1 Departments of Medicine and Pathology, Boston University School of Medicine and Boston City Hospital, Boston, Massachusetts 02118; 2 Vanderbilt University School of Medicine, Nashville, Tennessee 37212; 3 Medical Research Council Immunochemistry Unit, Department of Biochemistry, Oxford University, Oxford OX1 3QU, United Kingdom; 4 Department of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus, Denmark; and 5 Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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The present study provides the first direct comparison of anti-influenza A virus (IAV) activities of the collectins surfactant protein (SP) A and SP-D, mannose-binding lectin (MBL), and conglutinin. SP-D, MBL, and conglutinin inhibited IAV hemagglutination activity with a greater potency than and by a distinct mechanism from SP-A. Although isolated trimeric SP-D carbohydrate recognition domains inhibited hemagglutination activity, preparations of SP-D also containing the collagen domain and NH2 terminus caused greater inhibition. In contrast to SP-A (or nonmultimerized SP-D), absence of the N-linked attachment did not effect interactions of multimerized SP-D with IAV. SP-D, SP-A, and conglutinin caused viral precipitation through formation of massive viral aggregates, whereas MBL formed aggregates of smaller size that did not precipitate. All of the collectins enhanced IAV binding to neutrophils; however, in the case of MBL, this effect was modest compared with the binding enhancement induced by SP-D or conglutinin. These studies clarify the structural requirements for viral inhibition by SP-D and reveal significant differences in the mechanisms of anti-IAV activity among the collectins.
influenza A virus; mannose-binding lectin; neutrophils
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INTRODUCTION |
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THE COLLECTINS ARE A GROUP of collagenous lectins present in mammalian serum and pulmonary secretions that are believed to play a role in first-line host defense against a variety of pathogens by binding to distinctive microbial carbohydrate determinants (17). The clearest evidence for this is a syndrome of increased propensity to childhood infections associated with low levels of the serum collectin mannose-binding lectin (MBL) (17, 25). More recently, low levels of MBL have also been found in patients with immunodeficiency persisting in adulthood (25). The collectins share certain basic structural properties. They contain globular carbohydrate recognition domains (CRDs) that attach to carbohydrates in a calcium-dependent manner. The CRDs are tethered together in trimeric subunits by collagen domains that, in turn, are bound together at the amino terminus by disulfide bonds into large multimeric structures containing multiple CRD heads.
Several lines of evidence suggest that collectins may play an important
role in host defense against viral infections. MBL and conglutinin have
been shown to inhibit infectivity of human immunodeficiency virus (HIV)
by attaching to carbohydrates on the viral envelope protein (6, 16).
The incidence of undetectable levels of MBL is greater in patients
infected with HIV than in the general population, suggesting that a
deficiency of MBL may predispose humans to HIV infection (8, 23). Low
levels of MBL are also associated with shorter survival in HIV-infected subjects (8). The first evidence of an interaction of collectins with
influenza A virus (IAV) arose from the determination by Anders et al.
(1) that the mammalian serum -inhibitors of IAV infectivity are
serum collectins. These inhibitors were shown to act by attaching in a
carbohydrate-dependent manner to virus-associated carbohydrates. Strains of IAV were shown to develop acquired resistance to the
-inhibitors by virtue of the loss of high mannose oligosaccharide attachments on the viral hemagglutinin (HA). We have confirmed that
purified human MBL and bovine conglutinin inhibit IAV HA activity and
infectivity and also act as opsonins, increasing respiratory burst
responses of neutrophils treated with IAV (14, 15). Both were found to
protect neutrophils from depressing the effects of IAV on neutrophil
functional responses to other stimuli. IAV-induced phagocyte
deactivation is a likely contributory cause to bacterial
superinfections that complicate IAV epidemics (12, 19).
Although interactions of serum collectins with IAV may be important in restricting bloodborne dissemination of IAV, the major locus of IAV replication is within the respiratory tract. We therefore investigated whether the collectins present in pulmonary surfactant, surfactant protein (SP) A and SP-D, also exhibit anti-IAV effects. SP-D in purified form, or as it occurs in human bronchoalveolar lavage (BAL) fluids, was shown to have similar antiviral and opsonic effects as the serum collectins (11). As with MBL and conglutinin, indirect evidence indicated that SP-D mediated its interactions with IAV via a Ca2+-dependent attachment to virus-associated carbohydrates. SP-D was found to be particularly active at causing viral aggregation and at protecting neutrophils from IAV-induced deactivation. Subsequent studies with various recombinant SP-D preparations suggested that these two functional properties of SP-D were related (3, 13). SP-A was also noted to inhibit IAV HA activity (11) and infectivity (4).
