated respiratory burst independent of
phospholipid turnover and Ca2⫹ transients. J. Immunol. 144:1411.
32. Lang, M. L., and M. A. Kerr. 2000. Characterization of Fc␣R-triggered Ca2⫹
signals: role in neutrophil NADPH oxidase activation. Biochem. Biophys. Res.
Commun. 276:749.
33. Cassidy, L. F., D. S. Lyles, and J. S. Abramson. 1988. Synthesis of viral proteins
in polymorphonuclear leukocytes infected with influenza A virus. J. Clin. Microbiol. 26:1267.
34. Monteiro, R. C., and J. G. J. van de Winkel. 2003. IgA Fc receptors. Annu. Rev.
Immunol. 21:177.
35. Fanger, N. A., K. Wardwell, L. Shen, T. F. Tedder, and P. M. Guyre. 1996. Type
I (CD64) and type II (CD32) Fc␥ receptor-mediated phagocytosis by human
blood dendritic cells. J. Immunol. 157:541.
36. Keler, T., P. M. Guyre, L. A. Vitale, K. Sundarapandiyan, J. G. J. van de Winkel,
Y. M. Deo, and R. F. Graziano. 2000. Targeting weak antigens to CD64 elicits
potent humoral responses in human CD64 transgenic mice. J. Immunol. 165:
6738.
37. Geissmann, F., P. Launay, B. Pasquier, Y. Lepelletier, M. Leborgne, A. Lehuen,
N. Brousse, and R. C. Monteiro. 2001. A subset of human dendritic cells expresses IgA Fc receptor (CD89), which mediates internalization and activation
upon cross-linking by IgA complexes. J. Immunol. 166:346.
38. Hamre, R., I. N. Farstad, P. Brandtzaeg, and H. C. Morton. 2003. Expression and
modulation of the human immunoglobulin A Fc receptor (CD89) and the FcR ␥
chain on myeloid cells in blood and tissue. Scand. J. Immunol. 57:506.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
13. Guyre, P. M., R. F. Graziano, B. A. Vance, P. M. Morganelli, and M. W. Fanger.
1989. Monoclonal antibodies that bind to distinct epitopes on Fc␥RI are able to
trigger receptor function. J. Immunol. 143:1650.
14. Morton, H. C., G. van Zandbergen, C. van Kooten, C. J. Howard,
J. G. J. van de Winkel, and P. Brandtzaeg. 1999. Immunoglobulin-binding sites
of human Fc␣RI (CD89) and bovine Fc␥2R are located in their membrane-distal
extracellular domains. J. Exp. Med. 189:1715.
15. van de Wetering, J. K., M. van Eijk, L. M. G. van Golde, T. Hartung,
J. A. G. van Strijp, and J. J. Batenburg. 2001. Characteristics of surfactant protein
A and D binding to lipoteichoic acid and peptidoglycan, 2 major cell wall components of Gram-positive bacteria. J. Infect. Dis. 184:1143.
16. Hartshorn, K., D. Chang, K. Rust, M. White, J. Heuser, and E. Crouch. 1996.
Interactions of recombinant human pulmonary surfactant protein D and SP-D
multimers with influenza A. Am. J. Physiol. 271:L753.
17. Madan, T., U. Kishore, M. Singh, P. Strong, H. Clark, E. M. Hussain,
K. B. M. Reid, and P. U. Sarma. 2001. Surfactant proteins A and D protect mice
against pulmonary hypersensitivity induced by Aspergillus fumigatus antigens
and allergens. J. Clin. Invest. 107:467.
18. Wright, J. R., D. F. Zlogar, J. C. Taylor, T. M. Zlogar, and C. I. Restrepo. 1999.
Effects of endotoxin on surfactant protein A and D stimulation of NO production
by alveolar macrophages. Am. J. Physiol. 276:L650.
19. Hartshorn, K. L., E. C. Crouch, M. R. White, P. Eggleton, A. I. Tauber, D. Chang,
and K. Sastry. 1994. Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses. J. Clin. Invest. 94:311.
20. Hartshorn, K. L., M. R. White, V. Shepherd, K. Reid, J. C. Jensenius, and
E. C. Crouch. 1997. Mechanisms of anti-influenza activity of surfactant proteins
A and D: comparison with serum collectins. Am. J. Physiol. 273:L1156.
21. Hartshorn, K. L., E. Crouch, M. R. White, M. L. Colamussi, A. Kakkanatt,
B. Tauber, V. Shepherd, and K. N. Sastry. 1998. Pulmonary surfactant proteins
A and D enhance neutrophil uptake of bacteria. Am. J. Physiol. 274:L958.
22. Hartshorn, K. L., M. Collamer, M. R. White, J. H. Schwartz, and A. I. Tauber.
1990. Characterization of influenza A virus activation of the human neutrophil.
Blood 75:218.
23. Shen, L., P. M. Guyre, and M. W. Fanger. 1987. Polymorphonuclear leukocyte
function triggered through the high affinity Fc receptor for monomeric IgG. J. Immunol. 139:534.
24. Persson, A., D. Chang, and E. Crouch. 1990. Surfactant protein D is a divalent
cation-dependent carbohydrate-binding protein. J. Biol. Chem. 265:5755.
25. van Rozendaal, B. A. W. M., A. B. van Spriel, J. G. J. van de Winkel, and
H. P. Haagsman. 2000. Role of pulmonary surfactant protein D in innate defense
against Candida albicans. J. Infect. Dis. 182:917.