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* 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
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Medical Research Council, Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom
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Abstract |
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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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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, 9, 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/anti-CD89 enhanced killing of C. albicans and E. coli by neutrophils in in vitro killing assays and enhanced neutrophil activation by IAV.
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Materials and Methods |
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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 (rhSP-D), 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, 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.
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 (Sigma-Aldrich) 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/µg 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.
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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 µg/ml rfSP-D/anti-CD89 or rfSP-D in a volume of 50 µl for 30 min at 37°C. Subsequently, samples were incubated with 4 x 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 96-well 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 x 105 neutrophils suspended in D-PBS (pathogen:neutrophil ratio, 4:1) were added to the samples to a total volume of 135 µl. Plates were shaken 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 x [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.
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Results |
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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 cross-linked 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 cross-reactivity 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).
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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. 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).
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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).
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Effect of blocking CD89 and CRD on rfSP-D/anti-CD89-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-CD89-mediated 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 CRD-pathogen interaction is essential for rfSP-D/anti-CD89-mediated pathogen uptake.
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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 µg/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 µg/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 µg/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 µg rfSP-D. In the presence of 0.88 µg/ml hSP-D, the uptake of C. albicans was 252 ± 20% of that without opsonization. Opsonization with 0.88 µg/ml hSP-D in combination with 8 µg/ml rfSP-D/anti-CD89 significantly increased uptake to 309 ± 26%, whereas opsonization with 8.88 µg/ml hSP-D stimulated uptake to a significantly lower extent than opsonization with 0.88 µg/ml hSP-D or with 0.88 µg/ml hSP-D plus 8 µg/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 µg/ml hSP-D had at best a minor effect on the uptake of the bacteria (Fig. 5C). The uptake with 0.88 µg/ml hSP-D was significantly increased by simultaneous addition of 2 µg/ml rfSP-D/anti-CD89, but not by increasing the hSP-D concentration from 0.88 to 2.88 µg/ml.
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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. Although 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).
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Discussion |
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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 (anti-CD89 Fab'), a molecule containing a different Fab' (rfSP-D/anti-CD64), 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 CRD-pathogen 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 rfSP-D/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 FcRI (CD64), has been shown to effectively protect CD64 transgenic mice from lethal invasive candidiasis (29).
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.
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Acknowledgments |
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Footnotes |
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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: j.j.batenburg{at}vet.uu.nl
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.
Received for publication October 7, 2003. Accepted for publication February 10, 2004.
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References |
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RI (CD89) as a novel trigger molecule for bispecific antibody therapy. Blood 90:4485.R). Crit. Rev. Immunol. 16:423.[Medline]
R expression and IgA-mediated function on neutrophils induced by chemoattractants. J. Immunol. 150:4532.[Abstract]
of Fc receptor expression and IgA-mediated superoxide generation and killing of Pseudomonas aeruginosa by polymorphonuclear leukocytes. J. Infect. Dis. 170:82.[Medline]
RI (CD64) or FcRI (CD89) on neutrophils. J. Infect. Dis. 179:661.[Medline]
receptors detected by receptor-specific monoclonal antibodies. J. Immunol. 148:1764.[Abstract]
RI are able to trigger receptor function. J. Immunol. 143:1650.[Abstract]
RI (CD89) and bovine Fc2R are located in their membrane-distal extracellular domains. J. Exp. Med. 189:1715.RI as target for immunotherapy of invasive candidiasis. J. Immunol. 166:7019.R-triggered Ca2+ signals: role in neutrophil NADPH oxidase activation. Biochem. Biophys. Res. Commun. 276:749.[Medline]
receptor-mediated phagocytosis by human blood dendritic cells. J. Immunol. 157:541.[Abstract]
chain on myeloid cells in blood and tissue. Scand. J. Immunol. 57:506.[Medline]This article has been cited by other articles:
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  M. White, P. Kingma, T. Tecle, N. Kacak, B. Linders, J. Heuser, E. Crouch, and K. Hartshorn Multimerization of Surfactant Protein D, but Not Its Collagen Domain, Is Required for Antiviral and Opsonic Activities Related to Influenza Virus J. Immunol., December 1, 2008; 181(11): 7936 - 7943. [Abstract] [Full Text] [PDF]
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 M. Duval, M. R. Posner, and L. A. Cavacini A Bispecific Antibody Composed of a Nonneutralizing Antibody to the gp41 Immunodominant Region and an Anti-CD89 Antibody Directs Broad Human Immunodeficiency Virus Destruction by Neutrophils J. Virol., May 1, 2008; 82(9): 4671 - 4674. [Abstract] [Full Text] [PDF]
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 M. R. White, E. Crouch, J. Vesona, P. J. Tacken, J. J. Batenburg, R. Leth-Larsen, U. Holmskov, and K. L. Hartshorn Respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza A virus Am J Physiol Lung Cell Mol Physiol, October 1, 2005; 289(4): L606 - L616. [Abstract] [Full Text] [PDF]
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) or rfSP-D/antiCD89 (•). 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: *, p < 0.05; **, p < 0.01.
), rfSP-D/anti-CD64 (
), anti-CD89 F(ab')2 (
), or anti-CD89 Fab' (
). 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: *, p < 0.01.
). C. albicans was opsonized with 0.88 µg/ml hSP-D (