Structural comparisons of the collectins predict that significant functional differences may have evolved between the collectins. In this paper, we demonstrate such functional differences between MBL, conglutinin, SP-A, and SP-D by comparing the ability of these collectins to inhibit IAV HA activity, to mediate virus aggregation and precipitation, and to modulate interactions of IAV with neutrophils. Also, we further characterize the mechanisms of anti-IAV activity of SP-D through the use of a panel of recombinant wild-type and mutant SP-D preparations, including several not previously tested against IAV.
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MATERIALS AND METHODS |
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Reagents
N-formyl-methionyl-leucyl-phenylalanine (FMLP), cytochalasin B, horseradish peroxidase type II, scopoletin, superoxide dismutase (SOD), cytochrome c, Ficoll, dextran, sodium citrate, and citric acid were purchased from Sigma Chemical (St. Louis, MO), and Hypaque was obtained from Winthrop Pharmaceuticals (Des Plaines, IL). Dulbecco's phosphate-buffered saline (PBS) was purchased from Flow Laboratories (Costa Mesa, CA).Collectin Preparations
The recombinant SP-D preparations used in these studies are summarized in Table 1. The methods of preparation and isolation are as described in the references listed in Table 1. Recombinant rat (rr) SP-D and the cysteine mutant form [Ser15,20]rrSP-D have a single N-linked oligosaccharide attachment at Asn70 in the collagen domain (3). In some experiments, we treated [Ser15,20]rrSP-D with 15 units/ml of recombinant peptide N-glycosidase F (Oxford Glycosystems, Bedford, MA). To retain the functional activity of the protein, N-glycosidase digestion was carried out in PBS without added sodium dodecyl sulfate (SDS) or dithiothreitol for 18 h at 37°C. Based on SDS-polyacrylamide gel electrophoresis (PAGE), this procedure caused the expected reduction in the size of [Ser15,20]rrSP-D after deglycosylation (data not shown).
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Native human MBL was purified on mannan Sepharose and anti-immunoglobulin G columns as previously described (7). Conglutinin was purified from bovine serum as previously described (14, 21). Native human SP-A was purified from BAL fluids of alveolar proteinosis patients as previously described (11). All of the collectin preparations used in this study were free of other contaminating proteins as judged by SDS-PAGE analysis.
Virus Preparation
IAVs were grown in the chorioallantoic fluid of 10-day-old embryonated hen's eggs. Allantoic fluid was harvested after 48 h of incubation and clarified by centrifugation at 1,000 g for 40 min followed by centrifugation at 135,000 g to precipitate the viruses. The virus-containing pellets were then resuspended and purified on a discontinuous sucrose density gradient as previously described (10). Virus stocks were dialyzed against PBS, separated into aliquots, and stored at 70°C until used. HA titers were determined by titration of virus samples in PBS followed by the addition of thoroughly washed human type O red blood cells. The A/PR/8/34/H1N1 (PR8) strain of IAV was a gracious gift from Dr. Jon Abramson (Bowman-Gray School of Medicine, Winston-Salem, NC). The A/Mem71H-BelN strains were kindly provided by Dr. E. Margot Anders (University of Melbourne, Melbourne, Australia). The A/Bangkok 79/H3N2 (Bangkok 79) strain was a gift from Dr. Robert Webster (St. Jude's Hospital, Memphis, TN). The potency of each virus stock was measured by HA and protein assays after samples were thawed from frozen storage atIncubation of collectins with IAV stocks was carried out at 37°C in tris(hydroxymethyl)aminomethane (Tris)-buffered saline with 2 mM Ca2+ except in indicated experiments in which maltose or other saccharides were added or in which the buffer contained 10 mM EDTA and no added Ca2+ (11).
