Collectins and Pulmonary Host Defense - ATS Journals

Minireview Collectins and Pulmonary Host Defense Erika C. Crouch Department of Pathology, Washington University School of Medicine, St. Louis, Missouri

The surfactant-associated proteins SP-A and SP-D are members of a family of collagenous host defense lectins, designated collectins. There is increasing evidence that these pulmonary epithelial-derived proteins are important components of the innate immune response to microbial challenge, and that they participate in other aspects of immune and inflammatory regulation within the lung. The collectins bind to glycoconjugates and/or lipid moieties expressed by a wide variety of microorganisms and certain other organic particles in vitro. Although binding may facilitate microbial clearance through aggregation or other direct effects on the organism, SP-A and SP-D also have the capacity to modulate leukocyte function and, in some circumstances, to enhance their killing of microorganisms. The biologic activity of cell wall components, such as gram-negative bacterial polysaccharides, may be altered by interactions with collectins. Complementary or cooperative interactions between SP-A and SP-D could contribute to the efficiency of this defense system. Collectins may play particularly important roles in settings of inadequate or impaired specific immunity. Acquired or genetic alterations in the levels of active proteins within the airspaces and distal airways may increase susceptibility to infection. Crouch, E. C. 1998. Collectins and pulmonary host defense. Am. J. Respir. Cell Mol. Biol. 19:177–201.

Introduction Pulmonary Host Defense The respiratory system is challenged by a constant onslaught of inhaled toxic substances and infectious agents. For this reason, the upper airways and lung have evolved a complex and multilayered system of defense that involves mechanical, reflex, and cellular mechanisms, as well as locally synthesized and systemically derived defense molecules (Table 1). It has been appreciated for many years that non-immune mechanisms must be important components of this defense system, particularly in early development, in the interval between exposure and the development of specific immunity, and in states of impaired (Received in original form April 22, 1997 and in revised form December 16, 1997) Address correspondence to: Dr. Erika C. Crouch, Department of Pathology, Barnes-Jewish Hospital at Washington University Medical Center, 216 S. Kingshighway, St. Louis, MO 63110. E-mail: [email protected]. edu Abbreviations: bovine serum albumin, BSA; carbohydrate recognition domain, CRD; dipalmitoylphosphatidylcholine, DPPC; ethylenediamenetetraacetic acid, EDTA; hemagglutinin, HA; influenza A virus, IAV; interleukin, IL; a trimeric coiled-coil linking domain, L; the linking peptide in conjunction with the CRD, L1CRD; lipopolysaccharide(s), LPS; a short cysteine-containing NH2-terminal cross-linking domain, N; phosphatidylinositol, PI; surfactant protein (A, D), SP(-A, -D); tumor necrosis factor alpha, TNF-a. Am. J. Respir. Cell Mol. Biol. Vol. 19, pp. 177–201, 1998 Internet address: www.atsjournals.org

immune function (1–3). It is also appreciated that individuals with similar immune status and exposure history can show marked differences in their susceptibility to pulmonary infection. Recent studies have drawn attention to the probable roles of surfactant-associated proteins, specifically surfactant protein A (SP-A) and surfactant protein D (SP-D), in this innate, natural, and nonclonal defense system (4–6). Collectins: A Family of Collagenous Lectins SP-A and SP-D are members of a family of collagenous carbohydrate binding proteins (collagenous C-type lectins), now commonly known as collectins (2, 7, 8). These include serum mannose binding protein (MBP or MBL) and two bovine serum lectins, conglutinin and CL-43. MBP and conglutinin are recognized acute-phase proteins that have been strongly implicated in various aspects of the systemic response to microbial challenge. Both SP-A and SP-D show specific interactions with various microorganisms and leukocytes in vitro. Recent data further suggest that lung collectins are a part of a more generalized response of the lung to acute lung injury, and that they modulate local inflammatory/immune responses (9–11). Collectins may also participate in the recognition or clearance of other complex organic materials, such as pollens (12) and dust mite allergens (13). Thus, acquired or genetically determined differences in pulmonary collectin activity may in part account for the varied suscep-

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 19 1998

TABLE 1

Biochemistry and biophysical properties of lung collectins Property

Gene products (human) Mature monomer size N-linked sugar Other major modifications Subunit composition Predominant state of oligomerization Stoke’s radius ƒ/ƒ0 Sedimentation coefficient Charge (pI)

Length of triple helical domain Maximum distance between trimeric CRDs (heads)

tibility of individuals to microbial challenge, particularly in the setting of inadequate or impaired specific immunity, or contribute to the pathogenesis of certain immunologically mediated lung disorders, such as allergic asthma and hypersensitivity pneumonitis. Some disorders associated with an increased risk of pneumonia (e.g., diffuse alveolar damage, chronic bronchitis, cystic fibrosis, diabetes mellitus, congestive heart failure) may also be associated with acquired defects in collectin function. This review focuses on the structure–function correlations of the two known pulmonary collectins. Biochemical properties of potential functional significance, and the interactions of these proteins with specific organisms and host cells, are systematically compared and contrasted. Potential methodologic difficulties are discussed, and questions for guiding future research are presented. For completeness, recent findings relating to the regulation of collectin biosynthesis and secretion and the molecular regulation of collectin gene expression are also concisely reviewed. The structural and functional properties of the serum collectins are thoroughly discussed in several recent reviews (2, 14–16).

Historic Perspectives Surfactant Protein A SP-A was first identified by King and colleagues in 1972 (17, 18). In the late 1980s, protein, cDNA, and genomic sequencing demonstrated the presence of collagenous sequences and a carboxyl-terminal C-type lectin motif (19– 22), predicting the subsequent demonstration of a triple helical collagen domain (18) and carbohydrate binding activity (23, 24). The best-characterized ligands for SP-A are lipid in nature. Purified SP-A binds to specific surfactant-associated phospholipids in vitro, primarily dipalmitoylphosphatidylcholine (DPPC) (25–27). These and numerous other observations have until recently suggested that SP-A’s major function in vivo is to regulate the production and/or metabolism of the airspace lining material. Novel studies by Tenner and coworkers published in

SP-A

SP-D

2 30–35 kD 1 at Asn187 in CRD Hypro Blood Group A antigen Trimer 6 trimers 5 18 chains 110–120 Å 2.1 14 S Acidic (4.2–5.0) SP-A1 5 4.6 SP-A2 5 4.8 z 20 nm z 20 nm

1 43 kD 1 at Asn70 in collagen domain Hypro Hylys, Hylysglycosides Trimer 4 trimers 5 12 chains 178 Å 3.3 s20,w 5 8.9 Basic (5.2–81) z 46 nm z 110 nm

1989 were the first to suggest a more diverse range of biologic functions (28). Purified SP-A was shown to enhance FcR and CR1-mediated phagocytosis by monocytes/ macrophages, indicating modulation of leukocyte function. Subsequent studies demonstrated a variety of interactions with microorganisms in vitro and effects on leukocyte function and antimicrobial activity (6). In fact, recent studies suggest that the primary function of SP-A relates to lung defense. The most compelling evidence to date is that otherwise healthy transgenic mice lacking a functional SP-A gene, SP-A (2/2), do not demonstrate obvious abnormalities in normal respiratory function or surfactant lipid metabolism (29, 30). Furthermore, these animals demonstrate increased bacterial proliferation, more intense lung inflammation, and an increased incidence of splenic dissemination following intratracheal inoculation with the Group B streptococcus, a major pulmonary pathogen in the neonatal period (31). These studies also suggested defective clearance of Staphylococcus aureus and Pseudomonas aeruginosa. Surfactant Protein D SP-D was originally characterized in 1988 as one of several collagenous glycoproteins (CP4) secreted in cultures of freshly isolated rat type II cells (32). Subsequent studies demonstrated the presence of SP-D in bronchoalveolar lavage (BAL), and in association with crude surfactant (33). In 1992 Kuan and coworkers demonstrated lectin-mediated binding of SP-D to gram-negative bacteria and resultant bacterial aggregation, first suggesting possible roles for SP-D in pulmonary host defense (34).

