Collectins are multimeric host defence lectins with trimeric CRDs (carbohydrate-recognition domains) and collagen and N-terminal domains that form higher-order structures composed of four or more trimers. Recombinant trimers composed of only the CRD and adjacent neck domain (termed NCRD) retain binding activity for some ligands and mediate some functional activities. The lung collectin SP-D (surfactant protein D) has strong neutralizing activity for IAVs (influenza A viruses) in vitro and in vivo, however, the NCRD derived from SP-D has weak viral-binding ability and lacks neutralizing activity. Using a panel of mAbs (monoclonal antibodies) directed against the NCRD in the present study we show that mAbs binding near the lectin site inhibit antiviral activity of full-length SP-D, but mAbs which bind other sites on the CRD do not. Two of the non-blocking mAbs significantly increased binding and antiviral activity of NCRDs as assessed by haemagglutination and neuraminidase inhibition and by viral neutralization. mAb-mediated cross-linking also enabled NCRDs to induce viral aggregation and to increase viral uptake by neutrophils and virus-induced respiratory burst responses by these cells. These results show that antiviral activities of SP-D can be reproduced without the N-terminal and collagen domains and that cross-linking of NCRDs is essential for antiviral activity of SP-D with respect to IAV.
- influenza A virus
- surfactant protein D
SP-D (surfactant protein D) plays important roles in innate defence against IAV (influenza A virus) and other viral and bacterial infections [1–6]. Trimeric NCRD [neck plus CRD (carbohydrate-recognition domain)] fragments of human or rodent SP-D are functional lectins and efficiently bind to some microbial ligands including mannan and bacterial lipopolysaccharides . Furthermore, NCRD preparations have been found to bind to correct many abnormalities of SP-D−/− mice [8–11]. These findings have raised hope that NCRDs may be employed as therapy for some infectious or inflammatory states. Unfortunately, the isolated trimeric NCRD of human SP-D binds weakly to IAV and does not inhibit viral infectivity, despite strong binding and neutralizing activity for full-length SP-D dodecamers [12,13]. hNCRDs (human NCRDs) and trimers also bind poorly to phosphatidylinositol, as compared with human dodecamers . Overall, these results suggest that wild-type SP-D has a remarkable dependence on co-operativity among NCRDs for binding to some ligands (e.g. IAV and phosphatidylinositol). Of interest, replacement of key residues around the lectin-binding site of SP-D results in the ability of some mutant NCRDs to efficiently bind to and neutralize IAV.
In the process of studying the epitopes and functional effects of mAbs (monoclonal antibodies) directed against the NCRD of human SP-D, we found that certain antibodies blocked the antiviral activity of wild-type SP-D, whereas others did not. Furthermore, among the non-blocking mAbs, we found that two mAbs increased viral binding and antiviral activity of hNCRDs. This provided us with a useful model system for testing the role of binding co-operativity in the antiviral activity of SP-D.
MATERIALS AND METHODS
Buffers and reagents
Dulbecco's PBS containing 0.9 mM calcium and 0.493 mM magnesium and PBS without calcium and magnesium were purchased from Invitrogen. PBS with added calcium and magnesium at pH 7.2 was used unless otherwise indicated.
The SP-D preparations used in the present study are summarized in Table 1. The methods for preparation of most of these constructs have been described previously. Dodecamers of wild-type recombinant human or rat SP-Ds were used as control preparations and were expressed in CHO (Chinese-hamster ovary) cells and purified as described in . NCRD preparations, including the RAK (hNCRD with the insertion of arginine, alanine and lysine between residues 324 and 325) and E321K mutants, were produced in Escherichia coli as described [12,13]. A neckless mutant 9 [hCRD (human CRD)] that cannot form trimers, was isolated and characterized as previously described . In the E321K mutant the glutamine residue at position 321 was replaced with lysine by site-directed mutagenesis.
