The lung scavenger receptor-rich protein glycoprotein-340 (gp-340) is present in bronchoalveolar lavage (BAL) fluids and saliva and mediates specific adhesion to and aggregation of bacteria. It also binds to surfactant proteins A and D (SP-A and -D). Prior studies demonstrated that SP-A and SP-D contribute to innate defense against influenza A virus (IAV). We now show that lung and salivary gp-340 inhibit the hemagglutination activity and infectivity of IAV and agglutinate the virions through a mechanism distinct from that of SP-D. As in the case of SP-A, the antiviral effects of gp-340 are mediated by noncalcium-dependent interactions between the virus and sialic acid-bearing carbohydrates on gp-340. Gp-340 inhibits IAV strains that are resistant to SP-D. Concentrations of gp-340 present in saliva and BAL fluid of healthy donors are sufficient to bind to IAV and inhibit viral infectivity. On the basis of competition experiments using competing saccharide ligands, it appears that SP-D does not entirely mediate that anti-IAV activity of BAL fluid and contributes little to that of saliva. Furthermore, removal of gp-340 from BAL fluid and saliva significantly reduced anti-IAV activity. Hence, gp-340 contributes to defense against IAV and may be particularly relevant to defense against SP-D-resistant viral strains.
- surfactant protein D
- salivary gland
influenza a viruses (IAVs) are a major cause of morbidity and mortality worldwide. In the United States alone influenza epidemics account for an average of ∼50,000 deaths per year (20). 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 (6-8, 10, 17, 18).
The role of SP-D appears to be particularly important in this regard. Among the known human collectins, SP-D has the strongest in vitro IAV neutralizing and aggregating activity (10). Removal of SP-D from bronchoalveolar lavage (BAL) significantly reduced antiviral activity (10). SP-D knockout mice (SP-D-/-) exhibit greatly increased viral titers, lung inflammation, and illness after infection with IAV (18). Several studies have demonstrated that SP-D levels in mouse lung lavage rise after IAV infection (18, 25). Replacement of SP-D by instillation of recombinant SP-D or by overexpression of modified forms of SP-D in the airway partially or completely normalizes the response of SP-D-/- mice to IAV infection (18, 30).
It is likely that other constituents of upper and lower airway fluids contribute to the innate host defense against IAV. Prior experiments demonstrate that depletion of SP-D from human BAL fluids did not entirely eliminate anti-IAV activity (10). Furthermore, although SP-D-resistant IAV strains developed in vitro replicate more highly and cause more illness in mice than wild-type viral strains, such resistant strains have not emerged as a major clinical problem in humans (25). One component of BAL fluid that contributes to anti-IAV activity through a distinct mechanism from SP-D is SP-A. SP-D-resistant strains remain sensitive or have increased sensitivity to inhibition by SP-A (7). Whereas SP-D-mediated viral inhibition depends on binding of SP-D to high-mannose oligosaccharides on the viral envelope proteins, SP-A-mediated inhibition is dependent on viral glycosylation and involves binding of the viral hemagglutinin to sialylated carbohydrates on SP-A (1, 7). However, the antiviral activity of SP-A is relatively modest compared with that of SP-D, and it is likely that other components of BAL fluid or upper airway secretions also contribute.
In the current study we demonstrate that another component of human lung and oral secretions, scavenger receptor-rich glycoprotein 340 (gp-340), has strong anti-IAV activities mediated through a mechanism resembling that of SP-A. Gp-340 was initially identified as a pulmonary SP-D-binding molecule that is present in BAL and associated with alveolar macrophages (13). Sequence analysis revealed gp-340 to be a member of the scavenger receptor family closely related to the putative tumor suppressor gene DMBT1 (14). Gp-340 colocalizes with SP-D lung lavage and intracellular compartments of alveolar macrophages and is expressed along with SP-D in lung, salivary glands, trachea, small intestine, and stomach. Gp-340 also binds to SP-A (28). Gp-340 has been identified as the “salivary agglutinin” (19, 24) that mediates specific adhesion to, and aggregation of, Streptococcus mutans and other bacteria (4, 23). We now demonstrate that gp-340 of lung or salivary origin has antiviral activity against IAV.
