Cooperative anti-influenza activities of respiratory innate immune proteins and neuraminidase inhibitor
Mitchell R. White,1
Erika Crouch,2
Martin van Eijk,3
Max Hartshorn,1
Lily Pemberton,1
Ida Tornoe,4
Uffe Holmskov,4 and
Kevan L. Hartshorn1
1Department of Medicine, Section of Hematology/Oncology, Boston University School of Medicine, Boston, Massachusetts 2Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 3Department of Biochemistry and Cell Biology and Graduate School of Animal Health, Utrecht University, Utrecht, The Netherlands 4Medical Biotechnology Center, University of Southern Denmark, Odense, Denmark
Submitted 27 September 2004
; accepted in final form 29 November 2004
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ABSTRACT
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The surfactant collectins, surfactant proteins A and D (SP-A and D), and scavenger receptor-rich glycoprotein 340 (gp340) inhibit influenza A virus (IAV) in the following order of potency: SP-D>gp340>SP-A. SP-D binds in a calcium-dependent manner to carbohydrate attachments on the viral hemagglutinin (HA) and neuraminidase (NA). By contrast, gp340 and SP-A act like mucins in that they provide sialic acid ligands that bind to the viral HA. In this study, SP-D, SP-A, and gp340 showed cooperative antiviral interactions. These cooperative effects were most evident in viral aggregation but were also observed in at least some hemagglutination inhibition and viral neutralization assays. The mechanism of binding between gp340 and SP-D was further characterized using monoclonal antibodies. Although gp340 can bind to SP-D at a site distinct from the mannan-binding site, binding of gp340 to SP-D did not contribute to cooperative antiviral interactions. SP-D and mucin showed cooperative interactions, apparently dependent on NA inhibition by SP-D. The commercial NA inhibitor oseltamivir had a similar effect and also enhanced the neutralizing activity of SP-A and bronchoalveolar lavage fluid. Hence, oseltamivir collaborates with innate immune proteins in inhibiting the initial infection of epithelial cells.
surfactant protein D; surfactant protein A; gp340; oseltamivir
INFLUENZA A VIRUS (IAV) infection is a major and worldwide cause of morbidity and mortality. In the United States alone influenza epidemics account for as many as 50,000 deaths per year (32). Innate immune mechanisms within the respiratory tract play an important role in the initial containment of IAV infection. There is strong evidence that the pulmonary collectins, surfactant proteins D and A (SP-D and SP-A), play important roles in the innate response to IAV infection (3, 9, 13, 14, 16, 21, 2628). In addition, our recent findings indicate that the lung and salivary scavenger receptor-rich glycoprotein 340 (gp340) contributes to innate defense against IAV (20).
The role of SP-D appears to be particularly important in innate defense against IAV. Among the known human collectins, SP-D has the strongest in vitro IAV neutralizing and aggregating activity (1618). Removal of SP-D from bronchoalveolar lavage (BAL) or the addition of monosaccharides that interfere with binding of SP-D significantly reduced antiviral activity (3, 16, 20). SP-D knockout mice (SP-D/) exhibit greatly increased viral titers, lung inflammation, and illness after infection with IAV (21, 27). Several studies have demonstrated that SP-D levels in mouse lung lavage rise after IAV infection (3, 21, 26, 27, 36). Replacement of SP-D by instillation of recombinant SP-D, or by overexpression of modified forms of SP-D in the distal lung, partially or completely normalizes the response of SP-D/ mice to IAV infection (21, 27, 45).
However, it is likely that other constituents of upper and lower airway fluids contribute to the innate host defense against IAV. Depletion of SP-D from human BAL fluids does not fully eliminate anti-IAV activity (16). SP-A is another component of BAL that may contribute to anti-IAV activity (21, 26, 28). A consistent finding is that inflammatory responses after IAV infection are increased in SP-A/ mice. IAV replication has been modestly increased in some studies of SP-A/ mice (21, 26), but not in others (28). SP-D-resistant strains remain sensitive, or have increased sensitivity, to inhibition by SP-A (13). SP-D-mediated viral inhibition results from calcium-dependent binding of SP-D to high-mannose oligosaccharides on the viral envelope proteins. Hence, SP-D is classified as a
-inhibitor of IAV [similar to mannose binding lectin (MBL) and bovine conglutinin] (17, 18). SP-A-mediated inhibition involves binding of the viral hemagglutinin (HA) to sialylated carbohydrates within the lectin domain of SP-A (1, 6, 13), and, therefore, SP-A is classified as a
-inhibitor of IAV (similar to
2-macroglobulin) (37). Interestingly, porcine SP-D (pSP-D), which is unique among SP-Ds in containing oligosaccharides within the lectin domain, shows distinctive interactions with IAV that are dependent on the N-linked sugar (57). Thus pSP-D acts as both a
- and
-inhibitor.