Molecular Structure Domain Structure The lung and serum collectins are assembled as oligomers of trimeric subunits. Each subunit consists of four major domains: a short cysteine-containing NH2-terminal cross-linking domain (N); a triple helical collagen domain of variable length; a trimeric coiled-coil linking domain (L; sometimes referred to as the neck); and a carboxyl-terminal, C-type

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Figure 1. Domain structures of SP-A and SP-D. Note the similar size and distribution of the amino-terminal, L-peptide, and carbohydrate recognition domains (CRD). Differences in monomer size result from differences in the length of the collagen domain (diagonal lines). The Asn-linked oligosaccharide in SP-D is located in the collagen domain, whereas the Asn-linked sugar in human SP-A is located within the CRD.

lectin carbohydrate recognition domain (CRD) (Figures 1 and 2; Table 1). Interactions between the amino-terminal domains of SP-D subunits have been shown to be stabilized by interchain disulfide bonds (35, 36), and similar mechanisms stabilize the oligomerization of SP-A (37) and most other collectins. SP-A structure. SP-A (26–35 kD, reduced) is predominantly assembled as octadecamers consisting of six trimeric subunits (18 chains) with relatively short collagen domains (Figure 2). In these respects, SP-A is very similar to serum MBP. However, SP-A lacks hydroxylysine, and shows no O-linked glycosylation of hydroxylysyl residues in the collagen domain. Although rat SP-A contains two Asn-linked oligosaccharides, one in the amino-terminal peptide domain and one in the CRD, human SP-A contains a single Asn-linked oligosaccharide within the CRD. Alternative proteolytic processing of the amino-terminal peptide of SP-A has been reported to influence glycosylation and the formation of disulfide cross-linked oligomers (38). Human SP-A molecules can be assembled as homotrimers or as heterotrimers derived from two genetically

Figure 2. Molecular organization of pulmonary collectins. The predominant molecular forms of SP-A and SP-D are compared assuming maximal spatial separation of the CRDs. The Asnlinked oligosaccharides of SP-D are probably less accessible to lectins expressed by microorganisms than the sugars of SP-A.

different chain types (39). The relative proportions of homoand heterotrimers accumulating in the lung have not been established, and it is not yet known whether the two forms are synthesized by the same cell type or accumulate at the same site in vivo. SP-D structure. SP-D (43 kD, reduced) is predominantly assembled as dodecamers consisting of four homotrimeric subunits (12 chains) with relatively long triple helical arms (35, 40–42) (Figure 2). With respect to these features, SP-D is most similar to conglutinin (8), which has no other known human homolog. However, SP-D is distinguished from conglutinin by an uninterrupted, cysteinefree collagen domain that contains the single site of Asnlinked glycosylation (Asn70). By contrast with SP-A, the collagen domain of SP-D contains hydroxylysine and hydroxylysyl-glycosides (33). Although natural and recombinant rat SP-Ds are almost exclusively assembled as dodecamers, preparations of natural human and bovine SP-D can include a high proportion of trimers (14, 42–44). It is unclear whether the accumulation of these forms reflects differences in the efficiency of intracellular multimerization or in the stability of secreted dodecamers. Higher-order Oligomerization of Lung Collectins SP-A multimers. In the setting of alveolar proteinosis, SP-A octadecamers can self-associate to form multimolecular complexes (45, 46). Under some circumstances the aggregated molecules may become cross-linked through disulfide interchange and the formation of other covalent bonds (47–49). The functional significance of SP-A multimerization is uncertain. However, differences have been observed between proteinosis SP-A and less highly multimerized preparations of natural or recombinant proteins in several biologic assays. For example, the multimerized form shows lower affinity binding to type II cells and is less potent as an inhibitor of lipid secretion (45). On the other hand, proteinosis SP-A is more potent than recombinant human SP-As in enhancing bacterial phagocytosis (50). Likewise, proteinosis SP-A is more effective than natural or recombinant SP-A in enhancing the adherence and phagocytosis of mycobacteria by macrophages (51).

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SP-D multimers. SP-D dodecamers can self-associate at their amino-termini to form much more highly ordered, stellate multimers with peripheral arrays of trimeric CRDs (35, 44, 52) (Figure 3). Natural SP-D from human alveolar proteinosis and bovine lavage, and recombinant human SP-D contain a high proportion of these multimers with up to eight (or possibly more) SP-D dodecamers. The multimers are not dissociated by ethylenediamenetetraacetic acid (EDTA) or competing sugars, and are cross-linked by disulfide and nondisulfide bonds. SP-D multimers show higher apparent binding affinity to a variety of ligands and are considerably more potent on a molar or weight basis in mediating microbial aggregation and aggregation-dependent interactions with leukocytes (44, 53).

Genomic Organization The human SP-A and SP-D genes have been localized to the region of 10q22.2–23.1 (54–58). SP-A is encoded by two genes, SP-A1 and SP-A2 (20, 59–61) with several alleles at each locus. Most species (e.g., rat, mouse, rabbit, dog) have only a single gene. Human SP-D is also encoded by a single gene (54). However, protein, cDNA, and genomic sequencing together suggest the existence of a number of SP-D alleles, some of which are characterized by amino-acid substitutions in the coding region (43, 54). Each SP-A gene encodes untranslated exons at the 59-end, and all but the first of these are subject to gene-specific alternative splicing (59, 61–63). The two human genes differ by approximately 21 nucleotides within the coding region but have more numerous base substitutions in the upstream and downstream untranslated regions (60). These changes result in only a few amino-acid substitutions, including the substitution of cys for arg at residue 85 within the collagen sequence, which has been implicated in the cross-linking of heterotrimers (63). The SP-D gene encodes at least one, and probably two, untranslated exons at the 59-end of the gene, similar to SP-A (54).

Figure 3. Alternative molecular forms of SP-D. Monomeric CRDs, trimeric CRDs, the single-arm RrSP-Dser15/20 mutant, and SP-D multimers are compared with natural or recombinant rat SP-D (RrSP-D) dodecamers. Monomeric CRDs show very weak binding affinity to various ligands. The minimum structure required for high-affinity binding is the trimeric CRD. Trimeric CRDs and single-arm mutant forms of SP-D can inhibit bridging interactions mediated by SP-D dodecamers or multimers. A similar situation exists for SP-A; however, SP-A multimers are less highly ordered.

Biosynthesis and Cellular Metabolism of SP-A and SP-D SP-A and SP-D are both synthesized and secreted by alveolar type II and nonciliated bronchiolar epithelial cells (64–68). However, there is evidence for considerable cellto-cell variation in the relative production or accumulation of SP-A and SP-D by these cells (69). Furthermore, SP-A and SP-D show differences in developmental expression and different patterns of regulation by glucocorticoids, cytokines, and other factors in rat and human lung explants. Developmental Regulation In the rat and human lung the accumulation of SP-A (relative to total protein or DNA) increases dramatically during late gestation, levels off or slightly declines in the early postnatal period, and then increases to reach maximal levels in the adult (70–72). The expression of the two human SP-A genes is differentially regulated during development with a predominance of SP-A1 transcripts (65%) in midgestation fetal lung, while the majority of the transcripts in the adult lung are derived from the SP-A2 gene (62). The administration of dexamethasone in utero accelerates the appearance of SP-A in rat lung (73–75). The accumulation of SP-D in the rat lung also increases abruptly in late gestation, slightly later than SP-A (65, 76, 77). Unlike SP-A, SP-D mRNA and protein levels continue to increase during the early postnatal period, eventually reaching their highest levels in the adult lung. As for SP-A, the administration of dexamethasone in utero accelerates the appearance of SP-D-producing cells and increases the cellular level of SP-D mRNA (76, 78, 79). In the human, SP-D mRNA is first detected at low levels (4 to 13% of adult) in the second trimester and message levels rise steadily during late fetal lung and postnatal lung development (80). Regulation of Collectin Production in Explant Culture Human fetal lung explants demonstrated stimulatory effects of cyclic adenosine monophosphate (cAMP) analogs,