Assessment of Viral Aggregation
Virus aggregation was assessed by several methods. First, we assessed aggregation by measuring the changes in light transmission through suspensions of IAV with an SLM/Aminco 8000C (SLM Instruments, Urbana, IL) spectrofluorometer after the addition of various concentrations of collectins as previously described (14). We also directly evaluated the presence of viral aggregates by fluorescent microscopy with fluorescein isothiocyanate (FITC)-labeled virus as previously described (13). Finally, an additional method was developed to assess the size of viral aggregates resulting from incubation with collectins that involved low-speed centrifugation (300 g) of control or collectin-treated viral samples followed by measurement of how much viral protein was precipitated (as judged by HA assay, protein assay, and SDS-PAGE analysis) as a result of incubation with collectins (see RESULTS for further details).Neutrophil Preparation
Neutrophils from healthy volunteer donors were isolated to >95% purity with dextran precipitation as previously described, followed by a Ficoll-Hypaque gradient separation for removal of mononuclear cells and hypotonic lysis to eliminate contaminating red blood cells (10). Cell viability was >98% as determined by trypan blue staining, and the cells were used within 5 h of isolation.Measurement of Neutrophil Activation
H2O2 production was measured by the oxidation of scopoletin, andMeasurement of Viral Binding to Neutrophils
Viral binding to neutrophils was measured by preparing FITC-labeled virus and incubating this preparation with neutrophils, followed by evaluation of the cell-associated fluorescence with a flow cytometer or fluorescence microscopy as previously described (13).Statistics
Statistical comparisons were made with Student's paired t-test. ![]() |
RESULTS |
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Inhibition of HA Activity of Various IAV Strains by Collectins
SP-A inhibits HA activity with much less potency than and by a different mechanism from SP-D, MBL, or conglutinin. Table 2 shows the comparative activity of wild-type collectin preparations at inhibiting the HA activity of several IAV strains. Some of the data are reproduced from prior publications (as noted) for purposes of comparison. Recombinant human (rh) SP-D and rrSP-D, native human MBL, and bovine conglutinin had roughly similar abilities to inhibit HA activity of the Bangkok 79 or Mem71H-BelN strains of IAV. We have previously shown that HA inhibition mediated by MBL, SP-D, or conglutinin is abrogated by the addition of EDTA or blocking sugar preparations during incubation of the lectin with the virus (11, 14, 15). These results are consistent with the hypothesis that these collectins inhibit IAV HA activity via binding to virus-associated carbohydrates. The markedly reduced ability of these collectins to inhibit HA activity of the PR8 strain probably results from the reduced glycosylation of the HA of this IAV strain (5).
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Findings obtained with SP-A were, however, distinct from those obtained with the other collectins. Not only were substantially higher concentrations of SP-A required to inhibit HA activity, but the inhibitory effects of SP-A were not abrogated in the presence of EDTA or mannan (Table 2). Furthermore, SP-A had actually more potent HA inhibitory activity against the PR8 strain than against the Bangkok 79 or Mem71H-BelN strains. In view of this enhanced activity against the PR-8 strain, we also tested the activity of SP-A against another IAV strain, Mem71H-BelN/BS, which, compared with its parent strain Mem71H-BelN, has lost a high mannose oligosaccharide attachment close to the sialic acid binding site of the HA. Note that, by virtue of this change, Mem71H-BelN/BS is largely resistant to HA inhibition by MBL or conglutinin (1, 14, 15). In contrast, SP-A actually was more efficient at inhibiting Mem71H-BelN/BS than the fully glycosylated parent strain (see Table 2).
As noted, Table 1 describes the various recombinant mutant SP-D preparations used in this paper. [Ala72]rrSP-D was specifically produced to test whether removal of the single N-linked glycosylation site on SP-D alters structural or functional activities of the molecule. As previously reported (2), [Ala72]rrSP-D is structurally identical (including the degree of multimerization) to rrSP-D. As shown in Table 3, the [Ala72]rrSP-D mutant had equal, if not greater, HA inhibitory activity compared with the wild-type protein. As expected, this HA inhibitory activity was calcium dependent. Note that, like wild-type SP-Ds, [Ala72]rrSP-D had markedly reduced HA inhibitory activity against the PR8 strain of IAV.