Gp340 has strong anti-IAV activities mediated through a mechanism resembling that of SP-A (20). Gp340 was initially identified as a pulmonary SP-D binding molecule that is present in BAL and associated with alveolar macrophages (22, 23). Sequence analysis revealed gp340 to be a member of the scavenger receptor family closely related to the putative tumor suppressor gene DMBT1. Gp340 colocalizes with SP-D lung lavage and intracellular compartments of alveolar macrophages (40). In addition, it is expressed along with SP-D in lung, salivary glands, trachea, small intestine, and stomach. Gp340 also binds to SP-A (40). A form of Gp340 has been identified as the "salivary agglutinin" (29, 35) that mediates specific adhesion to, and aggregation of, Streptococcus mutans and other bacteria (2, 8, 33).
The goal of this study was to examine the functional interactions of the
-inhibitor SP-D with the
-inhibitors SP-A, lung gp340, and mucin in the host defense against IAV. We demonstrate cooperative interactions between these proteins with respect to aggregation or inhibition of hemagglutination and infectivity of IAV. Cooperative activity was most pronounced on aggregation assays. Of interest, the cooperative interactions of SP-D with gp340 did not appear to involve binding of the two proteins to each other. SP-D exerted the dominant activity in HA inhibition assays, even when combined with other inhibitors. Mucin and SP-D showed strong cooperative interactions on viral aggregation and HA inhibition assays, likely as a result of inhibition of neuraminidase (NA) activity by SP-D. Inhibition of viral NA activity with oseltamivir increased viral aggregation and HA inhibition activity of mucin and SP-A, but not of SP-D, consistent with distinct modes of viral inhibition by these proteins.
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MATERIALS AND METHODS
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Buffers.
Dulbecco's phosphate-buffered saline with (PBS++) and without (PBS) calcium and magnesium were purchased from GIBCO-BRL (Grand Island, NY).
Virus preparation.
IAV was grown in the chorioallantoic fluid of 10-day-old chicken eggs and purified on a discontinuous sucrose gradient as previously described (15). The virus was dialyzed against PBS to remove sucrose, aliquotted, and stored at 80°C until needed. The Philippines 82/H3N2 (Phil82) strain was kindly provided by Dr. E. Margot Anders (Univ. of Melbourne, Melbourne, Australia). The hemagglutination (HA) titers of virus preparations were determined by titration of virus samples in PBS with thoroughly washed human type O, Rh() red blood cells as described (19). Postthawing the viral stocks contained
5 x 108 plaque-forming units/ml.
Lung gp340, SP-D, and SP-A preparations.
Table 1 summarizes the different collectin and collectin-binding proteins utilized in this study. Recombinant human SP-D (RhSP-D) was produced in Chinese hamster ovary (CHO)-K1 cells as previously described (9). The chimeric collectin containing the NH2 terminus and collagen domain of recombinant human SP-D (RhSP-D) and the neck and carbohydrate recognition domain (CRD) domains of human MBL (SP-D/MBLneck+CRD) was constructed and expressed as described (11, 42, 43). Using gel filtration, we fractionated RhSP-D and the SP-D/MBLneck+CRD chimera into preparations predominantly composed of trimers, dodecamers, and multimers as described in detail elsewhere (43). For these studies the dodecameric fraction of RhSP-D and SP-D/MBLneck+CRD chimera was used unless otherwise specified. The RrSP-Dser15,20 form of recombinant rat SP-D was also generated in CHO cells and exists only in the trimer configuration due to site-directed mutagenesis of NH2-terminal cysteines normally involved in disulfide bonding between trimeric subunits of SP-D (3). SP-A was purified from human alveolar proteinosis fluid as described (26).