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g-interferon, and epidermal growth factor; and inhibitory effects of indomethacin, insulin, phorbol myristate acetate (PMA), tumor necrosis factor alpha (TNF-a), tumor growth factor beta, and lipopolysaccharides (LPS) (70, 80–82). There are also concentration-dependent effects of glucocorticoids on SP-A mRNA levels in human lung explants (80). The effects of hormones and various cytokines appear to involve both transcriptional and/or post-transcriptional regulatory mechanisms. In human fetal lung organ culture, SP-A2 is preferentially upregulated by cAMP and inhibited by glucocorticoids, whereas SP-A1 appears to be constitutively expressed (62). Sequences upstream from the start site of SP-D transcription include a conserved canonical AP-1 element, several AP-1 and CRE-like sequences, as well as sequences similar to those identified in conglutinin and other acute phase proteins (NF-IL6, PEA3, APF-1). Studies using human fetal lung explants have confirmed the upregulatory effects of glucocorticoids (80), but show no evidence of modulation by several agents known to alter SP-A expression, including g-interferon, PMA, LPS, and TNF-a (80).

with differential, time-dependent alterations in expression of SP-A and SP-D by type II and Clara cells (97, 98). Thus, regional concentrations of these molecules may be influenced by the specific cellular responses to various forms of injury. The production and accumulation of both SP-A and SP-D is rapidly increased following intratracheal instillation of LPS (99). Because the mRNAs for the lung collectins are increased within several hours to a few days following injury, McIntosh and coworkers suggested that they are pulmonary acute-phase proteins, similar to liver-derived MBL and conglutinin, which are systemic acute-phase reactants (99). The mechanism of LPS-mediated increases in lung collectin production is unknown. However, as previously noted, SP-A and SP-D mRNA levels are decreased or unchanged, respectively, by LPS in human lung explants. This suggests the involvement of other inflammatory mediators or cytokines in vivo. As indicated previously, SP-A production is upregulated by g-interferon in lung explants (81).

Cellular Pathway of Assembly and Secretion Collectin biosynthesis and assembly are complex processes. Recent studies of recombinant SP-D assembly by CHO-K1 cells suggest that folding of the CRD, trimerization of monomers, triple helix formation, the amino-terminal association of trimeric subunits, and the formation of interchain disulfide cross-links occur in the rough endoplasmic reticulum, and that oligosaccharide maturation occurs in the Golgi immediately prior to secretion (83). Although the pathway of SP-A secretion has not been fully elucidated, the maturation of the N-linked sugars in SP-A also occurs late and shortly prior to secretion (84). Investigators initially assumed that SP-A was subject to regulated secretion in association with lamellar bodies (LB). However, in human lung explants, all but a small fraction of the newly synthesized protein is secreted via a constitutive pathway and independent of LB (85, 86). It is unclear to what extent bronchiolar cells contribute to the non-LBassociated fraction, and whether there are structural differences in the molecules targeted to these organelles.

Very little SP-A is identified in BAL supernatants following high-speed centrifugation. Although detergents or organic solvents have usually been employed for the extraction of SP-A from the surfactant pellet, EDTA has recently been shown to solubilize the majority of the protein (100), suggesting that the conformation of the C-type lectin domain is important for its association with surfactant lipid. Immuno-electron microscopic studies have shown that SP-A is associated with lipid-rich components, particularly tubular myelin (101, 102). Tubular myelin formation is also nearly absent from the lungs of SP-A (2/2) transgenic mice (29). The molecular orientation of SP-A in relation to the surfactant layer in vivo is not known. By contrast, the majority of the total immunoreactive SP-D remains in the BAL supernatant following high-speed centrifugation (50 to 90%, depending on the species) (33, 103). Immunologic studies have shown that the insoluble fraction is associated with amorphous granular material, and that the immunoreactive material can be efficiently solubilized with EDTA or specific saccharides (93). No significant amounts of SP-D are associated with surfactant that has been collected or washed in the presence of chelators or purified by sucrose density centrifugation. Early studies reported that there was much less total SP-D than SP-A in the airspace of rats and in human alveolar proteinosis lavage. However, recent comparative assays by Honda and coworkers gave 3.1 6 0.4 mg/ml for SP-A and 1.3 6 0.2 mg/ml for lavage from healthy nonsmokers (104). These studies employed a standard saline lavage procedure, a very brief 250 3 g centrifugation to remove cells, and wellcharacterized immunoassays for both proteins.

Collectin Degradation Little is currently known about the physiologic turnover of lung collectins or their susceptibility to degradation following injury. Instillation of SP-A into rabbit lungs has shown that the half-life of the solubilized protein is approximately 4.5–6.5 h (87, 88). SP-A can also be rapidly internalized and degraded by macrophages in vitro (89), and accumulates in alveolar macrophages in vivo (90–92). SP-D is found in both endosomal vesicles and lysosomal granules of alveolar macrophages, indicating that these cells can also internalize SP-D (4). Modulation of Collectin Production and Accumulation In Vivo Immunohistochemical and in situ hybridization studies strongly suggest that the production of these molecules is increased in association with acute injury and epithelial activation (69, 93–96). Acute hyperoxia in rats is associated

Airspace Distribution of Lung Collectins

Biochemical Properties As previously suggested, there is evidence that the number and spatial distribution of CRDs can influence the binding activities of collagenous lectins. Thus, solubility or other chemical parameters that influence protein aggregation or multimerization could be important determinants of collectin function or influence binding activity in vitro. Bio-

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logic activity may also be influenced by changes in the conformation of the CRDs that influence calcium or ligand binding. Charge Properties Both SP-A and SP-D are secreted as several distinct isoforms that can be resolved by 2-D isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (32, 105). The SP-A isoforms are relatively acidic (pI 4.2–5), whereas the SP-D isoforms are basic (pI 5–9) (Table 1). Because the predicted pI of the CRD for both proteins is in the range of 4 to 5, differences in net charge between SP-A and SP-D predominantly reflect differences in the collagen domain, which is very basic (pI z 10) in SP-D but weakly acidic in SP-A. At least part of the heterogeneity of human SP-A results from slight charge differences between the two SP-A gene products (39). However, the remainder of the heterogeneity for both proteins can be attributed to variations in terminal sialylation (105), heterogeneity in the charge of the collagen domain (as observed for other collagenous proteins), and perhaps acetylation (106). Resolution of newly synthesized or natural rat SP-D by diethylaminoethyl chromatography under nondenaturing conditions also partially resolves species of different monomer size attributable to differences in terminal sialylation (32). This observation suggests that, at least for SP-D, similarly modified monomers are preferentially associated to form multimers. The functional significance of this heterogeneity and differences in charge is not known. The sialylated sugars of SP-A, which are located on the CRD, are able to interact with carbohydrate binding proteins associated with certain viruses, and may also contribute to the calciumdependent self-aggregation of SP-A (44, 107, 108). However, the N-linked oligosaccharides of SP-D dodecamers do not appear to be similarly accessible, and mutant proteins lacking the consensus for N-linked glycosylation are indistinguishable from wild-type SP-D with respect to the aggregation and neutralization of influenza A virus (IAV) and the agglutination of gram-negative bacteria (K. Hartshorn, manuscript submitted). Solubility Purified SP-A and SP-D show marked differences in solubility at neutral pH that probably reflect differences in charge. SP-A tends to precipitate at physiologic ionic strength (even in the absence of calcium) and is usually stored in low-salt (e.g., 5 mM Tris) buffers. By contrast, SP-D is fully soluble in saline at neutral pH and precipitates in low-salt buffers. This is consistent with the supposition that the vast majority of SP-A is insoluble and associated with surfactant lipids in vivo, whereas SP-D is preferentially distributed within the “aqueous hypophase.” Bound surfactant lipids, or partial denaturation of the protein by organic solvents used to remove these lipids, may influence the solubility of SP-A in vitro. For example, butanol-extracted proteinosis SP-A has been reported to show a greater propensity for self-aggregation than normal SP-A solubilized with EDTA (23).