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Sequences amino terminal to the CRD and neck region contribute to HA inhibition by SP-D. HA inhibition results with another mutant form of rrSP-D, termed [Ser15,20]rrSP-D, are shown in Table 3. This preparation lacks NH2-terminal cysteines and is unable to form multimeric structures beyond the basic trimer that comprises a single trimeric CRD and collagen stalk of SP-D (3). Although wild-type rrSP-D forms large structures containing from 4 to 32 trimeric CRDs/molecule, [Ser15,20]rrSP-D forms only single-armed structures with one trimeric CRD. For the experiments presented in Table 3, [Ser15,20]rrSP-D was incubated in PBS alone or PBS containing N-glycosidase F for 18 h at 37°C (as described in Collectin Preparations) to remove the single N-linked oligosaccharide attachment on the collagen domain of the protein. As shown in Table 3, [Ser15,20]rrSP-D had HA inhibitory activity against both Bangkok 79 and PR8 IAV strains (as previously reported). Although [Ser15,20]rrSP-D was less potent at mediating HA inhibition of Bangkok 79 than either wild-type rrSP-D or [Ala72]rrSP-D mutant, it had a greater ability to inhibit HA activity of PR8 than any of the other preparations. Removal of the N-linked sugar from [Ser15,20]rrSP-D had no effect on HA inhibition against Bangkok 79 but abolished activity against PR8. Hence, although the N-linked glycan on normally multimerized SP-D did not interact with IAV, it did so in the case of trimeric [Ser15,20]rrSP-D.
The glutathione S-transferase (GST)-SP-D CRD preparation consists of isolated trimeric CRD portions of the SP-D molecule attached to GST as previously described (13) and lacks the collagen domain of the molecule and along with it the ability to form multimers. The HA-inhibiting effects of GST-SP-D CRD or [Ala72]rrSP-D [like those of native human and rat SP-Ds (11)] were abrogated by EDTA or maltose. The concentration of GST-SP-D CRD required to inhibit IAV HA activity was >10 times higher than those of multimerized SP-D preparations (e.g., wild-type rrSP-D or [Ala72]rrSP-D) and ~4 times higher than that of [Ser15,20]rrSP-D needed to achieve a similar effect.
We also tested human "SP-D head and neck CRD" and "SP-D head
CRD" preparations containing only the head and neck region of the
CRD (without the associated GST domain) for HA inhibitory activity. The
SP-D head and neck CRD preparation forms the basic CRD
trimer by virtue of containing the -helical neck region of the
molecule, whereas the SP-D head CRD preparation lacks this region and
exists in monomeric form. The trimeric SP-D head and neck CRD
preparation had weak but reproducible HA inhibitory activity, whereas
no HA inhibitory activity could be demonstrated for the monomeric SP-D
head CRD preparation. The HA inhibitory activity of the SP-D head and
neck CRD preparation was less than that of either the GST-SP-D CRD or
[Ser15,20]rrSP-D
preparations, suggesting that the presence of collagen (or GST) domains
enhanced the ability of these molecules to inhibit viral HA activity.
SP-D, SP-A, and Conglutinin Cause Gross Aggregation and Precipitation of IAV Particles
Using a light-transmission assay, we previously found that the optimal concentrations of SP-D and conglutinin that cause aggregation of a stirred suspension of IAV are ~0.5-1.5 µg/ml. Figure 1 shows that wild-type rrSP-D and [Ala72]rrSP-D have a similar ability to aggregate IAVs. Although the results depicted in Fig. 1 show aggregation of the Bangkok 79 IAV strain, similar results were obtained in three experiments with the Mem71H-BelN strain of IAV as well (data not shown). As in prior experiments with conglutinin or other SP-D preparations (11, 14), viral aggregates were frequently visible after treatment with rrSP-Ds. Although MBL also induced viral aggregation in this assay, no visible viral aggregates were evident after incubation with MBL (data not shown).
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Although unaggregated viral particles remain in solution unless they are subjected to ultracentrifugation, we reasoned that aggregates of sufficient size would precipitate out of solution with low-speed centrifugation. To test this, we incubated Bangkok 79 IAV with rrSP-D or control buffer and then subjected the samples to centrifugation at 300 g. As shown in Fig. 2, incubation of IAV (Bangkok 79 strain) with rrSP-D caused nearly complete precipitation of viral protein under these conditions. SDS-PAGE gels of supernatants and pellets from these experiments confirmed that nearly all of the viral protein and rrSP-D were present in the precipitated fraction (data not shown). As expected, the untreated virus sample remained nearly entirely in the supernatant. Viral aggregates and pellets were grossly visible in these experiments. In fact, in the SP-D-treated samples, the viral aggregates visibly precipitated before centrifugation, forming an amorphous cloudy material in the bottom part of the tube. The results in Fig. 2 were confirmed by HA assay that showed nearly complete clearance of HA activity from the supernatant in samples treated with rrSP-D (Fig. 3).