MATERIALS AND METHODS
Dulbecco's phosphate-buffered saline with (PBS++) and without (PBS) calcium and magnesium were purchased from GIBCO-BRL (Grand Island, NY).
IAV was grown in the chorioallantoic fluid of 10-day-old chicken eggs and purified on a discontinuous sucrose gradient as previously described (9). The virus was dialyzed against PBS to remove sucrose, aliquoted, and stored at -80°C until needed. Philippines 82/H3N2 (Phil82) strain and its bovine serum-β inhibitor resistant variant, Phil82/BS, were kindly provided by Dr. E. Margot Anders (University of Melbourne, Melbourne, Australia). The A/PR/8/34/H1N1 (PR-8) strain was a gift of Dr. Jon Abramson (Bowman Gray School of Medicine, Winston-Salem, NC). The hemagglutinin titer of each virus preparation was determined by titration of virus samples in PBS with thoroughly washed human type O, Rh(-) red blood cells as described (9). Postthawing the viral stocks contained ∼5 × 108 plaque forming U/ml.
Hemagglutination Inhibition Assay
The hemagglutination (HA) inhibition assay was carried out in round-bottom 96-well plates (Costar) (12). First, 50 μl of PBS were added to all wells. After this, 50 μl of purified gp-340, SP-A, SP-D, saliva, or BAL fluid were added to the first well in a row. After being mixed, 50-μl aliquots were removed from the first well and titrated across the plate with a multichannel device. Hence, each sample underwent a 1:1 dilution in each subsequent well. After this, a fixed dose of virus was added in 50 μl to all wells, and then 100 μl of a solution containing type O human erythrocytes were added to all wells (final volume per well was thus 200 μl). The plates were then read after 1 h to determine which wells had visible erythrocyte pellets. Individual wells were scored as being fully pelleted or half-pelleted. A pellet indicates that the HA activity of the virus has been inhibited in that well. (When the virus agglutinates the cells, they form a lattice that cannot fall into the bottom of the well). In Table 1, the results are expressed according to the amount of a specific purified collectin or gp-340 that was present in the most diluted well in which a pellet was noted. In Tables 2, 3, and 5 the results are expressed simply as the number of wells (hence, number of 50% dilutions) in which inhibition was observed for either saliva or BAL fluid. We expressed the results in this way since saliva and BAL are mixtures of various proteins and other substances. The effect of adding various sugars or of removing gp-340 or IgA from BAL fluid or saliva was tested by the HA inhibition assay in Tables 3 and 5. In these cases, a decrease of one well in HA inhibition activity indicates a 50% decrease in activity.
Lung gp-340, Salivary gp-340, Microfibril-Associated Protein 4 and Collectin Preparations
Recombinant human SP-D (RhSP-D) was produced in CHO-K1 cells as previously described (6). For these studies the dodecameric fraction of RhSP-D was used unless otherwise specified. SP-A was kindly provided by Dr. Jeffrey Whitsett (Children's Hospital and Medical Center, Cincinnati, OH). The collectin preparations used in this report were tested for degree of contamination with endotoxin using 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 ∼20-100 pg/ml (or 6-12 endotoxin units/ml using internal assay standard).
Gp-340 was isolated as described (13). In brief, BAL was centrifuged at 300 g for 10 min, then at 10,000 g for 30 min. The precipitate was dissolved in Tris-buffered saline containing 0.05% emulphogen (polyoxyethylene tricdecyl ether, BC 720; Sigma, St. Louis, MO) and 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 0-1 M NaCl. The gp-340-containing fractions were separated by gel permeation chromatography on a Superose 6 prep-grade column. The purity of the resulting gp-340 was analyzed by chromatography and SDS-PAGE. The gp-340 preparation was free of SP-D and SP-A or other major contaminants.
The salivary gp-340 preparation was prepared as described (19). Parotid saliva was precipitated at 4°C, centrifuged at 5,000 g for 15 min at 4°C and resuspended in Tris with 10 mM EDTA and 0.05% [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Samples were applied to a Uno Q-6 column (Bio-Rad, Hercules, CA) and eluted with a linear gradient of 0-0.5 M NaCl.
