Mitchell R. White

Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses.

We tested the hypothesis that pulmonary surfactant-associated lectins--surfactant proteins A and D (SP-A, and -D)--contribute to initial protective mechanisms against influenza A viruses (IAVs). SP-D potently inhibited hemagglutination activity of several strains of IAV as well as causing viral aggregation. SP-D enhanced neutrophil binding of IAV and neutrophil respiratory burst responses to the virus. Neutrophil dysfunction resulting from IAV exposure was diminished when the virus was pre-incubated with SP-D. Each of these effects was mediated by the calcium-dependent carbohydrate-binding property of SP-D. Native SP-D preparations of both human and rat origin, as well as recombinant rat SP-D, had similar activity. SP-A also inhibited IAV hemagglutination activity. We have previously reported that related mammalian serum lectins (mannose-binding lectin [MBL] and conglutinin) have similar effects. SP-D was at least 10-fold more potent at causing hemagglutination inhibition than were SP-A or MBL. SP-D was shown to contribute to potent anti-IAV activity of human bronchoalveolar lavage fluid. These results suggest that SP-D--alone, and in conjunction with SP-A and phagocytic cells--constitutes an important component of the natural immune response to IAV infection within the respiratory tract.

Pulmonary surfactant proteins A and D enhance neutrophil uptake of bacteria

The collectins are a class of collagenous lectin proteins present in serum and pulmonary secretions [pulmonary surfactant protein (SP) A and SP-D] that are believed to participate in innate immune responses to various pathogens. With the use of flow cytometric and fluorescent-microscopic assays, SP-A and SP-D were shown to increase calcium-dependent neutrophil uptake of Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus. Evidence is provided that the collectins enhanced bacterial uptake through a mechanism that involved both bacterial aggregation and direct actions on neutrophils. The degree of multimerization of SP-D preparations was a critical determinant of both aggregating activity and potency in enhancing bacterial uptake. The mechanisms of opsonizing activity of SP-D and SP-A differed in important respects from those of opsonizing antibodies. These results provide the first evidence that surfactant collectins may promote neutrophil-mediated clearance of bacteria in the lung independently of opsonizing antibody.

Human mannose-binding protein functions as an opsonin for influenza A viruses.

Influenza A viruses (IAVs) cause substantial morbidity and mortality in yearly epidemics, which result from the ability of the virus to alter the antigenicity of its envelope proteins. Despite the rapid replication of this virus and its ability to infect a wide variety of cell types, viremia is rare and the infection is generally limited to the upper respiratory tract. The preimmune host defense response against IAV is generally, therefore, successful. We have previously provided (and summarized) evidence that neutrophils contribute to defense against IAV, although neutrophil dysfunction and local tissue damage may be less salutory byproducts of this response. Here we provide evidence that the serum lectin mannose-binding protein directly inhibits hemagglutinin activity and infectivity of several strains of IAV. In addition mannose-binding protein acts as an opsonin, enhancing neutrophil reactivity against IAV. Opsonization of IAV by mannose-binding protein also protects the neutrophil from IAV-induced dysfunction. These effects are observed with physiologically relevant concentrations of mannose-binding protein. Two different allelic forms of recombinant mannose-binding protein are found to have similar effects. We believe, on the basis of these data, that mannose-binding protein alone and in conjunction with phagocytic cells is an important constituent of natural immunity (i.e., preimmune defense) against IAV.

Human defensins and LL-37 in mucosal immunity

Defensins are widespread in nature and have activity against a broad range of pathogens. Defensins have direct antimicrobial effects and also modulate innate and adaptive immune responses. We consider the role of human defensins and the cathelicidin LL‐37 in defense of respiratory, gastrointestinal, and genitourinary tracts and the oral cavity, skin, and eye. Human β‐defensins (hBDs) and human defensins 5 and 6 (HD5 and −6) are involved most obviously in mucosal responses, as they are produced principally by epithelial cells. Human α‐defensins 1–4 (or HNPs 1–4) are produced principally by neutrophils recruited to the mucosa. Understanding the biology of defensins and LL‐37 is the beginning to clarify the pathophysiology of mucosal inflammatory and infectious diseases (e.g., Crohns disease, atopic dermatitis, lung or urinary infections). Challenges for these studies are the redundancy of innate defense mechanisms and the presence and interactions of many innate defense proteins in mucosal secretions.

Mechanisms of anti-influenza activity of surfactant proteins A and D: comparison with serum collectins

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.

Mechanism of binding of surfactant protein D to influenza A viruses: importance of binding to haemagglutinin to antiviral activity.