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Moderately higher concentrations of conglutinin also caused essentially complete precipitation of viral protein (Figs. 2 and 3). In contrast, [Ser15,20]rrSP-D completely lacked the ability to cause viral precipitation. This result implies that formation of higher order multimers is necessary for SP-D to form large viral aggregates. Preincubation of the virus with 100 µg/ml of [Ser15,20]rrSP-D did, however, substantially reduce the amount of viral protein precipitated by the subsequent addition of 8 µg/ml of wild-type rrSP-D by 77 ± 7% (mean of 2 experiments; data not shown). Hence, although [Ser15,20]rrSP-D was not able to cause viral precipitation on its own, at a sufficient concentration, it could inhibit the precipitating effect of wild-type rrSP-D.
Of further interest, MBL was totally inactive at causing viral precipitation (Fig. 2). Thus, although MBL was able to cause viral aggregation by the light-transmission assay, these aggregates were of insufficient size to result in precipitation. Also, as shown in Fig. 3, the HA inhibitory activities of MBL or [Ser15,20]rrSP-D were substantially less than those of rrSP-D and conglutinin in these experiments. These results differ markedly from the results shown in Table 2 in which MBL had HA inhibitory activity comparable to SP-D. This discrepancy likely reflects the inability of MBL to induce viral precipitation. Note that the concentration of IAV used in Fig. 2 was considerably higher than that employed in the HA inhibition assays shown in Table 2.
rhSP-D had viral-precipitating activity similar to wild-type rrSP-D (e.g., 4 and 8 µg/ml of rhSP-D caused precipitation of ~50 and 80% of viral protein, respectively; data not shown). The ability of wild-type rrSP-D or rhSP-D to cause precipitation of the Bangkok 79 strain was lost when the assay was carried out in EDTA-containing buffer (data not shown). Also note that rrSP-D caused no appreciable precipitation of the PR8 strain of IAV (data not shown).
SP-A also caused substantial precipitation of Bangkok 79 IAV (Fig. 2), although ~10 times higher concentrations of SP-A than of SP-D were necessary to induce precipitation. In contrast to the case of SP-D, SDS-PAGE analysis of SP-A-treated samples showed that even at 76 µg/ml of SP-A, a significant amount (~30%) of viral protein and SP-A remained in the supernatant. Also in contrast to SP-D, precipitation by SP-A was not inhibited by EDTA. As shown in Fig. 3B, the degree of reduction in supernatant HA activity was nearly identical in the presence or absence of EDTA. Similar results were obtained when assaying for supernatant protein in the presence or absence of EDTA in SP-A-treated samples (data not shown).
Differences Among Collectins in Modulation of Neutrophil Interactions With IAV
SP-D and conglutinin induce binding of large viral aggregates to neutrophils. Hartshorn et al. (13) previously demonstrated an association between the ability of SP-D to aggregate IAV particles and SP-D-induced enhancement of viral binding to neutrophils. In view of the differences in extent of viral aggregation between MBL and the other collectins, we compared the ability of the collectins to enhance IAV binding to neutrophils. Preincubation of FITC-labeled IAV with either SP-A or MBL enhanced the mean viral binding to neutrophils to a modest extent as assessed by flow cytometry. The maximal increase in viral binding was 151 ± 13 and 150 ± 8% of control values after incubation with 16 µg/ml of SP-A or MBL, respectively (P < 0.05; n = 4 preparations for each). This degree of binding enhancement was modest compared with the enhancement previously reported by Hartshorn and colleagues (11, 13) after preincubation of the virus with SP-D. Also, the concentrations required to achieve optimal binding enhancement with these collectins were higher than those previously needed for SP-Ds. To clarify the reasons for these differences, we examined the binding of FITC-labeled virus to adherent neutrophils by fluorescent microscopy. As shown in Fig. 4, preincubation of FITC-labeled IAV with optimal concentrations of MBL resulted in binding of small viral aggregates to the neutrophil surface. As shown in Fig. 5, the size of the aggregates formed in the presence of SP-D or conglutinin was much greater than was the case for MBL. Wild-type rrSP-D and [Ala72]rrSP-D gave similar results to rhSP-D in this assay (data not shown).