pSP-D was purified from BAL fluid as described previously (57). pSP-D was composed of a mixture of dodecameric and more highly multimerized forms. N-deglycosylated pSP-D (pSP-Ddeg) was obtained by treatment of pSP-D with recombinant N-glycanase (from Flavobacterium meningosepticum; Glyko, Novato, CA). Purified pSP-D (50 µg/100 µl), dissolved in 20 mM Tris·HCl (pH 7.4), 150 mM NaCl, and 5 mM EDTA, was mixed with 15 mU N-glycanase and incubated at 37°C for 3 h. Sham-treated pSP-D (pSP-Dcon) was obtained by incubation in the absence of enzyme. After incubation, we purified and separated pSP-Ddeg and pSP-Dcon from any N-glycanase by adding mannan-sepharose (0.5-ml bed volume per 0.5-ml incubation mixture) and a final concentration of 10 mM CaCl2 to the incubation mixture. Mannan-bound pSP-Ddeg was then isolated as described for the isolation of pSP-D, with the exception of gel filtration chromatography. The state of multimerization of pSP-Ddeg was not different from that of untreated or sham-treated pSP-D as determined by gel filtration analysis.
The collectin preparations used in this report were tested for degree of contamination with endotoxin with a quantitative endotoxin assay (Limulus amebocyte lysate; Bio-Whittaker, Walkersville, MD). The final concentrations of endotoxin in samples containing the highest concentrations of collectins were
20100 pg/ml (or 612 endotoxin units/ml using internal assay standard).
Gp340 was isolated as described (20). In brief, BAL was centrifuged at 300 g for 10 min and then 10,000 g for 30 min, and the 10,000 g and the precipitate were dissolved in tris-buffered saline containing 10 mM EDTA and incubated for 18 h at 4°C with gentle stirring. Insoluble material was removed by centrifugation at 10,000 g. The supernatant was applied to a Mono-Q fast flow column (Pharmacia), and retained proteins were eluted with a linear gradient of NaCl 01 M. The gp340 containing fractions were separated by gel permeation chromatography on a Superose 6 Prepgrade column. The purity of the resulting gp340 was analyzed by chromatography and SDS-PAGE. The gp340 preparation was free of SP-D and SP-A or other major contaminants.
Production of monoclonal antibodies against SP-D.
Monoclonal antibodies (MAbs) were raised against SP-D purified from amniotic fluid as described (31). In short, mice were immunized with 10 µg of purified human SP-D adsorbed onto aluminum hydroxide gel and emulsified with 0.25 ml of Freund's incomplete adjuvant. Fusions were performed with the cell line X63Ag8.6.5.3, and positive clones were identified by ELISA using purified SP-D coated onto the wells. Cells from wells with antibodies against SP-D were recloned three times by the limiting dilution method. The specificity of the MAbs for SP-D was checked by calcium- and maltose-dependent binding to SP-D by ELISA and by Western blotting of partially purified SP-D. A panel of nine antibodies was obtained.
Measurement of aggregation of IAV particles.
We assessed aggregation of IAV particles following addition of various concentrations of collectins by monitoring changes in light transmission on a highly sensitive SLM/Aminco 8000C (SLM Instrument, Urbana, IL) spectrofluorometer as described (17). The aggregation of viral particles or liposomes is demonstrated by a decline in light transmission (i.e., increased turbidity).
Assessment of binding of SP-D to gp340.
Binding was determined with solid-phase ELISAs in lung gp340 or PBS containing fatty acid-free BSA (Sigma) were allowed to adhere overnight to ELISA plates (4). The plates were then washed and incubated with PBS containing 25% fatty acid-free BSA for blocking. After further washes, various preparations of RhSP-D, pSP-D, or SP-D/MBLneck+CRD chimera were added for 30 min at 37°C. After further washing the bound collectins were detected with appropriate antibodies. RhSP-D was detected with a rabbit polyclonal antibody. pSP-D was detected with rabbit polyclonal antibody raised specifically against pSP-D. Deglycosylation of pSP-D was previously shown not to alter recognition of pSP-D by this antibody preparation (5). The SP-D/MBLneck+CRD chimera was detected with the 131-1 MAb against human MBL (graciously provided by R. A. B. Ezekowitz, Massachusetts General Hospital, Boston, MA). The bound antibodies were detected with goat anti-rabbit or donkey anti-mouse fAb2 fragments coupled to horseradish peroxidase (Jackson Immunochemicals), and bound secondary antibodies were detected with 3,3',5,5'-tetramethylbenzidine peroxidase substrates (Bio-Rad Labs, Hercules, CA). The reaction was stopped using 1 N sulfuric acid. The optical density was measured on an ELISA plate reader at 450 nm. Each data point was performed in duplicate.