mAbs 245-01, 245-02 and 246-02–246–08 were raised against SP-D by inoculating mice with 10 μg/ml human SP-D as previously described . The 245-01 antibody, which was raised against a recombinant fragment of human SP-D composed of only the neck and CRD, recognizes human SP-D by Western blot analysis in the reduced and unreduced form, and was shown to be effective for immunolocalization of SP-D in human tissues. The 246-07 mAb probably binds to the primary calcium and saccharide-binding region of the CRD because it blocks binding of SP-D to mannan . In contrast, the 246-04 mAb binds to a distinct region of the CRD and does not block binding to mannan. The 246-04 mAb does, however, block binding of human SP-D to lung gp-340, a secreted member of the scavenger receptor superfamily that can co-isolate with SP-D .
Binding of mAbs to SP-D (wild-type and mutants)
SP-D preparations were diluted in coating buffer [15 mM Na2CO3 and 35 mM NaHCO3, (pH 9.6)] to a concentration of 2 μg/ml and coated on to ELISA plates overnight, followed by washing and addition of mAbs. The final concentration of mAbs used for the ELISA assay was 1 μg/ml. Bound mAbs were detected with HRP (horseradish peroxidase)-conjugated donkey anti-mouse antibodies followed by TMB (3,3′,5,5′-tetramethylbenzidene) peroxidase. A450 values were measured on a POLARstar OPTIMA plate reader (BMG Labtech).
IAV was grown in the chorioallantoic fluid of 10-day-old chicken eggs and purified on a discontinuous sucrose gradient as previously described . The virus was dialysed against PBS to remove sucrose, aliquoted and stored at −80 °C until needed. Philippines 82/H3N2 (Phil82) strain was kindly provided by Dr E. Margot Anders (Department of Microbiology, University of Melbourne, Melbourne, Australia). Post-thawing, the viral stocks contained ∼5×108 plaque-forming units/ml.
HA (haemagglutinin) inhibition assay
HA inhibition was measured by serially diluting collectin or other host defence protein preparations in round-bottomed 96-well plates (Serocluster U-Vinyl plates; Costar) using PBS as a diluent. After adding 25 μl of IAV, giving a final concentration of 40 HA units/ml or 4 HA units/well, the IAV–protein mixture was incubated for 15 min at room temperature (25 °C), followed by addition of 50 μl of a type O human erythrocyte suspension (containing ∼108 erythrocytes/ml). The minimum concentration of protein required to inhibit fully the haemagglutinating activity of the viral suspension was determined by noting the highest dilution of protein that still inhibited haemagglutination. Inhibition of HA activity in a given well is demonstrated by the absence of formation of an erythrocyte pellet. If no inhibition of HA activity was observed at the highest protein concentration used then the value is expressed as >the maximal protein concentration.
Measurement of viral aggregation by collectins
Viral aggregation was measured by light transmission and EM (electron microscopy) as previously described .
Fluorescent focus assay of IAV infectivity
MDCK (Madin–Darby canine kidney) cell monolayers were prepared in 96-well plates and grown to confluency. These layers were then infected with diluted IAV preparations for 45 min at 37 °C in PBS and tested for the presence of IAV-infected cells after 7 h using a monoclonal antibody directed against the influenza A viral nucleoprotein (provided by Dr Nancy Cox, CDC, Atlanta, GA, U.S.A.) as previously described . IAV was pre-incubated for 30 min at 37 °C with SP-D or control buffer (PBS), followed by addition of these viral samples to the MDCK cells. Where indicated, SP-Ds were first incubated with mAbs prior to adding them to IAV.
Human neutrophil preparation
Neutrophils from healthy volunteers were isolated to >95% purity using dextran precipitation, followed by Ficoll–Paque gradient separation for the removal of mononuclear cells, and then hypotonic lysis to eliminate any contaminating erythrocytes, as previously described . Cell viability was determined to be >98% by Trypan Blue staining. The isolated neutrophils were resuspended at the appropriate concentrations in control buffer (PBS) and used within 2 h. Neutrophil collection was performed with informed consent as approved by the Institutional Review Board of Boston University School of Medicine, Boston, MA, U.S.A.