Collectins are important in the initial containment of a variety of pathogens, including influenza A virus (IAV). We provide the first systematic evaluation of the oligosaccharide-binding sites for pulmonary surfactant protein D (SP-D) on specific IAV coat glycoproteins and define the relationship between this binding and antiviral activity. With the use of several techniques, SP-D was found to bind via its carbohydrate-recognition domain (CRD) to mannosylated, N-linked carbohydrates on the HA(1) domain of the haemagglutinin (HA) and on the neuraminidase of IAV. Using a set of IAV strains that differed in the level and site of glycosylation, and a panel of recombinant collectins, we found that binding of SP-D to the globular domain of the HA was critical in mediating the inhibition of viral haemagglutination activity and infectivity. We also demonstrated that the pattern of binding of a collectin to IAV glycoproteins can be modified by altering the monosaccharide-binding affinity of its CRD or by linking the CRD to a different N-terminal/collagen domain. These studies clarify the mechanisms of viral neutralization by collectins and might be useful in engineering collectins for enhanced antiviral activity.

Human Neutrophil Defensins Increase Neutrophil Uptake of Influenza A Virus and Bacteria and Modify Virus-Induced Respiratory Burst Responses

Human neutrophil peptides (HNPs) are released from granules of neutrophils in response to various activating stimuli and they participate in the killing of bacteria and the stimulation of various inflammatory responses. HNPs also inhibit infectivity of enveloped viruses, including influenza A virus (IAV). In this study, we demonstrate that HNPs increase the uptake of IAV and bacteria by neutrophils. The dimeric HNPs also induced aggregation of IAV and bacterial particles, which may, in part, explain their ability to increase uptake. HNPs did not increase neutrophil respiratory burst responses to IAV. We have recently demonstrated direct interactions of HNPs with surfactant protein D (SP-D), another important effector of innate immunity and antimicrobial host defense. Although HNPs did not alter SP-D-dependent uptake of IAV, they counteracted the ability of SP-D to increase IAV-induced neutrophil H2O2 generation. Our studies reveal previously unappreciated functional effects of HNPs, expand our understanding of the antiviral properties of HNPs, and suggest important interactions between collectins and HNPs in the host response to viruses and bacteria.

Innate Defense against Influenza A Virus: Activity of Human Neutrophil Defensins and Interactions of Defensins with Surfactant Protein D

Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some instances when relatively SP-D-resistant strains were treated with SP-D and HNPs. HNPs were found to bind to the neck and/or carbohydrate recognition domain of SP-D. This binding was specific because no, or minimal, binding to other collectins was found. HNPs precipitated SP-D from bronchoalveolar lavage fluid and reduced the antiviral activity of bronchoalveolar lavage fluid. HNP-1 and -2 differed somewhat in their independent antiviral activity and their binding to SP-D. These results are relevant to the early phase of host defense against IAV, and suggest a complex interplay between SP-D and HNPs at sites of active inflammation.

Neutrophil survival is markedly reduced by incubation with influenza virus and Streptococcus pneumoniae: role of respiratory burst

Bacterial superinfections are an important cause of morbidity and mortality during influenza A virus (IAV) epidemics. We demonstrate that incubation with the combination of IAV and Streptococcus pneumoniae caused marked reductions in survival of neutrophils in vitro compared with treatment with control buffer or IAV or S. pneumoniae alone. This cooperative effect was in part mediated by acceleration of neutrophil apoptosis as evidenced by increases in annexin‐V binding and caspase‐3 activation. However, GM‐CSF did not increase survival of neutrophils exposed to IAV and S. pneumoniae. IAV enhanced neutrophil uptake of S. pneumoniae significantly. Furthermore, the combination of IAV and S. pneumoniae caused significantly more hydrogen peroxide production than IAV or S. pneumoniae alone. This increased respiratory burst activity contributed to the diminished neutrophil survival caused by IAV and S. pneumoniae. The NADPH oxidase inhibitor, diphenyleneiodonium, significantly improved survival of neutrophils treated with IAV and S. pneumoniae. These findings may help to explain the increased susceptibility of IAV‐infected patients to infections with S. pneumoniae.

Interactions of α-, β-, and θ-Defensins with Influenza A Virus and Surfactant Protein D

We have reported that the α-defensins human neutrophil peptides (HNP)-1 and HNP-2 neutralize and aggregate influenza A virus (IAV) and promote uptake of IAV by neutrophils. These α-defensins were also shown to bind to surfactant protein (SP)-D and reduce its antiviral activity. In this study, we examined retrocyclin (RC)1 and RC2, humanized versions of the antiviral θ-defensins found in the leukocytes of certain nonhuman primates. RC1 was just as effective as HNP-1–3 in neutralizing IAV, and RC2 and RC101 (an analog of RC1) were more effective. In contrast, human β-defensins (HBDs) showed less neutralizing activity. Human defensins 5 and 6 (mainly produced by intestinal Paneth cells) had viral neutralizing activity similar to HNP-1–3. Like HNP-1–3, RCs induced viral aggregation and promoted the uptake of IAV by neutrophils. We used surface plasmon resonance to evaluate binding of defensins to SP-D. HBDs, HD6, and HNP-4 bound minimally to SP-D. HNP-1–3 and RCs bound SP-D with high affinity; however, unlike HNP-1 and HNP-2, RCs did not inhibit SP-D antiviral activity. HBDs also did not inhibit antiviral activity of SP-D. Given their strong neutralizing activity and compatibility with SP-D, RCs may provide attractive prototypes for designing therapeutics that can prevent or treat respiratory infections caused by IAV.