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Given the size of viral aggregates induced by SP-D and conglutinin, we also wanted to determine whether such aggregates could be internalized by the cells. As shown in Fig. 6, neutrophils incubated with SP-D-treated virus at 4°C showed attachment of large fluorescent viral aggregates on their external surface. In contrast, when the cells were incubated at 37°C for 30 min, most of the fluorescence became internalized. These results were confirmed with flow cytometry (i.e., neutrophils incubated with SP-D-treated IAV at 37°C showed a 260 ± 15% increase in cell-associated fluorescence compared with cells treated with unopsonized IAV).
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Similar dose-response dependence for collectin-induced viral aggregation and enhancement of IAV-stimulated neutrophil respiratory burst responses. Hartshorn and colleagues previously reported that IAV alone stimulates a neutrophil respiratory burst response and that preincubation of IAV with SP-D (11) or conglutinin (14) causes enhancement of these responses. As shown in Fig. 7, the ability of IAV to stimulate neutrophil H2O2 responses was also enhanced when the virus was preincubated with either rrSP-D or [Ala72]rrSP-D at concentrations that caused aggregation. [Note that Brown-Augsburger et al. (3) previously found that preincubation of IAV with [Ser15,20]rrSP-D did not cause viral aggregation or enhancement of neutrophil H2O2 responses.] Preincubation of IAV with MBL or SP-A also enhanced the ability of the virus to stimulate H2O2 responses with a concentration dependence that suggested that viral aggregation contributed to this effect. Although 2.6 or 3.3 µg/ml of MBL or SP-A, respectively, did not cause enhanced H2O2 production, 5.2 or 6.7 µg/ml, respectively, of these collectins did (data not shown; n = 2 preparations for each).
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SP-A does not protect neutrophils against IAV-induced deactivation. As shown in Table 4, wild-type rrSP-D and [Ala72]rrSP-D were able to protect neutrophils against IAV-induced depression of FMLP-stimulated superoxide responses to a similar extent. These results are similar to those previously reported for native SP-Ds (11), conglutinin (14), or MBL (15). In contrast, SP-A was not protective in this assay even at considerably higher concentrations.
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DISCUSSION |
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IAV continues to cause considerable morbidity and mortality, to a large extent because of the ability of the virus to continually alter the antigenic properties of its surface proteins (19). It is likely that nonspecific host defense mechanisms such as phagocytes (12) and collectins play a role in the initial containment of IAV. Hartshorn et al. (11) reported that SP-D present in BAL fluid inhibits IAV HA activity. Anders et al. (1) recently showed, using a mouse model of IAV infection, that the degree of in vitro inhibition of particular IAV strains by SP-D correlates strongly with replication in the mouse lung. In this paper, we clarify the mechanism of anti-IAV activity of SP-D by the use of a panel of recombinant SP-D preparations and by comparison of SP-D with other collectins with respect to HA inhibition, viral aggregation and precipitation, and modulation of IAV interactions with neutrophils.
SP-D, MBL, and conglutinin were highly potent at mediating HA inhibition in a limiting-dilution assay (i.e., with minimal concentrations of virus). In this assay, MBL and conglutinin had slightly greater potency than SP-D. However, SP-A had substantially less potency than the other collectins in this assay. In addition, HA inhibition by SP-A was not calcium dependent and not inhibited by mannan. Also, the activity of SP-A was actually greater against IAV strains that have reduced glycosylation on their HA molecule. These results are consistent with those of Benne et al. (4), who showed that the ability of SP-A to inhibit infectivity of IAV (or herpes simplex virus; see Ref. 18) depends on attachment of the virus to carbohydrates on SP-A. The lectin activity of the IAV HA is not Ca2+ dependent. Our results suggest that for IAV strains with less extensive carbohydrate modification (particularly of the high mannose type) on the viral HA, viral binding to SP-A-associated carbohydrates may be facilitated. This is of interest because Anders et al. (1) showed that such strains replicate to a greater degree after inoculation in the murine airway. This may indicate that SP-D is more critical than SP-A in limiting IAV replication in vivo.