Binding of Divalent Cations Calcium binding to the CRD. The saccharide binding activities of SP-A and SP-D are abolished with 2 mM EDTA and can be restored following readdition of excess calcium (24, 109). Biophysical studies of SP-A have identified two calcium binding sites: one low affinity (10 23 M) and one high affinity (1025 M). The high-affinity binding site has been localized to the collagenase resistant fragment (L1CRD) (110). Calcium binding alters the conformation of this domain as detected by intrinsic fluorescence assays, increased resistance to proteolytic degradation, and alterations in antibody binding. Although similar studies have not been performed for SP-D, neoglycoprotein binding assays are consistent with the presence of at least one highaffinity binding site (1026 M) (109). Finally, sequence studies of the CRDs of both proteins and comparison with MBL are consistent with two calcium coordination sites per monomer (111, 112). Together these observations suggest that the calcium binding sites in the CRD are fully occupied at the estimated alveolar fluid calcium concentration of 1.5 mM (110) and help maintain a conformation suitable for lectin activity. Interestingly, other divalent cations show disparate effects on the saccharide binding activities of SP-D and SP-A, presumably through subtle effects on the conformation of the carbohydrate binding site. Although magnesium, strontium, manganese, and barium inhibit the binding of SP-D to a-glucosyl sugars, the latter three divalent cations can substitute for calcium in the binding of SP-A to mannose (24). Calcium-dependent precipitation. SP-A can undergo calcium-dependent self-aggregation (108, 113). Precipitation readily occurs with low calcium concentrations with NaCl concentrations in excess of 20 mM in the presence of nonionic detergents (24). Because the self-association of human proteinosis SP-A and SP-A-mediated phospholipid vesicle aggregation were blocked following glycosidase digestion, Haagsman and coworkers concluded that the CRD binds to the N-linked oligosaccharide within the CRD of another SP-A molecule, thereby leading to aggregation (113). This conclusion was subsequently questioned because deletion of the consensus for N-linked glycosylation in recombinant rat SP-A synthesized by insect cells did not prevent calcium-dependent lipid aggregation (114). However, this apparent discrepancy could reflect differences in assay conditions and the state of protein oligomerization. For example, the former studies were performed at low ionic strength, whereas the latter experiments were performed in the presence of 0.15 M NaCl. SP-D can precipitate in the presence of calcium at physiologic ionic strength and pH. This results from lectindependent aggregation, presumably mediated by binding of the CRD to N- or O-linked glycoconjugates within the collagen domain. The precipitates are readily resolubilized with competing sugars or EDTA. Interestingly, ordered multimers of dodecamers account for the majority of the SP-D that precipitates with calcium, and there is little precipitation of purified dodecamers. Thus, human proteinosis SP-D, which contains a high proportion of multimers, can show . 70% precipitation within a few hours following the addition of calcium; whereas less than 10% of sili-

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cotic rat SP-D precipitates following even more extended periods of incubation. This is a particularly important methodologic consideration when working with SP-D at concentrations greater than a few hundred nanograms per milliliter. The protein is typically stored in the presence of EDTA with the addition of excess calcium immediately prior to performing a binding essay. SP-D also selectively and efficiently precipitates from lavage in the presence of barium sulfate; however, the mechanism has not been elucidated (109). Effects of pH Many C-type lectins show reversible loss of binding at mildly acidic pH. This property is critical to the function of endocytic receptors that must release their ligands following internalization. It has been suggested that protonation of carboxylate groups involved in calcium binding decreases calcium binding and is necessary (although not always sufficient) for carbohydrate ligand release. Interestingly, both SP-A and SP-D show relative preservation of saccharide binding activity at pH 5 (24, 109). Intrachain Disulfide Bonds within the CRD Intrachain bonds stabilize the conformation of the CRD. Peptide mapping studies of SP-A have shown that the second and third cysteines (cys204–cys218), and the first and fourth cysteines (cys135–cys226), are cross-linked (110), consistent with the crystal structure of serum mannose binding protein. Analogous cross-linking of the cysteines of SP-D (cys261–cys353 and cys331–cys345) is predicted. A variety of observations suggest the intrachain bonds are required for calcium-dependent saccharide binding and confer resistance to thermal denaturation or proteolytic degradation. For example, natural SP-D retains lectin activity in solid-phase neoglycoprotein binding assays following boiling for 10 min at neutral pH. However, activity is irreversibly lost when the protein is heated briefly in the presence of low concentrations of dithiothreitol (DTT). Interchain Disulfide Bonds within the Collagen or Amino-terminal Domains Interchain disulfide bonds are required for the formation of stable trimeric subunits and higher-order oligomers. Biochemical studies are consistent with two classes of interchain disulfide bonds, intersubunit (i.e., between trimers) and intrasubunit (within trimers) (35). The latter are comparatively stable and can form and re-form spontaneously following reduction (37). Thus, low concentrations of sulfhydryl reducing agents can liberate trimeric subunits from collectin molecules, sometimes with partial preservation of intratrimeric disulfide bonds, depending on the concentration of reducing agent and the incubation temperature (35, 37). The interchain bonds (inter- and/or intrasubunit) are also required for normal thermal stability of the collagen triple helix. Complete reduction of SP-A with DTT decreases the melting temperature of the short collagen helix from 41.5 to 28.58C (37). Elimination of the amino-terminal cysteines of rat SP-D shows a much more modest decrease in melting temperature (36), presumably

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reflecting an intrinsically greater stability of the long and uninterrupted SP-D helix. Susceptibility to Proteolytic Degradation In Vitro SP-A can be degraded in vitro by human leukocyte elastase (115, 116). However, in the presence of physiologic calcium concentrations purified SP-D is not degraded by elastase or a variety of mammalian neutral proteinases (36). Apparently, disulfide cross-linking of the amino-terminal domain, tight folding of the collagen triple helix and coiledcoil domain, and disulfide-stabilized folding of the CRD together exclude proteases from potential internal cleavage sites in natural SP-D at 378C. Multimerization of collectin subunits may also interfere with proteolytic degradation within the amino-terminal peptide and collagen domains. Oxidant treatments have been shown to enhance the susceptibility of SP-A to cleavage by elastase or trypsin (115– 117), suggesting that lung injury might result in protein modifications that could enhance the susceptibility of collectins to degradation in vivo.