Measurement of IAV uptake by neutrophils
FITC-labelled IAV (Phil82 strain) was prepared and the uptake of the virus by neutrophils was measured as previously described . In brief, IAV was incubated with neutrophils for 30 min at 37 °C in the presence of control buffer. Trypan Blue (0.2 mg/ml) was added to these samples to quench extracellular fluorescence. Following washing, the neutrophils were fixed with paraformaldehyde and neutrophil-associated fluorescence was measured using flow cytometry. The mean neutrophil fluorescence (>1000 cells counted per sample) was measured.
Measurement of neutrophil H2O2 production
H2O2 production was measured by assessing the reduction in scopoletin fluorescence as previously described . Measurements were made using the plate reader as described above.
Statistical comparisons were made using Student's paired, two-tailed t test or ANOVA with post-hoc test (Tukey's). ANOVA was used for multiple comparisons with a single control.
Interactions of mAbs with SP-D
We compared binding of a panel of mAbs raised against human SP-D with full-length human SP-D and NCRDs derived from rat and mouse SP-D (Figure 1). Note that some epitopes were conserved between human and rodent SP-Ds (e.g. 246-03); whereas other epitopes were partially retained in rat SP-D but not mouse SP-D (246-04 and 246-08). In Figure 2 (left-hand panel) we next compared binding of mAbs with hNCRD or hCRD (which differ in that the latter lacks the neck domain). All mAbs bound to the hNCRD trimer preparation. mAb 245-01 did not bind to hCRD, strongly suggesting that this mAb recognizes the neck region of SP-D. Because 245-01 had reduced binding to full-length SP-D (Figure 1) the epitope that it recognizes may be less accessible in the full-length molecule than in the NCRD. Monoclonal 245-02 may bind to the junction of the neck and CRD because binding was greatly, but not completely, reduced to hCRD. Of interest, binding of 246-07 was also greatly reduced in hCRD. We have previously reported that this mAb blocks antiviral activity of SP-D and binding to mannan and very probably recognizes an epitope near or overlapping the carbohydrate-binding site [23,24].
We also compared binding of the mAbs with two mutant versions of hNCRD, RAK and E321K (Figure 2, right-hand panel). The RAK mutant has a three-amino-acid insertion adjacent to Asp325 and slightly increased antiviral activity compared with wild-type hNCRD . Asp325 is found on the N-terminal ridge adjoining the primary calcium and saccharide-binding site and the interspecies substitution of asparagine at position 325 was shown to alter the affinity for ManNAc. The mAbs 246-02, 246-03, 246-05 and 246-07 had reduced binding to RAK (Figure 2, right-hand panel); hence, it is likely that the epitopes recognized by the mAbs overlap with the saccharide-binding site. Binding of mAbs 245-01, 245-02, 246-04 and 246-08 was not reduced by the RAK insertion implying that they do not bind near Asp325. Glu321 is one of the amino acids that co-ordinates with the primary calcium and replacement of this residue with a lysine residue results in loss of lectin activity. As shown in Figure 2 (right-hand panel) mAbs 246-03, 246-05 and 246-07 had greatly reduced binding to E321K again indicating that these mAbs recognize epitopes overlapping the saccharide-binding site of the CRD. Binding of mAb 246-06 was also reduced to some extent in both RAK and E321K mutants.
In Figure 3 we show that 246-07 and three other mAbs (246-02, 246-03 and 246-05) strongly inhibited binding of full-length SP-D to IAV. In contrast, mAbs 246-04 and 246-08 caused minimal inhibition of binding to IAV, and 246-06 had an intermediate effect. As shown in Table 2 (first column of results), the 246-02, 246-03 and 246-05 mAbs also interfered with HA inhibitory activity of SP-D; whereas 245-01, 245-02, 246-04, 246-06 and 246-08 did not. These results support the concept that the former antibodies bind near the saccharide-binding site, whereas the latter do not.
