A systems biology analysis of the Drosophila phagosome.

Phagocytes have a critical function in remodelling tissues during embryogenesis and thereafter are central effectors of immune defence. During phagocytosis, particles are internalized into 'phagosomes', organelles from which immune processes such as microbial destruction and antigen presentation are initiated. Certain pathogens have evolved mechanisms to evade the immune system and persist undetected within phagocytes, and it is therefore evident that a detailed knowledge of this process is essential to an understanding of many aspects of innate and adaptive immunity. However, despite the crucial role of phagosomes in immunity, their components and organization are not fully defined. Here we present a systems biology analysis of phagosomes isolated from cells derived from the genetically tractable model organism Drosophila melanogaster and address the complex dynamic interactions between proteins within this organelle and their involvement in particle engulfment. Proteomic analysis identified 617 proteins potentially associated with Drosophila phagosomes; these were organized by protein-protein interactions to generate the 'phagosome interactome', a detailed protein-protein interaction network of this subcellular compartment. These networks predicted both the architecture of the phagosome and putative biomodules. The contribution of each protein and complex to bacterial internalization was tested by RNA-mediated interference and identified known components of the phagocytic machinery. In addition, the prediction and validation of regulators of phagocytosis such as the 'exocyst', a macromolecular complex required for exocytosis but not previously implicated in phagocytosis, validates this strategy. In generating this 'systems-based model', we show the power of applying this approach to the study of complex cellular processes and organelles and expect that this detailed model of the phagosome will provide a new framework for studying host-pathogen interactions and innate immunity.

Distinct and overlapping functions of allelic forms of human mannose binding protein.

Human mannose binding protein (MBP) is a C-type serum lectin involved in first-line host defense against a variety of bacterial, fungal and viral pathogens. Recently an association was found between low levels of serum MBP and an increased frequency of recurrent infections in infants. A particular genotype, in which glycine is substituted by aspartic acid at codon 54 of MBP in the fifth collagen repeat, shows apparent concordance with the clinical phenotype. We report, however, that this genotype occurs in 5% of the population and encodes a functional protein. Our results indicate that the Gly54Asp allele does not account for a deficiency state, but instead suggest that MBP may have two predominant allelic forms that have overlapping function and differ only in their ability to activate the classical pathway of complement.

Down-regulation of mannosyl receptor-mediated endocytosis and antigen F4/80 in bacillus Calmette-Guérin-activated mouse macrophages. Role of T lymphocytes and lymphokines.

Bacillus Calmette-Guerin (BCG) infection alters the surface and endocytic properties of mouse peritoneal macrophages (PM) compared with thioglycollate- elicited (TPM) or resident PM (RPM). Expression of Ia antigen (Ag) is enhanced up to fourfold, but plasma membrane receptors that mediate binding and uptake of mannosyl/fucosyl-terminated glycoconjugates (MFR), Fc receptors, and the macrophage (mphi)-specific Ag F4/80 are reduced by 50-80 percent. Levels of Mac-1 remain relatively stable. These changes are accompanied by enhanced secretion of O(2)(-), after further stimulation with phorbyl myristate acetate, and of plasminogen activator. Both these products are released by TPM, but not RPM. The characteristic surface phenotype of BCG-PM can also be induced by injection of C. parvum, another mphi- activating agent, but not by thioglycollate broth, lipopolysaccharide, or proteose peptone. Purified protein derivative (PPD) and N-acetylmuramyl-L- alanyl-D-isoglutamine. 2H(2)0 are soluble agents with partial activity. Alteration of mphi markers by BCG infection depends on T lymphocyte function, although studies with nude mice indicate that other pathways may also serve to modify the surface of the mphi. Mphi from uninfected animals displayed all markers of activation after adoptive transfer of specifically-sensitised lymphocytes with PPD, intraperitoneally, or after co- cultivation. Treatment of primed lymphocytes with anti-Thy-1 antibody and complement ablated this effect. Lymphokines obtaned by Ag or mitogen stimulation induced similar changes in TPM and RPM. Mannose-specific endocytosis decayed rapidly, time 1/2 approximately equal to 16 h and stabilized at approximately 25 percent of control values. Single-cell analysis showed that residual MFR activity was uniform in the target population. Loss of Ag F4/80 after activation by lymphocyte and PPD was less marked than after infection (35 percent vs 80 percent), unlike MFR activity, which declined to a similar extent. Induction of mphi Ia by lymphokine reached a peak after 2-3 d and was lost within 2 d of its removal. Recovery of MFR and F4/80 was incomplete under these conditions. These studies establish that activated mphi known to display enhanced antimicrobial/anticellular activity express markedly different surface properties distinct from elicited or resident cells. The role of antigen- stimulated T cell products in regulating mphi function is confirmed, and down-regulation of mannosyl-receptor-mediated endocytosis provides a sensitive, quantitative, and cell-specific new marker to study their properties and mechanism of action. Extensive, but selective remodeling of mphi plasma membrane structure could play an important role in controlling recognition and effector mechanisms of the activated mphi.