In Vitro Interactions with Carbohydrate and Lipid Ligands Structural Requirement for Ligand Binding For all of the collectins, the major requirements for specific carbohydrate binding include the conserved C-type lectin motif in the context of a tertiary structure stabilized by calcium binding and intrachain disulfide cross-linking, and the formation of a trimeric molecule with an appropriate spatial distribution of the constituent CRDs. The C-type lectin domain. Protein and cDNA sequencing studies have shown that the primary sequence of the carboxy-terminal domains of SP-A and SP-D are homologous (z 40%) and that they both contain characteristic elements of the mannose-type C-type lectin motif (19, 21, 43, 118, 119). Various biochemical and molecular studies have definitively established that these domains are primarily responsible for the carbohydrate binding activity. Comparisons of the predicted secondary structure of the carboxy-terminal domains of SP-A and SP-D with the published crystal structure of MBP (111, 112), and mapping of disulfide cross-links in SP-A (110), further suggest that the tertiary structures of their CRDs are generally similar to MBP. Thus, these molecules are each predicted to have two hydrophobic cores, a similar linear distribution of a-helices and b-sheets, two potential calcium coordination sites, and a major disulfide stabilized loop that apposes the carboxy-terminal end of the CRD with the amino-terminal end of the CRD in proximity to the linking peptide. In MBP, charged and polar residues in the vicinity of the short internal disulfide loop (Glu185 and Asn187) directly participate in calcium and form hydrogen bonds with the 3-hydroxyl group of mannose. Analogous studies have confirmed the involvement of the corresponding residues in SP-A (120) and SP-D (121). The distribution of nonconserved residues in and around the generic ligand binding site, and their associated subtle alterations in CRD structure, presumably contribute to ligand specificity. Trimeric clusters of CRDs. The linking peptide in con-

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junction with the CRD domain (L1CRD) can form trimers, and interactions between hydrophobic sequences on L and the CRD determine the spatial distribution of the three CRDs, thereby generating a single, trimeric, high-affinity ligand binding site (122). Approximately 100- to 200-fold higher concentrations of saccharide competitor are required to block the binding of trimeric CRDs (10–20 mM), as compared to monomers (0.1 mM), to neoglycoprotein ligands in solid-phase binding assays. Thus, trimeric clustering of CRD permits collectin binding to complex ligands in the physiologic range of free “sugar” concentrations. At least in the case of MBP, the three saccharide binding sites form a relatively planar array perpendicular to the axis of the trimeric linking peptide. Thus, high-affinity binding usually requires the simultaneous occupancy of two to three saccharide binding sites within a single trimeric subunit in apposition to a surface with a comparable spatial distribution of saccharide ligands. Spatial distribution of trimeric CRDs. As implied in the previous paragraph, the assembly of collectin monomers to form trimeric clusters of C-type CRDs is necessary and sufficient for high-affinity binding to specific saccharide and lipid ligands. However, the capacity for bridging interactions between spatially separated ligands depends on an appropriate oligomerization of trimeric subunits, resulting in a characteristic spatial distribution of the trimeric CRDs. Thus, trimeric CRDs appear to be functionally univalent with regard to their capacity to participate in bridging interactions between large particulate ligands. The concept of a functionally univalent trimer is consistent with the inability of a single-arm mutant, RrSP-Dser15/20, and bacterially expressed trimeric L1CRDs to cause significant bacterial aggregation and viral precipitation (36). In fact, trimeric CRDs and single-arm forms of the protein can function as competitive inhibitors of SP-D-mediated microbial aggregation. On the other hand, higher orders of oligomerization can also influence apparent binding affinity to multivalent ligands, and perhaps influence ligand selectivity (123). The apparent dissociation constant for the binding of collectins to highly substituted ligands (affinity matrices, intact organisms) is typically orders of magnitude higher than observed with simple test ligands and mono- or disaccharide competitors. For example, the apparent kD for SP-D binding to maltosyl30-albumin in solid-phase binding assays is approximately 3 3 1028 M (109). However, the kD for binding to Escherichia coli is approximately 2 3 10211 M.

An important methodologic implication is that lectin-dependent binding is not always freely reversible in the presence of simple sugars. In Vitro Binding Activities SP-A and SP-D show calcium-dependent and saccharideinhibitable interactions with a wide variety of carbohydrates or carbohydrate-containing ligands, as well as specific phospho- and glycolipids (Tables 2 and 3). The ligands include various neoglycoproteins or saccharide-substituted affinity matrices, purified microbial glycoconjugates, and whole organisms. Given that these studies have employed many different assay systems, from solid-phase or solution binding assays to light-scattering assays of aggregation or precipitation, it is not surprising that there are some apparent discrepancies in the literature. Nevertheless, important generalizations can be made based on the available data, and the remaining discrepancies only serve to emphasize the importance of exercising caution when extrapolating from assay systems to the possible situation in vivo. Some of the interactions of collectins with lipids involve interactions with saccharide components of complex lipids (e.g., the binding of SP-D to glucosyl-ceramide, or SP-A with galactosyl-ceramide). However, it is likely that interactions with lipid-containing structures in the pulmonary airspace, or hydrophobic interactions with nonpolar moieties expressed on the surface of various microorganisms (e.g., SP-A binding to lipid A), constitute distinct biologic activities of these proteins in vivo. For this reason, carbohydrate and lipid binding activities are discussed separately below. Carbohydrate Binding SP-A. Purified human SP-A binds specifically to carbohydrates as assessed by saccharide competition in solid phase-binding assays using adsorbed mannan as the ligand (23) (Table 2). The order of preference was approximately N-acetylmannosamine . L-fucose . maltose . glucose . mannose with no inhibition by galactose, N-acetylglucosamine, and N-acetylgalactosamine. These results were independent of the source or method of extraction of SP-A, and generally consistent with earlier studies that examined binding to various saccharide-substituted supports (24). However, they are in obvious disagreement with another study that characterized the binding of iodinated butanolextracted human and dog SP-A to various neoglycopro-

TABLE 2

Carbohydrate selectivity of collectins in vitro Human SP-A ManNAc . Fuc . Mal . Glc . Man (Gal, GlcNAc no inhibition) Man, Glc, Gal, Fuc .. GlcNAc, GalNAc Man, Fuc . Glc, Gal .. GlcNAc Rat SP-D Mal, Inositol . Glc .. Man . Gal, Fuc, Lac (GlcNAc, GalNAc no inhibition) a-Glc-BSA ... b-Glc-BSA Human SP-D Mal . Fuc, Man . Glc . Gal, Lac . GlcNAc (GalNAc no inhibition)

IC50, solid-phase to mannan Affinity chromatography Direct binding to neoglycoproteins IC50, solid-phase to Mal-BSA Direct binding—solid phase assay IC50, solid-phase to Mal-BSA

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TABLE 3

Known “ligands” for lung collectins Ligand

Gram (2) lipopolysaccharides (LPS) Gram (2) capsular polysaccharides, K. pneumoniae Gram (1) lipotechoic acids (LTA) Influenza A hemagglutinins (HA) Influenza A neuraminidase (NA) Fungal cell wall glycoconjugates Pneumocystis gpA Leukocyte surface glycoconjugates Pollen glycoconjugates Dust mite allergen glycoconjugates Lipids Dipalmitoylphosphatidylcholine (DPPC) Phosphatidylinositol (PI) Nonglycosylated ceramides Glucosylceramide Galactosylceramide Gangliotriosylceramide (asialo-GM2) Extracellular matrix components Inflammatory mediators

teins, glycolipids, and neoglycolipids (124). Notably, the latter studies demonstrated a preferential recognition of galactose and an inability to compete for binding with free monosaccharides. The basis for these discrepancies is unclear, but they almost certainly reflect differences in the assay systems and possibly the physical state of the isolated SP-A. Although residues corresponding to Glu185 and Asn187 are found in essentially all members of the mannose binding subgroup, SP-A has a substitution in the position corresponding to Asn187 (Arg in dog and rat and Ala in human). Drickamer has suggested that this correlates with the capacity of SP-A to bind to a variety of sugars with comparatively weak affinity (125). In any case, mutagenesis of corresponding residues in the putative carbohydrate binding site of SP-A (Glu195 to Gln and Arg197 to Asp) reversed the preference from mannose to galactose in affinity chromatography assays using mannose- and galactose-substituted supports (120). Deletion of the consensus sequences for N-linked glycosylation showed no obvious effect on lectin activity (114). SP-D. Purified natural rat, human, and bovine SP-D, as well as recombinant rat and human molecules, preferentially recognize the a-anomeric configuration of nonreducing glucopyranosides (109). The order of preference of human SP-D in solid-phase competition assays using maltosyl-bovine serum albumin (BSA) as the ligand is approximately: maltose (inositol) . glucose, mannose, fucose . galactose, lactose, glucosamine . N-acetylglucosamine (Table 2). This binding specificity is consistent with known interactions of SP-D with the glucose-containing core oligosaccharides of LPS, the mannose-rich N-linked oligosaccharides of the hemagglutinin of influenza A, and the gpA of Pneumocystis carinii (see below). Saccharide specificity may be entirely determined by the trimeric L1CRD domains, given that bacterially ex-

SP-A

SP-D

Lipid A Binds some types ? Bound by HA ? Binds N-linked sugars 6 Binds Binds Binds

Core oligosaccharides No binding No binding N-linked sugars on HA N-linked sugars on NA of some strains Binds N-linked sugars Binds Binds Binds

Binds No binding Binds No binding Binds Binds ? ?