For some experiments, purified gp-340 was pretreated with neuraminidase (1.28 U/ml neuraminidase Type X; Sigma) for 1 h at 37°C. When examined by Western blot analysis, neuraminidase treatment did not cause any apparent degradation of gp-340 (data not shown).
Recombinant human microfibril-associated protein 4 (MFAP4) was produced in Escherichia coli and purified as previously described (16).
Source of Saliva and BAL Fluids
BAL fluids were obtained from healthy volunteer donors. Between 150 and 200 cc of normal saline were instilled for the lavage. Fluids obtained by this procedure were subjected to an initial centrifugation (150 g) to remove cells and large particulate matter. Saliva samples were obtained from healthy donors by simple expectoration into sterile 50-ml tubes, followed by centrifugation of saliva at 10,000 g to remove the mucinous precipitate and addition of 1% penicillin and streptomycin to inhibit bacterial growth. BAL fluids and saliva were obtained after informed consent as approved by the Boston University School of Medicine Institutional Review Board for Human Research.
Measurement of gp-340 Levels in BAL and Saliva
Gp-340 levels were measured using a sandwich ELISA. First, ELISA plates were coated with the anti-gp-340 monoclonal 213-01 overnight. To test for nonspecific binding, we coated some wells with fatty acid-free BSA (Sigma). After washing and further incubation of the wells with PBS containing 2.5% BSA, we added aliquots of purified gp-340, BAL fluid, or saliva samples 30 min at 37°C. After further washing, wells were incubated with a different anti-gp-340 MAb (Hyb 213-06) that had been prelabeled with biotin (13). Bound MAb 213-06 was detected using streptavidin-horseradish peroxidase. A concentration of 500 ng/ml of the MAbs was used. Bound secondary antibodies were detected with 3,3',5,5'-tetramethylbenzidine peroxidase substrates (Bio-Rad Labs). The reaction was stopped with 1 N sulfuric acid. The optical density was measured on an ELISA plate reader at 450 nm (OD450). Each data point was performed in duplicate. The ELISA detected concentrations of gp-340 between 12.5 and 1,000 ng/ml in a linear manner, with minimal background binding of antibodies to wells not containing BAL or saliva.
Assessment of Binding of gp-340 in BAL or Saliva to IAV
Binding of salivary or BAL proteins to IAV was tested via an ELISA in which 1 μg/ml of IAV was allowed to adhere onto 96-well plates by incubation overnight at 4°C in coating buffer. After centrifugation of the plates at 2,500 rpm for 10 min and washing with PBS, the plates were blocked with PBS containing 2.5% BSA for 3 h. These virus-coated plates were then incubated with BAL samples, followed by the addition of anti-gp-340 MAbs and other reagents as noted above (see Measurement of gp-340 Levels in BAL and Saliva).
Western Immunoblotting of BAL Fluid and Saliva
BAL fluid and saliva samples 4-15% Tris-glycine gels (Jule, Milford, CT) at 100 V in 25 mM Tris and 192 mM glycine. Molecular mass standards were run on all gels. The gels were transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA) with a minitransblot cell (Bio-Rad) run at 100 V for 1 h. After transfer, the blots were washed with 0.05% (vol/vol) PBS-Tween and then blocked with Carnation nonfat dry milk (5% wt/vol in PBS-Tween) overnight at 4°C. The presence of gp-340 was detected with a 1:5,000 dilution of the 213-06 MAb against gp-340, followed by a 1:10,000 dilution of donkey anti-mouse antibodies conjugated with horseradish peroxidase (HRP; Jackson Immunologicals, Bar Harbor, ME). IgA was detected with a 1:10,000 dilution of anti-human IgA polyclonal antibodies conjugated to HRP (Sigma). SP-D was detected with a 1:5,000 dilution of P13B12 rabbit polyclonal antibodies against human SP-D, followed by a 1:10,000 dilution of goat-anti-rabbit-HRP (Jackson Immunologicals). The proteins were finally visualized using chemiluminescence.