Standard curves for detection of the different collectins by their respective antibodies were generated, and results of gp340 binding assays were adjusted for differences in detection of known amounts of the collectins (see Table 1). However, since different antibodies were used for detection of RhSP-D, SP-D/MBLneck+CRD, and pSP-D, direct comparisons of binding of these proteins to gp340 are only approximate.
Assessment of binding of SP-A to IAV or SP-D.
Binding of SP-A to either RhSP-D dodecamers or IAV (PR-8 strain) was measured by ELISA as described above. Plates were coated with 1 µg/ml of RhSP-D or IAV (PR-8 strain) overnight followed by washing and blocking as described above. Various concentrations of SP-A were added, and bound SP-D was detected using the 238-01 MAb directed against SP-A.
Fluorescent focus assay of IAV infectivity.
Madin-Darby canine kidney monolayers were prepared in 96-well plates and grown to confluence. These cell layers were then infected with diluted IAV preparations (Phil82 strain) for 30 min at 37°C, followed by washing of the monolayer three times in serum-free Dulbecco's modified Eagle's medium (DMEM) containing 1% penicillin and streptomycin. The monolayers were then incubated for 7 h at 37°C with 5% CO2 in DMEM. The monolayers were subsequently washed three times with PBS++ and fixed with 80% acetone (vol/vol) for 10 min at 4°C. The monolayers were then labeled by incubating with monoclonal antibody directed against the IAV nucleoprotein (provided by Dr. Nancy Cox, Centers for Disease Control and Prevention, Atlanta, GA) in PBS with 0.1% BSA, 1% heat-inactivated human serum, and 0.02% NaN3 for 30 min at 4°C. The monolayers were washed three times in PBS and incubated with FITC-labeled goat anti-mouse IgG. The fluorescent foci were counted directly by fluorescence microscopy. Initially, various dilutions of virus were used to find the dose yielding
50 fluorescent foci per high-powered (x40) field. These foci usually appeared to correspond to single infected cells. Subsequently, this dose of virus was incubated with different doses of gp340 or SP-D to assess the effect of these treatments on infectivity.
Statistics.
Statistical comparisons were made using Student's paired, two-tailed t-test or ANOVA with post hoc test (Tukey's).
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RESULTS
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RhSP-D and lung gp340 have cooperative functional interactions with respect to IAV.
Suspensions of the SP-D-sensitive wild-type Phil82 strain of IAV were treated with RhSP-D dodecamers alone or in combination with gp340 (Fig. 1). Concentrations of the proteins that caused submaximal aggregation were chosen based on prior studies, so that cooperative effects could be observed. As shown in Fig. 1, strong cooperative effects were observed for combinations of RhSP-D and gp340. Prior studies have demonstrated that SP-D and lung gp340 have very strong IAV-neutralizing and HA-inhibiting activity. The cooperative effect between RhSP-D and gp340 was also observed when HA activity of the viral aggregation samples was tested (Table 2). At some concentrations RhSP-D and gp340 also had cooperative neutralizing activity (Table 3).
Evaluation of mechanism of binding between SP-D and gp340.
Binding of RhSP-D to gp340 was inhibited by EDTA but not by maltose, whereas binding of RhSP-D to mannan was inhibited by both (Fig. 2). Two MAbs directed against distinct epitopes on the CRD of SP-D had different effects on binding activities of RhSP-D (Fig. 2). The 245-1 MAb inhibited binding of RhSP-D to gp340 but not to mannan, whereas the reverse result was obtained with the 246-7 MAb (i.e., strong inhibition of binding to mannan with slight inhibition of binding to gp340). These results indicate that the specific regions of the SP-D CRD involved in binding to gp340 and mannan are distinct.