No specific binding Binds No binding Binds No binding No binding Binds type IV collagen Binds C1q

pressed trimeric CRDs show the same saccharide-inhibition profile as natural human SP-D (126). Site-directed mutagenesis of conserved residues of the mannose-type saccharide binding site (Glu321 to Gln and Asn323 to Asp) reversed the relative carbohydrate binding specificity from maltose, glucose . galactose to galactose . maltose, glucose (121). The profile of carbohydrate selectivity in solidphase binding assays suggests that binding is influenced by the nature of the substitution and anomeric configuration at carbons 2, 3, and 4, and particularly by the size of the substitution at carbon 2 (e.g., glucose . glucosamine . N-acetylglucosamine). Summary and important caveats. As shown in Table 2, the carbohydrate selectivities of the two proteins are generally consistent with their subclassification as mannosetype C-type lectins. However, somewhat different profiles in saccharide selectivity have been reported by different investigators for human SP-A, and there are subtle differences between rat and human SP-D. There are notable differences in primary structure of the SP-A and SP-D carboxy-terminal domains that include differences in conserved elements of the CRD which could account for differences in ligand carbohydrate binding. On the other hand, differences in protein solubility and in the valency of trimeric CRDs complicates the comparison of binding affinity and other parameters. Although there is evidence for a small soluble fraction of SP-A, the physiologic relevance of solution phase assays for studies of SP-A function remains uncertain. Lipid Binding SP-A. As indicated in the previous section, SP-A specifically binds to DPPC (127, 128) and aggregates surfactant phospholipids in the presence of calcium (108, 113, 129). SP-A also binds to several glycolipids and neutral glycosphingolipids including galactosyl-ceramide, lactosyl-cera-

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mide, and asialo-GM2, but not to glucosyl-ceramide (124, 130, 131). These interactions appear to involve recognition of both the ceramide and saccharide moieties (124, 131). Ross and coworkers showed that the collagenase-resistant domain of SP-A (containing the L1CRD) can bind to DPPC, whereas a smaller proteolytic fragment lacking the L peptide region lacks binding activity (132). On the other hand, synthetic peptides related to the L peptide or contiguous hydrophobic sequences in the CRD can show interactions with lipid molecules in vitro (133, 134). Consistent with these results, recent studies employing various SP-D/ SP-A chimeras have shown that both the L domain and CRD of SP-A are required for optimal binding (135). SP-A-mediated lipid aggregation is a more complex phenomenon that appears to require oligomerization of trimers (114, 135, 136). For example, the isolated collagenase-resistant peptide lacks the ability to aggregate phospholipids, and preferential reduction of amino-terminal interchain disulfide cross-links inhibits the aggregation of phospholipid vesicles (136). Interestingly, site-directed mutagenesis of conserved residues in the putative carbohydrate binding site (Glu195 to Gln and Arg197 to Asp) of rat SP-A expressed in insect cells does not inhibit binding to DPPC-containing liposomes but does inhibit their aggregation (120). Given the proximity of the saccharide binding site to the Asn-linked sugar, the mutation conceivably could have altered the propensity for lectin-mediated self-association. However, as suggested above, it remains controversial whether or not the Asn-linked sugars contribute to lipid aggregation (108, 114). Alternatively, conformational changes resulting from this mutation of the calcium binding site may influence the spatial relationship of the CRD to the L peptide region. SP-D. Purified SP-D shows high-affinity binding to phosphatidylinositol (PI) resolved on thin-layer chromatography (TLC) plates or presented in liposomes (135, 137, 138). PI is also the major surfactant-associated ligand of SP-D. In addition, SP-D binds to glucosyl-ceramide when displayed on TLC plates, but not to galactosyl-ceramide, asialo-GM2, or other major pulmonary glycolipids (103). The interactions of SP-D with PI and glucosyl-ceramide are calcium-dependent and inhibited by competing sugars, consistent with lectin-dependent binding. PI recognition may predominantly involve the inositol moiety. In this regard, myo-inositol is an efficient competitor of SP-D binding to maltosyl-BSA, and inositol shows homology to a-D-glucose in its distribution of hydroxyl groups (138). However, there is evidence that PI also interacts with the L peptide domain or other sites in the CRD not directly involved in saccharide binding. Site-directed mutagenesis of the putative saccharide binding site (Glu321 to Gln and Asn323 to Asp) of rat SP-D blocked interactions with glycosyl-ceramide but showed only a partial (and calcium-dependent) inhibition of binding to PI-liposomes (121). These same studies demonstrated contributions of the hydrophobic moiety to interactions with various glycolipids. Interestingly, the isolated L peptide of SP-D also binds to PI (and DPPC) in the presence of calcium when spread on plastic microtiter wells (122). However, it is unclear whether these lipid binding sites are exposed in the native molecule.

In other studies, surface balance techniques were used to demonstrate limited interactions of recombinant rat SP-D with monomolecular layers of phospholipids (139). In these experiments, there was no detectable phospholipid head-group preference and interactions of SP-D with the lipid were attributed to hydrophobic interactions. Calcium diminished the intrinsic surface activity of SP-D and its insertion into lipid monolayers. The reason for the apparent discrepancy with earlier studies is unknown, but could reflect the comparatively low sensitivity of these physical measurements. Summary. Phospholipid binding by the lung collectins involves interactions with the CRD but also requires the L peptide. It is not known whether the L peptide directly participates in binding in the intact protein, or whether hydrophobic interactions between the L peptide and CRD are required to maintain a specific conformation or spatial distribution of the CRDs. Differences in lipid binding preferences of SP-A and SP-D probably reflect differences in the structure of the CRD, as well as differences in the L peptide, which include a highly hydrophobic sequence following the amphipathic a-helix in SP-A (118). Phospholipid aggregation requires the L1CRD, as well as the oligomerization of trimeric subunits.

Interactions of Lung Collectins with Microbial Ligands The lining material of the alveoli and distal airways is ideally positioned to participate in the neutralization and clearance of inhaled microorganisms. Because the large serum collectins are presumed to be present at very low concentrations in the extravascular space in uninjured tissues, SP-A and SP-D probably constitute the major collectin defenses of the lung. Consistent with this hypothesis, lung collectins have been shown to interact with a wide range of microorganisms in vitro (Table 3), and dissemination of at least one organism is enhanced in SP-A (2/2) mice. In some cases specific microbial ligands have been defined. Viral Glycoconjugates The interactions of lung collectins with influenza A virus (IAV) have been extensively characterized, and provide a reasonable model system to examine structure–function relationships. IAV attaches to and infects cells by binding through its hemagglutinin to sialic acid-bearing components on the cell surface, while the neuraminidase is involved in viral production and perhaps inactivation of sialylated host proteins. The collectins are potent inhibitors of hemagglutinin (HA)-mediated agglutination, and may also inhibit neuraminidase activity. For example, the levels of SP-D in lavage fluids are sufficient to account for a significant fraction of the total HA inhibitory activity of the fluids as demonstrated by prior adsorption of SP-D using maltosylagarose affinity chromatography (140). Binding of SP-D to IAV. HA inhibition by SP-D involves the binding of SP-D through its CRD to glycoconjugates expressed near the sialic acid binding site on the hemagglutinin (or neuraminidase) of specific strains of IAV (53). HA inhibition assay is inhibited by EDTA or