Depletion of gp-340 from BAL Fluid and Saliva
The 213-06 MAb directed against gp-340 was coupled to Sepharose using the AminoLink Immobilization Kit (Pierce) following the manufacturer's instructions. After coupling overnight, remaining active sites were blocked with quenching buffer and then washed. The column was pretreated with PBS containing 5 mM EDTA, followed by incubation of BAL fluid or saliva (also containing 5 mM EDTA) with the column for 1 h at room temperature. EDTA was used to minimize binding of SP-D to gp-340 (13). The BAL fluid or saliva was then eluted from the column and assayed for content of SP-D, SP-A, and gp-340 by Western blot analysis or ELISA.
In initial experiments, passage of the 100-g supernatant of BAL fluid through this column caused reduction of both gp-340 and SP-A and was noted by Western blot analysis (data not shown). Therefore, the BAL fluid was subjected to an initial 10,000-g centrifugation for 30 min to remove SP-A (see Fig. 5). The 10,000-g supernatant of BAL (largely lacking SP-A) was then passed through the anti-gp-340 column, which did result in marked reduction of gp-340 without apparent alteration of either IgA or SP-D concentrations (Fig. 5). Incubation of saliva with the anti-gp-340 column effectively reduced gp-340 from saliva without altering levels of SP-D or IgA (Fig. 7).
Fluorescent Focus Assay of IAV Infectivity
Madin-Darby canine kidney (MDCK) monolayers were prepared in 96-well plates and grown to confluence. These 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 incubation with MAb 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 under fluorescent microscopy. Initially, various dilutions of virus were used to find the dose yielding ∼50 fluorescent foci per high powered (×40) field. These foci appeared to be single infected cells in general. Subsequently, this dose of virus was incubated with different doses of gp-340 or SP-D to assess the effect of these treatments on infectivity.
Measurement of Aggregation of IAV Particles
We assessed aggregation of IAV particles following the addition of various concentrations of collectins by monitoring changes in light transmission on a highly sensitive SLM/Aminco 8000C (SLM Instruments, Urbana, IL) spectrofluorometer as described (11). The aggregation of viral particles or liposomes is demonstrated by a decline in light transmission (i.e., increased turbidity).
Statistical comparisons were made by Student's paired t-test.
Lung and Salivary gp-340 Exert Significant Anti-Influenza Activity
Lung and salivary gp-340 inhibit HA activity of IAV through a mechanism distinct from that of SP-D or serum collectins. As shown in Table 1, lung and salivary gp-340 inhibited the erythrocyte HA activity of several strains of IAV. Lung gp-340 had significant inhibitory activity against all IAV strains tested. The HA inhibitory activity of lung gp-340 was not significantly altered when the assay was run in EDTA-containing buffer. Addition of monosaccharides (glucose, GlucNAc, mannose, or galactose; final concentration 42 mM) also did not alter HA inhibitory activity of lung gp-340 (data not shown). This concentration of mannose or glucose markedly reduced HA inhibitory activity of SP-D. Furthermore, gp-340 had similar or increased activity against SP-D- and serum collectin-resistant strains of IAV (e.g., Phil82/BS or PR-8) compared with that against a representative SP-D- and serum collectin-sensitive strain (Phil82). Phil82/BS and PR-8 are laboratory-derived strains that lack oligosaccharide attachments on their hemagglutinin (3, 5). Hence, the mechanism through which gp-340 inhibits IAV does not appear to involve binding of gp-340 to carbohydrate attachments on the viral HA.
Table 1 compares lung gp-340 with gp-340 purified from saliva and several other lung lavage proteins with known or possible host defense functions. Both gp-340 preparations had greater antiviral activity than SP-A against wild-type and SP-D-resistant strains of IAV. Although SP-D had greater activity against the wild-type Phil82 strain, lung or salivary gp-340 had, respectively, comparable or greater activity against the PhilBS or PR-8 strains of IAV compared with SP-D. Salivary gp-340 had reduced activity against Phil82 compared with lung gp-340, but greater activity against PR-8. MFAP4 is another SP-D-binding protein with lectin activity in human lung lavage (16). MFAP4 had no discernable activity against any IAV in these experiments (Table 1).