The human mannose-binding protein functions as an opsonin.

The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains: a cysteine-rich NH2-terminal domain that stabilizes the alpha-helix of the second collagen-like domain, and a third COOH-terminal carbohydrate binding region. The function of MBP is unknown, although a role in host defense is suggested by its ability to bind yeast mannans. In this report we show that native and recombinant human MBP can serve in an opsonic role in serum and thereby enhance clearance of mannose rich pathogens by phagocytes. MBP binds to wild-type virulent Salmonella montevideo that express a mannose-rich O-polysaccharide. Interaction of MBP with these organisms results in attachment, uptake, and killing of the opsonized bacteria by phagocytes. These results demonstrate that MBP plays a role in first line host defense against certain pathogenic organisms.

Macrophage activation selectively enhances expression of Fc receptors for IgG2a.

After infection with bacillus Calmette-Guérin, peritoneal macrophages (Mø) display enhanced expression of FcR for both monomeric and complexed IgG2a, but not IgG2b. Isotype specificity of FcR can be reversed on nonactivated Mø by immune lymphokines, and IgG2a immune complexes are more effective triggers of the respiratory burst in activated Mø. Selective enhancement of IgG2a FcR by Mø activation could account for efficacy of homologous ab in mediating cytotoxicity in some systems.

A human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins.

Mannose-binding proteins have been isolated from the liver of rats and humans and subsequently been found in the serum of rats, rabbits, and humans. We report the isolation of cDNA clones isolated from a human liver cDNA library that encodes a human mannose-binding protein. The primary structure has three domains: (a) an NH2-terminal cysteine-rich segment of 19 amino acids which appears to be involved in the formation of interchain disulfide bonds that would stabilize multimeric forms of the protein; (b) a collagen-like region consisting of 19 repeats of the sequence Gly-x-y; and (c) a COOH-terminal putative carbohydrate-binding domain consisting of 148 residues. This human mannose-binding protein bears 51% overall homology (allowing three gaps) with a rat mannose-binding protein C and 48% homology (allowing seven gaps) with a rat mannose-binding protein A. Like these homologous rat proteins, the human mannose-binding protein COOH-terminal sequences are homologous to the carbohydrate recognition portion of several other lectin-like proteins including mammalian hepatic receptors, an insect-soluble hemolymph, and a sea urchin lectin found in coelomic fluid. The apoproteins of dog and human surfactant and the human lymphocyte IgE Ec receptor have not been shown to have lectin-like properties, yet by homology are members of this family of lectin-like proteins. The human mannose-binding protein is preceded by a typical hydrophobic signal sequence and its hepatic secretion is induced as part of the acute-phase response consistent with its probable role in host defense.

Molecular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and phagocytosis of yeasts in Cos-1 cells.

The macrophage mannose receptor is an integral membrane protein expressed on the surface of tissue macrophages. After ligation of mannose-rich glycoconjugates or pathogens, the receptor mediates endocytosis and phagocytosis of the bound ligands by macrophages. The cDNA-derived primary structure of the mannose receptor predicts a cysteine-rich NH2-terminal domain, followed by a fibronectin type II region. The remainder of the ectodomain is comprised of eight carbohydrate recognition-like domains, followed by a transmembrane region, and a cytoplasmic tail. Transfection of the mannose receptor cDNA into Cos-I cells is necessary for receptor-mediated endocytosis of mannose-rich glycoconjugate as well as phagocytosis of yeasts. Deletion of the cytoplasmic tail results in a mutant receptor that is able to bind but not ingest the ligated pathogens, suggesting that the signal for phagocytosis is contained in the cytoplasmic tail.