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Fig. 2. Distinct MAbs directed against SP-D inhibit binding to SP-D to mannan and to gp340. Binding of SP-D to gp340 (top) and mannan (bottom) was tested by ELISA. Note that binding of SP-D to gp340 was inhibited by EDTA but not maltose, whereas binding of SP-D to mannan was inhibited by EDTA and maltose. Two MAbs directed against the CRD of SP-D were tested for their ability to block binding. The 245-1 MAb (left) blocked binding of SP-D to gp340 but not to mannan, whereas the 246-7 MAb (right) blocked binding to mannan but not to gp340. Hyb, hybridoma; OD405, optical density at 405 nm.
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As noted above, pSP-D is unique among SP-Ds in having a highly sialylated N-linked carbohydrate attachment to its CRD. Binding of pSP-D to gp340 was significantly reduced compared with binding of RhSP-D: means ± SE optical density at 450 nm for binding to RhSP-D to gp340 was 0.75 ± 0.1 compared with 0.14 ± 0.02 for binding of pSP-D (n = 4, P < 0.003). This effect was at least partially the result of the negatively charged carbohydrate on pSP-D, because treatment of pSP-D with N-glycanase resulted in significantly increased binding to gp340 (i.e., binding of deglycosylated pSP-D was 0.35 ± 0.05; P < 0.004 compared with binding of pSP-D or RhSP-D).
The SP-D/MBLneck+CRD chimera differs from RhSP-D in that the neck and CRD of SP-D are substituted by the corresponding domains of MBL (43). This chimera forms multimers in a similar manner as wild-type SP-D but has distinctive carbohydrate binding specificity and enhanced antiviral activity compared with RhSP-D (42, 43). As shown in Fig. 3, SP-D/MBLneck+CRD bound to gp340; however, binding was reduced compared with binding of RhSP-D at least at the lower concentrations of the proteins tested.

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Fig. 3. SP-D/neck and carbohydrate recognition domain domains of human mannose binding lectin (MBLneck+CRD) binds less strongly to gp340 than RhSP-D. Binding of SP-D/MBLneck+CRD and SP-D to gp340 were measured by ELISA using MAbs and directed against MBL or SP-D, respectively. Results are means ± SE of 3 experiments. Binding of SP-D/MBLneck+CRD was significantly less than binding of SP-D (P < 0.05) at all the concentrations shown except 125 ng/ml.
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Cooperative viral aggregating activity between SP-D preparations and gp340 predominantly reflects independent aggregating activities of the proteins.
Despite reduced ability of pSP-D to bind to gp340, the combination of pSP-D and gp340 caused greater viral aggregation than the combination of RhSP-D and gp340. Furthermore, deglycosylation of pSP-D resulted in cooperative viral aggregating activity that was nearly identical to that of RhSP-D (Fig. 4). As previously reported, SP-D/MBLneck+CRD dodecamers had greater ability to aggregate IAV compared with wild-type SP-D (Fig. 5) [e.g., 200 ng/ml of SP-D/MBLneck+CRD had approximately the same aggregating activity as 400 ng/ml of RhSP-D (Fig. 1)]. Despite reduced ability to bind to gp340, SP-D/MBLneck+CRD had strong cooperative viral aggregating activity when combined with gp340 (Fig. 5). RrSP-Dser15,20 had no viral aggregating on its own as previously reported (3). Combining RrSP-Dser15,20 with gp340 caused no greater viral aggregation than gp340 alone, despite binding to gp340 to an equal extent as RhSP-D dodecamers (data not shown). Overall these results suggest that cooperative aggregating effects observed with SP-D preparations and gp340 result from independent aggregating activities of the proteins.

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Fig. 4. Lung gp340 (Lgp340) and porcine SP-D (pSP-D) have stronger cooperative virus-aggregating effect than Lgp340 combined with either RhSP-D or deglycosylated pSP-D (pSP-Ddeg). Experiments (n = 46) were performed as in Fig. 1. Lgp340, pSP-D, pSP-Ddeg, or RhSP-D were used alone or in combination as indicated. Combining either SP-D preparation with Lgp340 caused greater aggregation than either SP-D or gp340 alone at all time points tested (P < 0.04). However, the combination of pSP-D with Lgp340 caused significantly greater viral aggregation than either the combination of pSP-Ddeg or RhSP-D with Lgp340 (P < 0.009).