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maltose (140), and binding to purified HA is blocked by prior glycosidase digestion. Notably, SP-D has negligible activity against the PR-8 strain of IAV, which lacks carbohydrate attachments on the head region of its HA molecule (141). As illustrated in Figure 4, higher degrees of valency or multimerization among the various SP-D preparations are associated with increased HA inhibitory activity. At least with some strains of IAV there is also binding to glycoconjugates associated with the viral neuraminidase, and it has been suggested that collectin binding to the neuraminidase can sterically interfere with HA activity (142, 143). The possibility that collectins can inhibit the neuraminidase requires further investigation. IAV binding to SP-A. Benne and coworkers demonstrated that the HA binds to sialic-acid residues on SP-A, presumably via the Asn-linked sugar associated with the CRD of SP-A (144). Herpes simplex virus can similarly interact with N-linked oligosaccharides on SP-A (107). By contrast with SP-D, human SP-A also has considerable HA inhibitory activity for PR8 (Hartshorn, manuscript submitted). The inhibitory activity for several strains, including PR8, is not inhibited by EDTA or by sugars expected to block collectin activity. On the other hand, binding of human SP-A to the neuraminidase of the A/X31 strain of IAV was partially inhibited by mannose and EDTA (142), suggesting both collectin-dependent and collectin-independent binding. Thus, it remains unclear as to the extent that the carbohydrate binding activity of SP-A contributes to its interactions with viral particles. Collectin-mediated viral aggregation. An important aspect of the interaction of collectins with IAV is their ability to cause viral aggregation and to enhance other aggregation-dependent activities (44, 53). Among the collectins, SP-D is the most potent at aggregating IAV particles, and multimers of dodecamers are much more potent than dodecamers (Figure 4). Approximately 10-fold lower con-

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centrations of SP-D dodecamers are needed to achieve maximal aggregation in light-scattering assays, as compared with SP-A or MBP octadecamers (53). SP-D-induced viral aggregates are also much larger than those obtained for SP-A or MBP. They can usually be visualized with the naked eye, and rapidly precipitate under unit gravity (Hartshorn, manuscript submitted). We speculate that the greater IAV-aggregating activity of SP-D results from its longer collagen domain, which theoretically allows binding between ligands separated by distances of approximately 100 nm, as compared with 20 nm for SP-A and MBP. The maximum distance between SP-D CRDs is approximately equal to the diameter of a single viral particle. Effects of SP-D on infectivity and viral multiplication in vivo. SP-D is a potent inhibitor of IAV infectivity as measured by egg inoculation assay, and more potent at inhibiting IAV infectivity than SP-A (or MBL) (44, 145, 146). Recent studies have also shown that the susceptibility of various IAV strains to neutralization by SP-D in vitro is inversely correlated with pulmonary viral replication in mice following nasal inoculation (Margot Anders, submitted manuscript). The susceptibility to neutralization directly correlates with specific differences in the number of glycoconjugates expressed on the hemagglutinin. Thus, strains with more oligosaccharide chains are uniformly more susceptible to neutralization by SP-D in vitro and show reduced replication in vivo. Strains such as PR-8, which lack sugars on the HA and do not bind to SP-D, show very high levels of replication. Inoculation of mice in the presence of mannan, a saccharide competitor of SP-D binding to IAV, also increases viral replication in mice consistent with involvement of a C-type lectin. Bacterial Glycoconjugates The lung collectins bind glycoconjugates expressed by a variety of gram-negative bacteria including specific strains

Figure 4. Effects of various molecular forms of SP-D on influenza A virus and its interactions with neutrophils. Trimers of CRDs and RrSP-Dser15/20s can bind to the virus as evidenced by inhibition of hemagglutination. However, these forms do not mediate viral aggregation or several activities dependent on viral aggregation, including viral binding to neutrophils, augmentation of the respiratory burst response to bound virus, and protection of neutrophils from viral deactivation. The latter activities involve aggregation-dependent enhancement of the association of IAV particles with their natural, neuraminidase-sensitive binding sites on the neutrophil surface (44, 53, 140).

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of such important pulmonary pathogens as Klebsiella pneumoniae, P. aeruginosa, Hemophilus influenzae, and E. coli (Table 3). However, their bacterial specificities overlap only partially, and their modes of interaction and the effects of binding on microbial interactions with host defense cells appear distinct. Gram-negative bacterial LPS. SP-A preferentially binds specifically to the lipid A domain of rough forms of LPS and to purified lipid A (147, 148). The binding to purified rough LPS is calcium-independent and is not inhibited by competing saccharides, but is inhibited or partially reversed by lipid A. Consistent with these findings, human SP-A binds to certain rough, but not smooth, strains of E. coli with resulting opsonization and enhanced phagocytosis and killing (149). However, because lipid A is presumed to be embedded within the bacterial cell wall, it is unclear to what extent SP-A binding to lipid A accounts for binding to the intact organism. By contrast, core sugars of LPS (glucose and/or heptose) have been identified have been identified as major ligands for rat or human SP-D on E. coli and Salmonella minnesota (34). SP-D also binds to isolated LPS from a variety of other gram-negative bacteria including K. pneumoniae and P. aeruginosa (34, 126, 150). Purified natural or recombinant dodecamers are potent agglutinins for

bacterial strains expressing O-antigen-deficient LPS molecules (e.g., rough strains of E. coli) and cause gross aggregation and precipitation of suspended organisms. By contrast with SP-A, SP-D binding to LPS and its effects on bacterial aggregation are blocked by EDTA, competing sugars, LPS, and rough mutant forms of LPS, but not by lipid A (34). Although SP-D also binds poorly to smooth (O-antigen-containing) LPS on lectin blots, SP-D can still bind to O-antigen-expressing bacteria, as evidenced by specific labeling and microaggregation in immunofluorescence assays (34). It is well known that O-antigens can mask the accessibility of core determinants to antibody, and likely that these structures can sufficiently interfere with collectin binding to limit aggregate size. Interestingly, SP-D was identified as the major E. coli binding protein in cell-free rat BAL (34). We have observed that growth conditions (e.g., aeration) can markedly influence the aggregation of specific gram-negative bacterial strains by SP-D, and that the extent of macroscopic aggregation inversely correlates with the size and complexity of the terminal O-antigen (Figure 5). Thus, phase variants that express a higher proportion of immature LPS may be preferentially aggregated with SP-D. Interestingly, immuno-electron microscopy studies have demonstrated preferential localization of binding

Figure 5. Modulation of surface expression of LPS- and SP-Dinduced agglutination of K. pneumoniae in response to growth conditions. K50 (cap-) strains of K. pneumoniae, which show agglutination by SP-D, were grown as aerated (A) or static (S) cultures. (Left panel) Outer membrane preparations from organisms grown under these conditions were resolved by SDS-PAGE and visualized by silver staining. Aeration is associated with a predominance of rapidly migrating rough forms, whereas conditions of static culture favor smooth forms with larger O-antigens. (Right panel) The minimal concentration of SP-D required for macroscopic aggregation of the organisms is given on the y-axis. Note that bacteria grown under aerated conditions (which show a predominance of rough LPS) are preferentially agglutinated by SP-D. These data were kindly provided by Dr. Itzhak Ofek.

Figure 6. Immunogold labeling of log phase cultures of E. coli. Bacteria were incubated with biotinylated SP-D and washed prior to incubation with streptavidin-gold and visualization by transmission electron microscopy. There was no significant labeling in the presence of EDTA or maltose. Note the preferential labeling near the site of cell division. This nonuniform labeling could reflect regional differences in the density of LPS, in LPS maturation, or in the expression of molecules that may interfere with SP-D binding. Thus, binding sites may be preferentially expressed during specific phases of the bacterial growth cycle.