A human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus.

In vitro infection by the human immunodeficiency virus (HIV) of CD4+ H9 lymphoblasts is inhibited by a mannose-binding protein (MBP) purified from human serum. In addition, MBP is able to selectively bind to HIV-infected H9 cells and HIV-infected cells from the monocyte cell line U937. These results indicate MBP most likely recognizes high mannose glycans known to be present on gp120 in the domain that is recognized by CD4 and thereby inhibits viral entry to susceptible cells. In support of this contention, recombinant gp120 binds directly to MBP; the binding is saturable, mannan inhibitable, removed by N-glycanase treatment, and dependent on divalent cations.

Surface properties of bacillus Calmette-Guérin-activated mouse macrophages. Reduced expression of mannose-specific endocytosis, Fc receptors, and antigen F4/80 accompanies induction of Ia.

Infection of the mouse peritoneal cavity by bacillus Calmette-Guérin (BCG) markedly alters the surface properties of the macrophages induced, compared with cells obtained from uninfected control animals or after injection of thioglycollate broth. Quantitative binding assays with radiolabeled ligands or antibodies showed that BCG-activated peritoneal macrophages (BCG-PM) expressed one-fourth or less receptor activity for mannose-terminal glycoconjugates as well as reduced levels of Fc receptors and of antigen F4/80 compared with nonactivated macrophages. Endocytosis mediated by mannose-specific receptors was reduced in parallel. In contrast, surface Ia antigen was increased threefold in the same adherent cell population. Radioautographic analysis confirmed that greater than 80% of adherent cells still expressed low levels of the macrophage-specific mannosyl receptor and antigen F4/80, and that I antigens had been induced on 64% of macrophages rather than on other cells. Control experiments established that only the BCG-PM macrophages released H2O2 after stimulation with phorbol myristate acetate, whereas both BCG-PM and thioglycollate-induced macrophages produced superoxide anion and plasminogen activator. The BCG-PM were viable, secreted normal levels of lysozyme, and displayed a stable phenotype after cultivation for 60 h. Inhibitors of oxygen products, prostaglandins, and proteases did not alter reduced endocytosis by BCG-PM. These studies indicated that expression of macrophage surface markers is reversed by BCG-activation, and that their known enhanced ability to lyse target cells extracellularly is associated with decreased endocytosis via specific receptors. Whether these changes are a result of an altered cell population or of modulation of selective surface properties is not known.

Local opsonization by secreted macrophage complement components. Role of receptors for complement in uptake of zymosan.

We have examined the role of macrophage (M phi plasma membrane receptors for the cleaved third complement component (iC3b; CR3) and mannosyl, fucosyl terminated glycoproteins (MFR) in uptake of unopsonized zymosan. Monoclonal antibodies against CR3, M1/70 (Mac-1) and MO1, each inhibited approximately 50% of uptake of 125I-zymosan by murine and human M phi, respectively. Yeast mannan inhibited 0-50% of zymosan uptake in various M phi, in parallel with their expression of MFR. We demonstrated that M phi were the source of C3 in our assay and that the activity of other components of the complement system, namely a C3 convertase, factor I, and a factor I cofactor were also present in serum-free cultures of human monocytes. Macrophage C3 was deposited rapidly, within 10 min, on the zymosan particles and mediated binding, ingestion, and stimulation of superoxide release in BCG-activated and thioglycollate-elicited peritoneal M phi via CR3. Local secretion of complement proteins by M phi themselves can therefore opsonize pathogens and cells able to activate the alternative pathway and effect their destruction.

Restriction of interferon gamma responsiveness and basal expression of the myeloid human Fc gamma R1b gene is mediated by a functional PU.1 site and a transcription initiator consensus.