Effects of non-inhibitory mAbs on HA-inhibiting and neutralizing activity of NCRDs for IAV
The ability of SP-D to neutralize or inhibit HA activity of IAV has been shown to depend on binding to glycans on the HA of the virus . Wild-type hNCRD has minimal binding to IAV using ELISA assays and does not cause any inhibition of HA activity or infectivity . As shown in Table 2 (middle column of results), addition of either 246-04 or 246-08 mAb enabled hNCRD to inhibit HA activity of IAV. The 246-08 was somewhat more potent than 246-04 at enhancing the HA-inhibitory activity of hNCRD. The E321K mutant lacked HA-inhibitory activity and this was not altered by the 246-08 mAb (Table 2, last column).
As shown in Figure 4, mAbs 246-04 and 246-08 enabled hNCRD to mediate viral-neutralizing activity. Although hNCRD caused no viral aggregation as measured by light transmission through a stirred viral suspension, addition of mAb 246-08 resulted in modest but statistically significant viral aggregation as assessed by light transmission (Figure 5A) or EM (Figure 5B).
Effect of mAbs on the ability of NCRDs to inhibit viral neuraminidase activity
Inhibition of neuraminidase activity of IAV appeared to be strongly dependent on co-operative binding effects, and could be largely mediated via steric effects following binding to the HA . hNCRD did not cause inhibition of neuraminidase activity of IAV; however, inhibition was observed when hNCRD was combined with mAb 246-04 or 246-08 (Figure 6).
Effect of mAbs on the ability of NCRDs to modify neutrophil responses to IAV
IAV binds to and is ingested by neutrophils resulting in a respiratory burst response characterized by H2O2 generation. Pre-incubation of IAV with dodecamers of full-length SP-D markedly potentiates viral uptake by neutrophils and virus-induced H2O2 generation [1,25]. These effects correlate in general with the ability of SP-D to induce viral aggregation . As expected, hNCRD did not cause any increase in viral uptake or respiratory burst responses by neutrophils; however, it did so when pre-incubated with mAb 246-08 (Figures 7A and 7B).
SP-D plays an important role in the early innate defence against common human strains of IAV. NCRD preparations of SP-D are relatively easy to produce in large quantities and conveniently assemble as trimers due to the coiled-coil structure of the neck domain. NCRDs are being evaluated as possible therapeutics for a variety of respiratory conditions in which natural functions of SP-D are insufficient [8–10]. They also have the potential advantage of lacking the collagen domain, which has been implicated as a trigger for pro-inflammatory effects of SP-D . Unfortunately, NCRD preparations of wild-type SP-D have a markedly diminished ability to bind to or inhibit IAV. Full- length multimeric SP-D or a collagen domain mutant form of SP-D, that retains the N-terminus and the ability to form small multimers, both exhibit strong viral-neutralizing activity in vitro and in vivo . This implies that binding to IAV by SP-D is strongly determined by co-operative interactions among CRD heads. We have also reported that modifications of residues around the lectin site of the hNCRD (e.g. as in the RAK mutant) can confer viral binding and neutralizing activity .