Lung and salivary gp-340 neutralize infectivity of IAV. In general HA inhibitory activity correlates with viral neutralizing activity. As shown in Fig. 1, lung and salivary gp-340 caused dose-related inhibition of infectivity of the IAV (Phil82 strain) for MDCK cells. The concentration of lung gp-340 that caused 50% neutralization of infectivity was ∼5.6 ng/ml compared with 108 ± 25 ng/ml (n = 4) for salivary gp-340. Considerably higher concentrations of SP-A (e.g., ∼3 μg/ml) were required to achieve 50% neutralization in this assay (Fig. 1). MFAP4 did not cause dose-dependent inhibition of infectivity in this assay. These results generally parallel HA inhibition results for the Phil82 strain shown in Table 1.
Gp-340 aggregates IAV particles. Gp-340 purified from either BAL or saliva aggregated IAV particles as judged by decreased light transmission through stirred viral suspensions (Fig. 2). Lung gp-340 had greater viral aggregating activity for the Phil82 strain of IAV than salivary gp-340 (compare results with 1.6 μg/ml of the proteins in Fig. 2). This finding is consistent with the greater HA inhibitory activity and neutralizing activity of the lung gp-340 preparation against Phil82 (Table 1 and Fig. 1). The ability of lung or salivary gp-340 to aggregate Phil82 IAV was compared with that of SP-D or SP-A (Fig. 2B). Gp-340 had greater aggregating activity than SP-A but less than SP-D.
In Fig. 2C the ability of salivary gp-340, SP-D, or SP-A to aggregate the PR-8 strain of IAV is compared. Salivary gp-340 aggregated this strain of virus to a significantly greater extent than comparable concentrations of SP-D or SP-A. SP-D caused no significant aggregation of PR-8, whereas SP-A caused a similar degree of aggregation of PR-8 as it caused with Phil82. Salivary gp-340 caused significantly greater aggregation of PR-8 than it did of the Phil82 strain (compare Fig. 2, B and C). This is consistent with the stronger HA inhibitory activity of salivary gp-340 against PR-8 than Phil82 (see Table 1).
Anti-IAV Activities of BAL Fluid and Saliva of Healthy Volunteers: Role of gp-340
Concentrations of gp-340 in saliva and BAL fluids of normal volunteers are sufficient to inhibit viral infectivity. Gp-340 levels in BAL fluids of normal volunteer donors were measured using a sandwich ELISA as described in materials and methods. The mean gp-340 concentration (130 ± 30 ng/ml or 1.06 ng of gp-340/μg of total BAL protein; n = 12) was in a range sufficient to inhibit HA activity or infectivity of IAV. Because these measurements were made of unconcentrated lavage fluids, local concentrations in the lung are presumably considerably higher. The mean level of gp-340 in saliva was 492 ± 40 ng/ml (n = 5), which was significantly higher than the concentration in BAL (P < 0.003). These results probably represent an underestimate of the actual salivary gp-340 content, since a significant proportion of the gp-340 may have been present in the gel phase of saliva that was removed by the initial 10,000-g centrifugation (29). In any case, the concentration of gp-340 in saliva was sufficient to inhibit infectivity of IAV.
Lower HA inhibitory activity of BAL fluid but not saliva against PR-8 strain of IAV. HA inhibitory activity of saliva and BAL fluids against Phil82, Phil82BS, and PR-8 strains of IAV was compared. BAL fluids had significantly reduced HA inhibitory activity against PR-8 compared with activity against Phil82 or Phil82BS (Table 2). There was a trend toward lower activity against PhilBS compared with Phil82 (i.e., 8 of 12 samples tested had lower activity); however, this was not statistically significant. Saliva samples were tested using concentrations of saliva that caused roughly similar degree of inhibition against Phil82. In contrast to BAL fluid, saliva did not have lower activity against either PhilBS or PR-8, rather, there was a trend toward increased activity against these strains compared with Phil82.