The restricted expression of the human Fc gamma R1b gene to myeloid cells is likely to be regulated by a combination of transcription factors that may not be solely expressed in myeloid cells, but act together to restrict the expression of the gene to myeloid cells. Low basal expression of the human Fc gamma R1b gene is specifically upregulated by interferon gamma (IFN-gamma). A 181-bp region of 5' flanking sequence contains several key regulatory motifs that include the extended gamma response region (XGRR) and the PIE region. The XGRR contains the 39-bp gamma response region originally defined in the highly homologous Fc gamma R1a gene. The XGRR is in close proximity to the 21-bp PIE motif that is conserved in the promoters of some other myeloid genes. The PIE motif contains a consensus site for the macrophage and B cell transcription factor, PU.1, and is adjacent to the cluster of transcription start sites. An active transcription initiator, Inr, consensus spans the start sites and appears to direct transcription initiation of this TATA-less gene. In this study, we demonstrate that the PIE region contains a functional PU.1 site that binds a human PU.1-like protein and that associated factors present in myeloid extracts also bind in this PIE region. Mutational analysis reveals an absolute requirement for an intact PU.1 box for both basal and IFN-gamma inducible expression of this gene. In addition, mutations in the Inr greatly reduce basal and inducible transcription. Insertion of a strong TATA box downstream from the Inr or at -30 bp from the transcription start sites restores basal and inducible activity in the presence of a mutated PU.1 site. We also demonstrate that indeed, when the XGRR is positioned in the context of a heterologous TATA containing promoter, it is able to respond equivalently to either IFN-alpha or IFN-gamma. However, IFN-alpha responsiveness does not occur in the context of the physiological Fc gamma R1b TATA-less basal promoter. Our results suggest that a human PU.1-like factor acts as a "bridging factor" between the upstream IFN-gamma enhancer and the Inr dependent preinitiation complex. These findings indicate that the structure of the basal promoter in combination with restricted activators like PU.1 are important in regulating the expression of this gene.

Phagocytic chimeric receptors require both transmembrane and cytoplasmic domains from the mannose receptor.

Phagocytosis has traditionally been viewed as a specialized function of myeloid and monocytic cells. The mannose receptor (MR) is an opsonin-independent phagocytic receptor expressed on tissue macrophages. When human MR cDNA is transfected into Cos cells, these usually non-phagocytic cells express cell surface MR and bind and ingest MR ligands such as zymosan, yeast, and Pneumocystis carinii. Expression of cDNA for Fc gamma RI (CD64), the high-affinity Fc receptor, in Cos cells confers binding but barely detectable phagocytosis of antibody-opsonized erythrocytes (EA). We report here that chimeric receptors containing the ligand-binding ectodomain of the Fc receptor and the transmembrane and cytoplasmic domains of the MR ingest bound EA very efficiently, whereas chimeras with the Fc receptor ecto- and transmembrane domains and the MR tail, or the Fc receptor ecto- and cytoplasmic domains and the MR transmembrane region, are significantly less phagocytic. All of the chimeric receptors bind ligand with equal avidity, but gain of functional phagocytosis is only conferred by the MR transmembrane and cytoplasmic domains. Endocytosis of monomeric immunoglobulin G by chimeric receptors demonstrates a similar pattern, with optimal uptake by the chimera containing both tail and transmembrane regions from the MR. The chimeric receptors with only the transmembrane or the cytoplasmic domain contributed by the MR were less efficient. Site-directed mutagenesis of the single tyrosine residue in the cytoplasmic tail (which is present in a motif homologous to an endocytosis consensus motif in the LDL receptor cytoplasmic tail [Chen, W.-J., J. L. Goldstein, and M. S. Brown. 1990. J. Biol. Chem. 265:3116]) reduces the efficiency of phagocytosis and endocytosis to a similar extent.

Macrophage complement and lectin-like receptors bind Leishmania in the absence of serum.