Although SP-A has an N-linked glycan on its CRD that may be readily accessible for interactions with pathogens, the only N-linked glycan on SP-D is on the collagen domain (18). We demonstrate conclusively using the mutant [Ala72]rrSP-D that this N-glycan does not contribute to HA inhibitory activity of multimerized SP-D. Of interest, however, in the case of trimeric [Ser15,20]rrSP-D, this N-glycan does appear to mediate HA inhibition of the PR8 strain. Brown-Augsburger et al. (3) previously reported that HA inhibition of the PR8 strain by [Ser15,20]rrSP-D was not calcium dependent and that the N-glycan on [Ser15,20]rrSP-D is sialylated. We now show that removal of the N-glycan from [Ser15,20]rrSP-D abolishes its HA inhibitory activity against PR8 without effecting activity against Bangkok 79 IAV. The obvious interpretation of these findings is that PR8 is able to bind to the N-glycan on [Ser15,20]rrSP-D. This N-glycan is most likely not accessible for such interactions in multimerized SP-D preparations, which, like MBL and conglutinin, have minimal HA inhibitory activity against PR8. Also of note is the fact that Bangkok 79 does not appear to attach to the N-glycan on [Ser15,20]rrSP-D, indicating that when there are sufficient glycans on the virus, attachment of the CRD of [Ser15,20]rrSP-D to the viral carbohydrates is preferred.
We also demonstrated that SP-D CRD trimers alone can inhibit IAV HA activity (Table 3). However, we showed that sequences amino terminal to the SP-D CRD contribute importantly to HA inhibition as well. Isolated SP-D CRD preparations (SP-D head and neck CRD and SP-D head CRD) were considerably less active at inhibiting IAV HA activity than the multimerized SP-D preparations (rrSP-D or [Ala72]rrSP-D). Although the SP-D head and neck CRD preparation had measurable HA inhibitory activity, the SP-D head CRD preparation had none. This implies that the ability to form a trimeric CRD head is a minimal requirement for HA inhibition. As demonstrated by Kishore et al. (20), the neck region of SP-D is necessary for trimerization and the SP-D head CRD exists in monomeric form. This monomeric preparation had significantly lower binding affinity for lipopolysaccharide or maltosyl-agarose than the trimeric SP-D head and neck CRD. This reduced binding affinity may, in part, account for the reduced ability to mediate HA inhibition.
Notably, the SP-D head and neck CRD preparation was ~20 times less potent than [Ser15,20]rrSP-D in the HA inhibition assay. The [Ser15,20]rrSP-D preparation has a full-length trimeric CRD head, neck, collagen domain, and NH2 terminus but lacks the ability to form higher order multimers by virtue of lacking NH2-terminal cysteine residues (3). [Ser15,20]rrSP-D had reduced HA inhibitory potency compared with fully multimerized preparations (rrSP-D or [Ala72]rrSP-D), indicating that multimerization contributes to HA inhibitory activity. However, the NH2 terminus or collagen domain per se may contribute in some way to HA inhibition (apart from its role in forming multimers) because the [Ser15,20]rrSP-D preparation had greater HA inhibitory activity than any of the isolated CRD preparations. Possible ways the collagen domain might contribute to HA inhibition include stabilization of the CRD trimer, steric interference, and/or exposure of sialylated N-linked sugars in the collagen domain that could serve as a binding site for IAV (see above).
It should be noted that some of the differences found among the isolated CRD preparations and [Ser15,20]rrSP-D or the multimerized rrSP-Ds could reflect differences in the species origin of these proteins (e.g., the SP-D head and neck CRD and SP-D head CRD preparations were derived from human SP-D, whereas [Ser15,20]rrSP-D and rrSP-D were of rat origin). However, previous data by Hartshorn et al. (11) showed no significant difference between the interactions of rat and human SP-Ds with IAV or neutrophils; hence it is unlikely that functional differences of the magnitude observed in this paper resulted from species differences.