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Fig. 5. Lung gp340 and SP-D/MBLneck+CRD have cooperative virus-aggregating activity. Experiments (n = 4) were performed as in Fig. 1 with the exception that SP-D/MBL was used in place of RhSP-D. Gp340 or SP-D/MBLneck+CRD alone caused significant aggregation of IAV (Phil82 strain) (P < 0.01 for either). Combination of lung gp340 with either 200 (top) or 400 (bottom) ng/ml of SP-D/MBLneck+CRD caused significantly more aggregation than either protein alone at 100700 s after addition of proteins (P 0.05).
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Cooperative viral aggregation caused by SP-A and SP-D or gp340.
RhSP-D and SP-A had cooperative viral aggregating activity when used in combination (Fig. 6). Other concentrations of RhSP-D and SP-A (e.g., 0.48 µg/ml of RhSP-D and 1.5 µg/ml SP-A) also showed significant cooperative aggregating activity (data not shown). No significant binding between SP-D and SP-A was noted, although SP-A bound to IAV in parallel assays (data not shown). Hence, cooperative viral aggregation induced by SP-D and SP-A does not appear to involve binding of the two proteins to each other.
Gp340 and SP-A also caused cooperative aggregation (Fig. 6). Combining SP-A with gp340 also caused additive viral neutralization at some concentrations (Table 4). However, SP-A did not cause significant cooperative effects when combined with SP-D or gp340 on HA inhibition assays (i.e., mean HA inhibitory concentrations of RhSP-D and gp340 alone were 8.6 ± 1.5 and 300 ± 58 ng/ml, compared with 9.4 ± 1.8 and 398 ± 124 in presence of 20 µg/ml of SP-A; n = 5).
Role of NA in HA inhibitory and virus-neutralizing activities of SP-D, SP-A, and mucin.
The NA inhibitor oseltamivir had no HA-inhibitory or virus-neutralizing effect on its own (Table 5 and data not shown). This is consistent with prior studies and the interpretation that the major role of the NA is in viral budding (30). The fluorescent focus assay measures the first round of viral replication and hence would not detect effects of oseltamivir on viral budding or propagation through the monolayer. However, oseltamivir significantly increased the HA inhibition of SP-A and mucin (Table 5). Oseltamivir increased the neutralizing activity of SP-A, but not of mucin (Table 5). It was striking that mucin caused both HA inhibition and viral aggregation but had no activity in the neutralization assay even in the presence of oseltamivir. Of interest, oseltamivir increased neutralizing activity of normal donor BAL fluid (Fig. 7). Oseltamivir did not increase the HA-inhibitory or -neutralizing activity of RhSP-D (consistent with the distinct mechanism of action of SP-D compared with SP-A and mucin).
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Table 5. Inhibition of neuraminidase activity by oseltamivir significantly increases HA inhibitory activity of SP-A and mucin
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Fig. 7. Oseltamivir increases the IAV neutralizing activity of SP-A and bronchoalveolar lavage fluid (BALF). Neutralization of IAV was assessed using the infectious focus assay. Results shown are means ± SE foci per well (n = 48 experiments) for Phil82 IAV in control buffer or after treatment with RhSP-D (44 ng/ml), SP-A (1.72 µg/ml), mucin (22 µg/ml), or BALF (25 µl). RhSP-D, SP-A, and BALF significantly reduced number of infectious foci compared with control buffer (P < 0.01 for all). Where indicated oseltamivir (10 µg/ml, solid bars) was added along with control buffer or the other inhibitors. *Addition of oseltamivir significantly reduced number of foci when added to SP-A or BALF by ANOVA.
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Mucin causes reversible viral aggregation: role of NA and cooperative effect of SP-D.