We have examined the relative roles of the macrophage (M phi) plasma membrane receptor for the cleaved third complement component (iC3b, CR3) and of the mannosyl/fucosyl receptor (MFR) in binding and ingestion of Leishmania donovani. In the absence of exogenous complement, the binding and ingestion of promastigotes, which are good activators of the alternative complement pathway, were inhibited by the anti-CR3 monoclonal antibody M1/70, by the Fab portion of an anti-C3 antibody, or by the nucleophile, sodium salicyl hydroxamate, an inhibitor of C3 fixation. This provides strong evidence that M phi-derived, cleaved C3 (iC3b) present on the promastigote surface mediates binding to CR3. Equivalent inhibition of promastigote binding and ingestion was also observed using the soluble inhibitors of MFR activity, mannan or ribonuclease B. No additive effect for blocking the two M phi receptors simultaneously was observed. For amastigotes, which are poor activators of the alternative pathway, a lesser but nevertheless equivalent effect was observed for the three soluble inhibitors of CR3-mediated binding vs. the two soluble inhibitors of MFR-mediated binding. Modulation experiments in which either CR3 or MFR had been rendered inaccessible demonstrated that both receptors must be present on the segment of M phi membrane to which the parasite binds. The combined function of these two distinct M phi receptors may provide a general mechanism for recognition and ingestion of other pathogenic protozoa known to activate the alternative pathway.

The human mannose-binding protein gene. Exon structure reveals its evolutionary relationship to a human pulmonary surfactant gene and localization to chromosome 10.

The human mannose-binding protein (MBP) plays a role in first line host defense against certain pathogens. It is an acute phase protein that exists in serum as a multimer of a 32-kD subunit. The NH2 terminus is rich in cysteines that mediate interchain disulphide bonds and stabilize the second collagen-like region. This is followed by a short intervening region, and the carbohydrate recognition domain is found in the COOH-terminal region. Analysis of the human MBP gene reveals that the coding region is interrupted by three introns, and all four exons appear to encode a distinct domain of the protein. It appears that the human MBP gene has evolved by recombination of an ancestral nonfibrillar collagen gene with a gene that encodes carbohydrate recognition, and is therefore similar to the human surfactant SP-A gene and the rat MBP gene. The gene for MBP is located on the long arm of chromosome 10 at 10q11.2-q21, a region that is included in the assignment for the gene for multiple endocrine neoplasia type 2A.

Definition of interferon gamma-response elements in a novel human Fc gamma receptor gene (Fc gamma RIb) and characterization of the gene structure.

The human Fc gamma RI (CD64) is a high affinity receptor for the Fc portion of immunoglobulin (Ig), and its constitutively low expression on the cell surface of monocyte/macrophage and neutrophils is selectively upregulated by interferon gamma (IFN-gamma) treatment (Perussia, B., E. T. Dayton, R. Lazarus, V. Fanning, and G. Trinchieri. 1983. J. Exp. Med. 158:1092). Three distinct cDNAs have been cloned and code for proteins that predict three extracellular Ig-like domains (Allen, J.M., and B. Seed. 1989. Science [Wash. DC]. 243:378). Several differences in the coding region of these cDNAs suggest that in addition to polymorphic differences a second Fc gamma RI gene could possibly exist. This alternative Fc gamma RI gene (Fc gamma RIb) was defined by the lack of a genomic HindIII restriction site (van der Winkel, J. G. J., L. U. Ernst, C. L. Anderson, and I. M. Chiu. 1991. J. Biol. Chem. 266:13449). We describe the characterization a second gene (Fc gamma RIb) that has a termination codon in the third extracellular domain and therefore predicts a soluble form of a termination codon in the third extracellular domain and therefore predicts a soluble form of the receptor. We also define two distinct IFN-gamma-responsive regions in the 5' flanking sequence of Fc gamma RIb that resemble motifs that have been defined in the class II major histocompatibility complex promoter. The Fc gamma RIb promoter does not possess canonical TATA or CCAAT boxes, but does possess a palindromic motif that closely resembles the initiator sequence identified in the terminal deoxynucleotidyl transferase/human leukocyte IFN/adeno-associated virus type II P5 gene promoters (Smale, S. T., and D. Baltimore. 1989. Cell. 57:103; Seto, E., Y. Shi, and T. Shenk. 1991. Nature [Lond.]. 354:241; Roy, A. L., M. Meisterernst, P. Pognonec, and R. C. Roeder. 1991. Nature [Lond.]. 354:245) virus type II P5 gene promoters raising interesting questions as to its role in the basal and myeloid-specific transcription of this gene.

Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila.