Competing sugars cause more interference with HA inhibitory activity of BAL than saliva. Addition of various monosaccharide competitors of mannose-type C-type lectins like SP-D interfered substantially with the ability of BAL to inhibit activity of Phil82 (Table 3). Among the monosaccharides tested, glucose and mannose caused significantly greater interference than GlucNAc or galactose, which are inefficient competitors of SP-D. The monosaccharides did not cause significant inhibition of HA inhibitory activity of BAL fluid against PR-8 (Table 3). These results are consistent with a significant contribution of C-type lectin activity to the HA inhibition caused by BAL fluids against the wild-type Phil82 strain. Note that there is residual activity of BAL despite addition of high concentrations of competing sugars, consistent with our prior finding that removal of SP-D from BAL fluid reduces but does not eliminate HA inhibiting activity (10). In contrast, competing monosaccharides caused much less inhibition of HA inhibitory activity of saliva against Phil82. Only mannose caused significant interference with this activity of saliva, although the degree of interference was much less than for BAL fluid. Hence, the HA inhibitory activity of saliva is much less dependent on C-type lectin activity than BAL fluid, implying a much reduced contribution of SP-D to this activity. This is consistent with the lack of reduction in HA inhibitory activity of saliva against PR-8 compared with Phil82 (Table 2).
Gp-340 in saliva or BAL binds to IAV. As shown in Fig. 3, gp-340 present in BAL and purified lung gp-340 (Fig. 3A) bound to the Phil82 strain of IAV in a concentration-dependent manner. Figure 3A shows results obtained with one representative BAL fluid sample. BAL fluid from 10 separate donors was tested, and all showed binding of BAL-associated gp-340 to IAV (means ± SE, OD450 was 0.32 ± 0.08 after subtraction of background binding to BSA; 50 μl of BAL fluid used). Binding of gp-340 to Phil82 IAV correlated strongly with the concentrations of gp-340 in the BAL fluids as measured using the sandwich ELISA (r = 0.97).
These results indicate that lung gp-340 can bind to IAV even in the presence of other components of BAL fluid and that binding correlates with the level of gp-340 in the BAL fluid. Because SP-D and gp-340 both bind to IAV and to each other, we tested directly whether SP-D modulates binding of lung gp-340 to IAV. IAV-coated ELISA plates were incubated simultaneously with various concentrations of both SP-D and lung gp-340 after which binding of gp-340 was measured (Fig. 3B). Note that SP-D was not found to significantly alter the binding of the 213-06 anti-gp-340 MAb to gp-340 (data not shown). SP-D at concentrations of either 250 or 500 ng/ml reduced binding of gp-340 to IAV significantly. However, gp-340 still bound significantly to IAV in presence of these concentrations of SP-D.
In parallel assays, binding of gp-340 in saliva to Phil82 IAV was significantly greater than binding of BAL-associated gp-340 (i.e., means ± SE OD450 was 1.2 ± 0.33; n = 5; P < 0.038 compared with BAL results; background binding to BSA subtracted before analysis).
Mechanism of binding of gp-340 to IAV. We hypothesized that the likely mechanism of binding of gp-340 to IAV involved noncalcium-dependent attachment of the IAV hemagglutinin to sialylated carbohydrates on gp-340. To test this we first preincubated the virus with the hemagglutinin ligand neuraminyl-lactose. This resulted in significantly reduced binding of BAL-associated gp-340 to the virus-coated plate, while not reducing background binding of gp-340 to plates not containing virus (Table 4). This supports the hypothesis that binding of gp-340 to IAV involves attachment of the HA to sialic acids on gp-340. This was further supported by the finding that neuraminidase treatment of purified gp-340 resulted in reduced binding to IAV. Before neuraminidase treatment, addition of 200 ng/ml of purified gp-340 to the virus-coated ELISA plate resulted in an OD450 of 0.310 ± 0.02. After neuraminidase treatment OD450 was reduced to 0.07 ± 0.03 (P < 0.004 compared with binding before neuraminidase treatment; n = 6).
The 213-06 MAb was used for detection of bound gp-340 in these assays. The 213-06 MAb binds to protein determinants on gp-340, whereas another MAb, 213-01, binds to sialylated carbohydrates attachments of gp-340 (2). Of interest is that, in preliminary experiments, preincubation of gp-340 with IAV significantly reduced binding of the 213-01 MAb to gp-340 (data not shown). This implies that the virus competes for binding with the 213-01 MAb. Overall, our results are consistent with the interpretation that IAV binds to sialic acids residues on gp-340.