Assays of viral aggregation revealed further important differences in activity among the collectins. Although MBL had a high potency at HA inhibition in the limiting-dilution assay, when tested against higher concentrations of IAV, it had greatly reduced activity compared with SP-D. Similarly, the HA inhibitory activity of [Ser15,20]rrSP-D was greatly reduced in this context. Our results indicate that HA inhibition in the setting of high concentrations of IAV is more related to viral precipitation. Multimerized SP-D preparations had the greatest viral precipitating activity. Although SP-A also mediated viral precipitation, its activity was considerably less than that of SP-D. The precipitating activity of SP-A was again distinctive in being non-calcium dependent.
Despite inducing some degree of viral aggregation by the light-transmission assay, MBL did not induce any precipitation over a wide range of concentrations. [Ser15,20]rrSP-D also induced no precipitation (presumably because of the inability to form multimers), although it was able to inhibit precipitation caused by wild-type SP-D. It is likely that the level of precipitating activity of the various collectins relates to collagen domain structure. Of the collectins, SP-D had the largest and most extended collagen domain. Conglutinin is highly related to SP-D, although it has a slightly smaller collagen domain than human SP-D (17). Also, conglutinin has not been reported to form multimers higher than dodecamers, whereas SP-D can form multimers containing up to 32 CRD heads (9). SP-A and MBL have much smaller collagen domains, with a configuration resembling a bouquet of tulips (17). Such a configuration may be less efficient at mediating massive aggregation.
Brown-Augsburger et al. (3) and Hartshorn et al. (13) reported that multimerized preparations of SP-D are able to form large aggregates of IAV particles and that such aggregates can bind to neutrophils. We show that the ability of SP-D and conglutinin to form such aggregates leads to a greater enhancement of IAV binding to neutrophils than was found with MBL. We also demonstrate for the first time that these large SP-D- or conglutinin-mediated aggregates are internalized by neutrophils. [Ala72]rrSP-D had activity similar to wild-type rrSP-D at enhancing IAV binding to neutrophils and enhancing neutrophil respiratory burst responses on exposure to IAV. Hence, for multimerized SP-D, the SP-D associated N-glycan does not contribute to the modulation of neutrophil interactions with IAV. SP-A was again distinct from the other collectins in that it did not protect neutrophils from the depressing effects of IAV on respiratory burst responses to other stimuli.
In summary, in this paper, we demonstrate major differences in functional interactions of the various collectins with IAV. The structural features of collectins of most importance in determining these distinctive functional attributes include glycosylation and differences in collagen domain size and multimerization. We also demonstrate that surfactant collectins have the ability to mediate viral precipitation, whereas MBL does not. We speculate that such precipitation may be important in the airway and may contribute to viral containment during early phases of infection by facilitating clearance of large numbers of viral particles through mucociliary or phagocytic mechanisms.
It is difficult to state precisely what levels of SP-A and SP-D are
present in the airway during IAV infection. We have measured SP-D
levels to be ~140 ng/ml in BAL fluids of healthy volunteers (11).
SP-A levels in similar samples have been found to be 10 times higher
than this (24). SP-D is largely found in the aqueous phase of pulmonary
secretions, whereas SP-A is largely lipid associated. BAL samples are
probably greatly diluted compared with actual airway fluids. Also,
levels of SP-A and SP-D may rise during IAV infection. In any case,
most of the functional effects we report in this paper occur at levels
of SP-D or SP-A that probably occur in the airway. Furthermore, it is
possible that the SP-A used in these studies is less active than that
present in situ in the airway because the extracted SP-A has been
exposed to lipid solvents. Experiments with SP-A in the presence of
lipid or with recombinant SP-A might show greater activity. It is also
likely that there are cooperative interactions among SP-A, SP-D, and
other surfactant components in the airway [e.g., we have reported
additive HA inhibitory effects between SP-D and SP-A (11)].
Further in vivo studies will be needed to clarify the relative
contributions of SP-D and SP-A to innate host defense against IAV.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Allergy and Infectious Diseases Grant AI-34897 (to K. L. Hartshorn); National Heart, Lung, and Blood Institute Grant HL-29594 (to E. C. Crouch); and the Boston City Hospital Fund for Excellence (to K. L. Hartshorn).
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FOOTNOTES |
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Address for reprint requests: K. L. Hartshorn, Boston Univ. School of Medicine, K-725, 80 East Concord St., Boston MA 02118.
Received 7 May 1997; accepted in final form 3 September 1997.
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