Like gp340 and SP-A, mucin presents sialic acid ligands to viral HA. However, the sialic acids of mucin are readily cleaved by the viral NA. Mucin caused viral aggregation, although the effect was largely reversible at concentrations up to 24 µg/ml (Fig. 8). Reversal of aggregation appeared to provide a real-time assay for viral NA activity, because the NA inhibitor oseltamivir caused a sustained aggregation response to mucin. Of note, adding SP-D to mucin caused a similar effect as oseltamivir (i.e., sustained aggregation response) (Fig. 8). The combination of SP-D and mucin caused markedly greater aggregation than either protein alone.

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Fig. 8. Mucin causes reversible aggregation of IAV: effect of adding oseltamivir or SP-D. A: dose-response effect of adding increasing concentrations of bovine submaxillary mucin to Phil82 strain of IAV. Note partial reversibility of aggregation caused by concentrations up to 24 µg/ml. B and C: effect of adding oseltamivir or RhSP-D (200 ng/ml) along with 24 µg/ml of mucin. The degree of aggregation caused by these combinations was significantly greater than aggregation caused by mucin alone at 300700 s (P < 0.05 by ANOVA).
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DISCUSSION
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Several innate inhibitors of IAV are constitutively present in respiratory secretions, including SP-D, SP-A, gp340, and mucins. Using several assays to compare the antiviral activities of these proteins we demonstrate the following hierarchy of activity for these proteins: SP-D>gp340>SP-A>mucin. The mechanisms of activity of these inhibitors differ as well. SP-D acts as a
-inhibitor, whereas gp340, SP-A, and mucin act as
-inhibitors. The anti-IAV activity of respiratory fluids represents the complex interactions of the different inhibitors. We demonstrate cooperative viral aggregating activities between all of these proteins. The concentrations of SP-D and gp340 in BAL fluids are
130 ng/ml and that of SP-A about tenfold higher (16, 20). Because BAL fluid is highly diluted compared with alveolar lining fluid, the local concentrations of these proteins are probably considerably higher than levels measured in BAL fluid. Hence, our studies involve concentrations of the proteins that are in the physiological range. In general results of HA inhibition or viral neutralization assays parallel the aggregation results, although not in all cases.
The interaction between SP-D and gp340 was evaluated in detail because these two proteins had the highest-potency antiviral activity against IAV and also because they bind to each other. The mechanism of binding of gp340 to SP-D was further characterized through the use of MAbs. Although gp340 binds in a calcium-dependent manner to the CRD of SP-D, the site of binding does not directly involve the carbohydrate binding site of SP-D (Fig. 2). Hence, these results suggest that gp340 and SP-D could both bind to each another and to a carbohydrate ligand (e.g., mannan or IAV glycoproteins) simultaneously.
However, our further studies suggest that binding of gp340 to SP-D does not play a role in their cooperative aggregating activity. This interpretation is supported by findings obtained with pSP-D, SP-D/MBLneck+CRD, and SP-A. First, pSP-D had markedly reduced ability to bind to gp340. This reduced binding is in part attributable to a highly sialylated, negatively charged carbohydrate attachment on the exposed binding surface of the CRD of pSP-D. This glycan may overlay the binding site of SP-D to gp340 or inhibit binding by charge repulsion (i.e., increased negative charge due to abundant sialic acids on the glycan of pSP-D). In any case, removal of the carbohydrate by N-glycanase reduced the cooperative aggregating activity of pSP-D and gp340 to a level comparable to that of hSP-D and gp340 and increased binding between the two proteins. SP-D/MBLneck+CRD had reduced ability to bind to gp340 compared with wild-type RhSP-D but retained strong cooperative activity on viral aggregation assays. Finally, SP-A also had cooperative aggregating activity when combined with SP-D despite the fact that these proteins did not bind to each other significantly. Overall these results are most consistent with the interpretation that the cooperative viral aggregating activity between SP-D, gp340, and SP-A largely reflect additive effects of the proteins. This was further supported by the fact that a trimeric form of SP-D (RrSP-Dser15,20) that lacks viral aggregating activity in its own right did not increase viral aggregation caused by gp340.