Neutralizing activity of BAL fluids: effect of depletion of gp-340. As shown in Fig. 4, BAL fluids from four tested donors had dose-related IAV neutralizing activity. Concentrations of gp-340 in these fluids are shown for reference. We next attempted selective removal of gp-340 from one of these BAL fluids (donor 3) to determine whether this would reduce viral neutralizing activity. Gp-340 has been reported to bind to or associate with several other proteins in mucosal secretions, including SP-A and SP-D, IgA (23), and mucins (27). Because these other proteins all may contribute to anti-IAV activity of BAL fluid, attempts to selectively remove gp-340 to determine its specific contribution to anti-IAV activity were expected to be problematic. Our approach involved preparation of a column in which the 213-06 MAb directed against gp-340 was coupled to Sepharose. After passage of BAL fluid through this column, reduction of both gp-340 and SP-A was noted by Western blot analysis (data not shown). Therefore, the BAL fluid was subjected to an initial 10,000-g centrifugation for 30 min to remove SP-A (see Fig. 5). This centrifugation procedure did not appear to significantly alter the level of gp-340, SP-D, or IgA (Fig. 5), nor did it alter the neutralizing activity of BAL against Phil82 (Fig. 6). This result suggests either that SP-A does not contribute substantially to the neutralizing activity of BAL fluid or, more likely, that removal of other components counterbalances the effect of removing SP-A. The 10,000-g supernatant of BAL (largely lacking SP-A) was then passed through the anti-gp-340 column, which did result in marked reduction of gp-340 without apparent alteration of either IgA or SP-D concentrations (Fig. 5). This procedure significantly reduced neutralizing activity of BAL fluid against Phil82 (Fig. 6).
A similar procedure was carried out using saliva (Fig. 7). Using the anti-gp-340 column, we found it possible to markedly reduce the gp-340 concentration in saliva. SP-D was detectable by Western blot analysis in saliva; however, the level of SP-D was not altered by passage through the anti-gp-340 column (Fig. 7). The level of IgA in saliva was also not altered by passage through the column (Fig. 7). For comparative purposes, IgA was removed using a similar column prepared with antibodies against IgA instead of the anti-gp-340 MAb. Removal of gp-340 from saliva caused significant reduction in HA inhibitory activity of saliva against IAV (Table 5). Of note, removal of gp-340 caused a comparable degree of reduction in HA inhibitory activity of saliva as removal of IgA. Removal of gp-340 caused greater reductions in activity against the PR-8 strain of IAV than against Phil82 (Table 5), which is consistent with the HA inhibitory activity of salivary gp-340 against these strains (Table 1). Removal of gp-340 also caused significant reduction in neutralizing activity against the Phil82/BS strain of IAV (Fig. 8). These results suggest that gp-340 contributes significantly to IAV inhibition by saliva and that gp-340 in saliva may be particularly important for protection against C-type lectin resistant strains of IAV.
Although prevention of IAV infection depends on the presence of respiratory antibodies, and resolution of IAV infection depends on cell-mediated immunity (15), innate immune mechanisms also appear to contribute importantly to host defense against the virus. We have previously demonstrated that SP-A and SP-D inhibit infectivity of IAV. SP-D was most potent in this regard, inhibiting infectivity of IAV by binding of its carbohydrate recognition domain (CRD) to oligosaccharide attachments on the viral envelope proteins (8). The current study demonstrates that another component of oral and respiratory secretions, gp-340, inhibits infectivity of IAV. Prior studies have suggested a role for salivary gp-340 in host defense against bacteria (24) and human immunodeficiency virus (21). This study demonstrates antiviral activity of gp-340 against influenza viruses.
Gp-340 exerts antiviral effects through a mechanism that differs from that of SP-D. HA inhibition by gp-340 is not calcium dependent, nor is it abrogated by addition of competing sugars. Furthermore, lung and salivary gp-340 have equal or greater inhibitory activity against strains of IAV that are resistant to SP-D (or to the serum collectins) due to loss of oligosaccharide attachments on the viral hemagglutinin. SP-A has a similar mechanism of action (1, 10); however, gp-340 is considerably more potent than SP-A at inhibiting HA activity and neutralizing activity (Table 1 and Fig. 1) and inducing viral aggregation (Fig. 2).