In the present study we initially sought to characterize binding of a panel of mAbs directed against the NCRD of human SP-D to wild-type or mutant forms of SP-D. Although the mAbs were generally highly specific for human SP-D, there was some overlap in binding to rodent SP-Ds. Using the RAK-, E321K- and hCRD-modified versions of hNCRD we provide partial mapping of epitopes of some of the mAbs. mAbs 246-02, 246-03, 246-05 and 246-07 bind near the lectin site of SP-D, whereas 245-01 and 245-02 bind to the neck region. Consistent with this interpretation, mAbs 246-02, 246-03, 246-05 and 246-07 blocked binding of full-length SP-D to IAV and interfered with HA inhibition caused by full-length SP-D. The mAbs 245-01, 245-02, 246-04, 246-06 and 246-08 did not interfere with these activities of SP-D. Based on moderately reduced binding to the RAK and E321K mutants, the 246-06 mAb may recognize a site which partially overlaps the saccharide-binding site or binding surface of the CRD, but it does not inhibit function.
Of interest, some of the non-blocking mAbs (246-04 and 246-08) were found to increase viral binding and various antiviral activities of hNCRD, including HA inhibition, virus neutralization and neuraminidase inhibition. This was not a non-specific effect of the mAbs, since cross-linking of E321K (which lacks lectin activity) did not result in antiviral activity. In addition the mAbs enabled hNCRD to cause viral aggregation, supporting the concept that the mAbs induced cross-linking of NCRDs. mAb 246-08 was more effective than 246-04 at enhancing antiviral activities of hNCRD on several of the assays. mAb 246-04 likely binds along the lateral surface of the CRD head. This conclusion is based on the prior findings that porcine SP-D has a glycan in this area which interferes with binding to lung gp-340 and mAb 246-04 blocks binding of gp-340 . Of note, lung-derived gp-340, like mAb 246-04, significantly potentiates antiviral activities of SP-D. Further studies to more precisely determine the binding sites of 246-04 and 246-08 are underway.
It is of interest that neither 245-01 nor 245-02 mAbs increased activity of the NCRDs since Ohya et al.  have reported that another mAb directed against the neck region of SP-D potentiated binding to Toll-like receptors . It is also notable that the mAbs did not increase antiviral activities of full-length dodecameric SP-D implying that co-operative binding is already maximal for dodecameric SP-D.
The ability of the mAbs to enhance neutrophil responses to IAV in the presence of the hNCRD will require further study. We have previously shown that the ability of full-length SP-D to induce viral aggregation correlates with its ability to promote viral uptake by, and virus-induced respiratory burst responses of, neutrophils [26,29]. Hence, the ability of hNCRD to aggregate virus particles in the presence of mAb 246-08 may account for the potentiation of neutrophil responses. It is also possible, however, that the Fc domain of some murine mAbs can engage neutrophil Fc receptors; future studies will involve preparation of Fab1 and Fab2 fragments of anti-SP-D mAbs to evaluate this possibility. Coupling of the SP-D NCRD to an Fab1 fragment of an antibody-directed against CD89 (the Fc receptor for IgA on neutrophils) results in dramatic increases in the uptake of IAV, bacteria and Candida by neutrophils and an increase in IAV-induced respiratory burst responses by these cells. Cross-linking of NCRD with 246-04 or 246-08 mAbs could modulate neutrophil responses in a similar manner .
We found that mAbs directed against the CRD of SP-D are able to block or enhance antiviral activities of SP-D, consistent with observed differences in the localization of their dominant epitopes. The observation that incubation of hNCRD with the non-blocking mAbs 246-04 and 246-08 allows hNCRD to mediate viral aggregation and enhance neutralization provides additional strong evidence that co-operative interactions between NCRD trimers are important, if not critical, for anti-influenza activity. The available results suggest that this is accomplished by promoting cross-linking interactions between NCRD trimers, thereby mimicking the normal multimeric structure of the native collectin. Cross-linking of NCRDs with mAbs or through other means may be a useful strategy in therapeutic applications of collectins.
Abbreviations: CRD, carbohydrate-recognition domain; EM, electron microscopy; HA, haemagglutinin; IAV, influenza A virus; mAb, monoclonal antibody; MDCK, Madin–Darby canine kidney; NCRD, neck plus CRD; SP-D, surfactant protein D
- © The Authors Journal compilation © 2008 Biochemical Society