Cooperative effects were also observed with combinations of SP-D, gp340, and SP-A on HA inhibition and viral neutralization assays in most cases. It should be noted that the HA inhibition assay and viral neutralization assay involve much more dilute viral preparations than the aggregation assay. In such circumstances the opportunities for the different proteins to simultaneously bind to viral particles may be reduced. This may explain why additive effects were not observed in some cases (e.g., HA inhibition assay combining SP-A with either RhSP-D or gp340). Viral aggregation assays are useful in that they provide a real-time assay in fluid phase of virus interaction with inhibitory proteins. This was illustrated in evaluation of the interactions of mucin and oseltamivir with IAV (see below). However, the results could also be relevant in vivo since aggregation could promote viral clearance through mucociliary or phagocytic means at times of peak viral replication (10).
The results obtained with mucin provide an informative contrast with those obtained with the other proteins. Although mucin acts as a
-inhibitor, its ability to aggregate the virus was transient, and it lacked viral neutralizing activity, despite HA inhibitory activity comparable to SP-A. These seemingly discordant results could also reflect differences in assay conditions. The sialic acids of mucin are rapidly hydrolyzed by IAV NA. This occurs rapidly as illustrated in the viral aggregation assay: reversal of aggregation occurred in minutes and was prevented by oseltamivir. In the case of the HA inhibition assay, the mucin could interfere with viral binding to red cells for sufficient time to allow the cells to pellet. In contrast, in the neutralization assay the viral NA has greater time to act. In any case, it is notable that SP-D had cooperative viral aggregating and HA inhibiting activity when combined with mucin. In particular, the ability of SP-D to prevent the reversal of aggregation observed with mucin alone may reflect its ability to bind to the viral NA and inhibit its activity (14, 36). Such an effect could be relevant in vivo both in early and later stages of viral infection (i.e., viral binding and entry and viral budding). Furthermore, it is interesting to speculate that pharmacological NA inhibitors may be most useful in treatment of IAV infection in settings where SP-D levels are reduced (e.g., smokers, subjects with concurrent bacterial pneumonia or cystic fibrosis) (24, 25, 34).
Our findings also provide further insight into the antiviral activity of oseltamivir. The major activity of NA inhibitors is believed to involve inhibition of viral budding. However, our results suggest that oseltamivir also inhibits initial infection of epithelial cells through interaction with innate
-inhibitors. This was clearly apparent when combining oseltamivir with SP-A or normal BAL fluid. Not surprisingly, oseltamivir had no additive effects when combined with SP-D (at least in the assays used in this study). Further studies of the interaction of NA inhibitors with innate inhibitory proteins will be of interest and may be relevant to the mechanism of action of NA inhibitors in vivo.
This study provides the first detailed evaluation of the antiviral interactions of SP-D, gp340, and SP-A. Studies of the interactions of these proteins with respect to other host defense functions (e.g., antibacterial or antifungal effects, opsonizing activity) will be of great interest. The ability of respiratory innate immune proteins to inhibit IAV through different mechanisms may provide protection against diverse viral strains. In particular
-inhibitors may have relatively greater importance in defense against viral strains resistant to SP-D or other
-inhibitors. It is noteworthy, for instance, that the 1918 pandemic strain of IAV lacked glycosylation near the sialic acid binding site of the HA that characterizes recently circulating human strains (39) and, hence, may have been resistant to SP-D. Glycosylation on the HA appears to increase over time in strains that establish themselves in the human population, perhaps in the process of adaptation to pressure from the adaptive immune system (i.e., glycosylation of the head region of the HA protects it from binding of antibodies) (38, 44). A similar phenomenon is observed in evolution of human immunodeficiency virus over time in a single host (41). It is of interest that evolution of these viruses to evade adaptive immune pressure renders them more vulnerable to inhibition by SP-D or MBL. In any case it is likely of value to have innate inhibitors operating via different mechanisms to provide protection against varied viral strains as well as varied pathogens of other types. Interactions among innate immune proteins provide an additional layer of protection in some settings.
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GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-69031 (K. L. Hartshorn) and HL-29594 and HL-44015 (E. C. Crouch). U. Holmskov was supported by grants from the Danish Medical Research Council (9902278), the Novo Nordic Foundation, the Fifth (EC) Framework Program (contract QLK2000-00325), and the Benzon Foundation.
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FOOTNOTES
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Address for reprint requests and other correspondence: K. L. Hartshorn, Boston Univ. School of Medicine, EBRC 414, 650 Albany St., Boston, MA 02118 (E-mail: Khartsho{at}bu.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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