We provide evidence that binding of gp-340 to influenza virus is mediated by attachment of the viral hemagglutinin to sialylated carbohydrates on gp-340, therefore acting as a γ-inhibitor akin to SP-A (1). γ-Inhibitors inhibit viral infectivity by competing with cell surface sialic acid-bearing ligands. The mechanism of binding of gp-340 to influenza differs, therefore, from the mechanism of binding to bacteria. Gp-340 binds to bacteria through interaction of bacteria with a specific peptide within the scavenger receptor domains (2).
Gp-340 from different individuals may vary in glycosylation or electrophoretic properties (19, 24), and such differences may account for differences we observed in antiviral activity of the salivary and lung gp-340 preparations. Alternatively, differences may exist between glycosylation or other properties of gp-340 produced in the lung and salivary glands. In preliminary studies we found that lung and salivary gp-340 differ in the type of sialic acid linkages to the penultimate galactose in their N-linked oligosaccharides (data not shown). The nature of these sialic acid linkages is a key determinant of binding affinity for diverse strains of influenza (22, 26). Hence, despite protein sequence identity, the salivary and lung forms of gp-340 could still differ considerably in their reactivity with specific strains of IAV. Further studies will be needed to clarify this. In any case, both lung and salivary preparations were strong inhibitors of all IAV strains tested, and each had greatest activity against SP-D-resistant strains. Hence, gp-340 appears to add another layer of protection against influenza virus infection that complements the activity of SP-D.
Our findings may in part explain why SP-D-resistant strains have not emerged as a significant threat in the human population. Of note, the neutralizing activity of saliva was strongest against SP-D-resistant strains and showed only minimal dependence on C-type lectin activity. BAL fluids, in contrast, showed strong dependence on C-type lectin-mediated inhibition (presumably mostly resulting from SP-D). Although we could demonstrate the presence of SP-D in saliva by Western blot analysis (Fig. 7), it appears likely that levels of SP-D in saliva are lower than in BAL fluid. By ELISA, the levels of SP-D were not clearly measurable (implying either interference by other proteins or levels <10 ng/ml). These observations suggest that IAV encounters different barriers to infection at different levels of entry into the host. Selective pressure on influenza viruses to maintain glycosylation of the hemagglutinin may be exerted not only by antiviral antibodies but by nonlectin innate inhibitors (like gp-340 or SP-A) as well.
Our studies indicate that in vitro findings with purified gp-340 are of physiological relevance for several reasons. BAL- and saliva-associated gp-340 bound to IAV. Also, the concentrations of gp-340 in BAL fluids or saliva of normal volunteer donors were sufficient to contribute to their IAV neutralizing activity based on in vitro activities of the purified proteins. Finally, removal of gp-340 from BAL fluid and saliva reduced neutralizing activity of these fluids. The last finding cannot be considered conclusive, since it is possible that reduction in other inhibitors occurred during removal of gp-340. However, the gp-340 depletion studies are compatible with an independent contribution of this protein to the overall anti-influenza activity of these fluids. Gp-340 appeared not to be strongly associated with lipids in normal BAL fluid (in contrast to SP-A) since centrifugation at 10,000 g did not appreciably change the amount of gp-340 detected by Western blot analysis. Hence, gp-340 may act largely in the aqueous phase along with SP-D.
Our results suggest that important interactions may occur between lung gp-340 and SP-A or SP-D with respect to their anti-influenza activities. This is an important area for further investigation. Results shown in Fig. 3 indicate that the ratio of SP-D to gp-340 in BAL fluid may affect binding of gp-340 to SP-D-sensitive strains of IAV. It is of interest that SP-D reduced binding of lung gp-340 to Phil82 IAV, since SP-D binds to gp-340. It is possible that the binding site for gp-340 on the CRD of SP-D is less accessible to bind to gp-340 after attachment to IAV. In any case our results confirm that BAL-associated gp-340 binds to IAV even in the presence of SP-D or other components of BAL and that the mechanism of binding most likely involves binding of free viral HA molecules to sialylated carbohydrates on gp-340. The greater binding of saliva-associated gp-340 to IAV may be accounted for by several factors, including higher levels of gp-340 in saliva, greater interference of SP-D with binding of gp-340 in the case of BAL, or differences in viral binding properties of lung vs. salivary gp-340. Future studies will also address this issue.
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.
- Copyright © 2003 the American Physiological Society