Macrophages in the Drosophila embryo are responsible for the phagocytosis of apoptotic cells and are competent to engulf bacteria. Croquemort (CRQ) is a CD36-related receptor expressed exclusively on these macrophages. Genetic evidence showed that crq was essential for efficient phagocytosis of apoptotic corpses but was not required for the engulfment of bacteria. The expression of CRQ was regulated by the amount of apoptosis. These data define distinct pathways for the phagocytosis of corpses and bacteria in Drosophila.

Phylogenetic perspectives in innate immunity.

The concept of innate immunity refers to the first-line host defense that serves to limit infection in the early hours after exposure to microorganisms. Recent data have highlighted similarities between pathogen recognition, signaling pathways, and effector mechanisms of innate immunity in Drosophila and mammals, pointing to a common ancestry of these defenses. In addition to its role in the early phase of defense, innate immunity in mammals appears to play a key role in stimulating the subsequent, clonal response of adaptive immunity.

Recombinant interferon gamma augments phagocyte superoxide production and X-chronic granulomatous disease gene expression in X-linked variant chronic granulomatous disease.

We examined the potential of interferon gamma (IFN-gamma) to ameliorate the physiologic defect of chronic granulomatous disease (CGD) by studying its effects on CGD phagocyte superoxide generation, NADPH oxidase kinetics, cytochrome b559 content, and expression of X-CGD (the gene for the X-linked disease). Granulocytes and macrophages from three patients in two kindreds with "variant" X-linked CGD (i.e., with very low, but detectable, baseline superoxide-generating activity) responded to IFN-gamma with enhanced nitroblue tetrazolium reduction and two- to eightfold increases in superoxide generation. IFN-gamma did not augment the respiratory burst activity of phagocytes from patients with "classic" CGD (i.e., no detectable baseline superoxide generation) or autosomal variant CGD. Incubation of a responding patient's granulocytes with IFN-gamma nearly doubled the maximal velocity for the NADPH oxidase, but did not change its abnormal Michaelis constant. Although the interferon-treated CGD granulocytes produced superoxide at a rate 40% of normal, the cytochrome b spectrum remained undetectable. IFN-gamma treatment of cultured monocytes from an IFN-gamma-responsive CGD patient increased the steady state level of RNA transcripts from the X-CGD gene from barely detectable up to approximately 5% of normal.

Interaction of human monocytes, macrophages, and polymorphonuclear leukocytes with zymosan in vitro. Role of type 3 complement receptors and macrophage-derived complement.

Macrophages take up zymosan in the absence of exogenous complement via receptors for iC3b (type 3 complement receptors) acting with or without lectin-like receptors for mannosyl-fucosyl-terminated glycoconjugates. We previously provided evidence that macrophages themselves secrete complement-alternative pathway components able to opsonize zymosan locally (Ezekowitz et al., J. Exp. Med. 1984. 159:244-260). We show here that covalently bound C3 cleavage products C3b and iC3b can be eluted from zymosan particles cultivated with 36-h adherent human monocytes in the absence of serum. The ligand binding site of type 3 complement receptors is involved in macrophage-zymosan interactions as shown by inhibition studies of zymosan binding and uptake with Fab fragments of anti-C3 antibodies and monoclonal antireceptor antibodies M01 and OKM10. In contrast, antibody IB4, which binds to a receptor epitope distinct from the binding site, does not inhibit zymosan uptake. Selective modulation of macrophage receptors onto anticomplement receptor antibody and mannose-rich yeast mannan, respectively, confirms that the complement and lectin-like receptors are distinct. Human polymorphonuclear leukocytes, which express receptors for complement, but are not known to secrete complement proteins, bind and ingest only exogenously opsonized zymosan. Unopsonized zymosan is a poor trigger of respiratory burst activity in neutrophils or 7-d adherent human macrophages, but induces cell aggregation and secretion of large amounts of superoxide anion when these cells are co-cultivated in serum-free medium and challenged with zymosan. Our studies indicate that complement and/or other products synthesized by macrophages at extravascular sites could play an important role in opsonization and lysis of pathogens able to activate the alternative pathway and mediate macrophage-neutrophil collaboration in first-line host defence.

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.