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sites in growth-phase cells near the sites of bacterial cell division (Figure 6). Gram-negative capsular polysaccharides. Recent studies have demonstrated specific binding of human proteinosis SP-A to the mannose-rich capsule of specific serovars and recombinant strains of K. pneumoniae (151). The binding of capsular polysaccharide to immobilized SP-A is also inhibited by mannan as well as purified capsular polysaccharides from a binding strain (K21a), but not by LPS or the capsular polysaccharides of a nonreactive strain (K2). SP-A promoted phagocytosis both as an opsonin and through mannan-inhibitable effects on macrophages, which appear to involve the macrophage mannose receptor. It is still unclear to what extent these effects involve the lectin activity of SP-A. Interestingly, epidemiologic studies have shown that among serotypes isolated with high frequency from patients with active infection, there was a preponderance of bacterial strains with capsular polysaccharides that are poorly recognized by SP-A and mannose receptor (152). In collaborative studies with Dr. Itzhak Ofek (University of Tel Aviv, Tel Aviv, Israel), we observed interactions of SP-D with specific strains of K. pneumoniae distinct from those recognized by SP-A. Specifically, SP-D showed lectindependent agglutination of two unencapsulated variants of K50 and K21a, and at least one weakly encapsulated strain (150). The binding of SP-D to the isolated Klebsiella LPS was inhibited by EDTA, competing sugars, and LPS from other gram-negative bacteria, but not by capsular polysaccharides. Although encapsulated strains can apparently bind SP-D, they do not undergo macroscopic aggregation. These observations suggest that SP-D does not recognize the capsular polysaccharides of K. pneumoniae, and that the presence of a well-formed capsule limits interactions of SP-D with underlying LPS molecules (Figure 7). Gram-positive cell wall. Systematic studies of collectin binding to various gram-positive organisms have not yet

Figure 7. Hypothetical comparison of the interactions of SP-A and SP-D with rough and smooth forms of encapsulated and unencapsulated gram-negative bacteria. SP-D binds to rough or short O-antigen forms of LPS expressed on unencapsulated or weakly encapsulated organisms, a variant phenotype that is likely required for bacterial colonization and invasion. Although encapsulation limits interactions with SP-D, the expression of specific capsular polysaccharides favors SP-A binding (151). In this scheme SP-A and SP-D serve complementary roles in interacting with gram-negative bacteria and modifying their interactions with host-defense cells.

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been described. Human proteinosis SP-A shows calciumdependent binding to clinical isolates of S. aureas and Streptococcus pneumoniae (153). Although S. aureas showed no evidence of calcium-dependent binding or aggregation under conditions leading to the aggregation of gram-negative bacteria (34), there was a higher background of EDTAinsensitive binding, suggesting involvement of other binding mechanisms. Nothing is currently known about the nature of the binding sites on gram-positive bacteria. Various capsular or cell wall glycoconjugates are certainly plausible candidates. In this regard, MBP has been shown to bind to lipotechoic acids (LTAs), and binding was restricted to LTAs with terminal sugars (154). However, hydrophobic interactions could also play an important role in the binding of collectins to gram-positive organisms. For example, LTAs contain fatty acids that could participate in hydrophobic interactions. A variety of other less well-characterized adhesive hydrophobic components (hydrophobins), and lectin-like proteins, are also expressed on gram-positive organisms (155). Fungal Glycoconjugates Pneumocystis carinii. Recent studies have demonstrated specific lectin-dependent interactions of both rat and human SP-A and SP-D with gpA, a mannose and glucoserich glycoprotein expressed on P. carinii cysts and trophozoites (156–160). Both proteins are associated with P. carinii in vivo, and are present on the surface of freshly isolated organisms. The proteins bind in a collectin-dependent mechanism that is inhibited by EDTA, competing sugars, or a specific antibody. Clusters of organisms in BAL can be partially disaggregated with EDTA or competing sugars, and “stripped” organisms can be agglutinated by purified SP-D, suggesting that SP-D contributes to the clustering of cysts observed in vivo. Interestingly,

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the production and accumulation of both proteins are increased in rats and immunosuppressed patients with pneumocystis pneumonia (156, 161). Other fungal organisms. Immunofluorescence studies using fluorescein isothiocyanate (FITC)-conjugated SP-D have demonstrated specific interactions of SP-D with other “fungal” organisms including such potential human pathogens as Histoplasma capsulatum, Aspergillus fumigatus, Candida albicans, and Blastomyces dermatitides (4) (Kuan and Crouch, unpublished observations). SP-D presumably recognized fungal cell wall glycoconjugates because labeling was blocked by EDTA or maltose, but not by lactose. Human proteinosis SP-A and recombinant rat and human SP-D bind to pathogenic unencapsulated, but not the capsulated, forms of Cryptococcus neoformans through a lectin-dependent mechanism (162). Although the unencapsulated organisms are readily agglutinated by SP-D, there is no significant aggregation by SP-A. More recently, human proteinosis SP-A and SP-D were shown to bind to Aspergillus fumigatus conidia in a calcium- and carbohydrate-dependent fashion consistent with binding of the CRD to cell wall glycoconjugates (163). Both SP-A and SP-D efficiently agglutinated the conidia.

Interactions with Nonmicrobial Particulates SP-A has been shown to bind specifically to various pollen grains through lectin-dependent binding to soluble glycoproteins (12). These interactions increased the binding of pollen grains to a lung epithelial cell line (A549). More recently SP-A and SP-D were shown to bind to dust mite allergens (13). Binding was calcium-dependent, inhibited by competing sugars, and prevented by prior deglycosylation of the mite extracts or purified proteins. Recombinant trimeric CRDs of SP-D were shown to inhibit the binding of the intact protein. There is also some evidence to suggest that coating of inorganic particles by SP-A and other surfactant components can enhance uptake by alveolar macrophages (164).

Interactions of Lung Collectins with Phagocytes There are at least three general mechanisms by which collectins can modify the host defense activities of pulmonary leukocytes (Table 4). These include opsonization or other effects mediated by the specific interactions of phagocytes with collectin-coated organisms, various direct effects of collectins on leukocyte function, and “indirect” effects involving lung collectin-mediated alterations in particle presentation secondary to aggregation. The first two mechanisms require collectin binding sites on the phagocyte cell surface, whereas the latter presumably uses pre-existing “receptors” (ligands) for microorganisms on the phagocyte cell surface. Particle opsonization may theoretically involve protein receptor-mediated binding of the collectin to the leukocyte, collectin-mediated bridging interactions between glycoconjugates expressed on the particle and host defense cell, and/or binding of the host cell to glycoconjugates expressed on the collectin. There is growing evidence that all of these mechanisms may be operative, alone or in combination, in specific microbial-phagocyte interactions.

TABLE 4

Modulation of host response to microorganisms* Collectin-mediated bridging between microorganisms and phagocytes Opsonization/phagocytosis, with or without enhanced killing Enhanced respiratory burst within cell or in an external microenvironment Direct effects of collectins on leukocyte function Enhanced leukocyte recruitment/chemotaxis Enhanced respiratory burst Enhanced phagocytosis and/or killing Enhanced expression of macrophage mannose receptors (SP-A binding to mycobacteria) Stimulation or inhibition of specific cytokine production Regulation of lymphocyte proliferation Indirect effects of collectins on leukocyte function Microbial aggregation Collectin binding to microbial glycoconjugates (SP-D binding to E. coli and IAV) Microbial lectin binding to collectin glycoconjugates (SP-A binding to IAV) Altered presentation of organisms secondary to an enhanced association with airspace lining material Modulation of leukocyte interactions with bacterial LPS Modulation of leukocyte apoptosis Modulation of host response independent of phagocytes Microbial aggregation with enhanced mucociliary clearance Microbial binding or aggregation with altered colonization of invasion Direct effects on microbial growth and metabolism *Examples in parentheses.

Collectin Receptors SP-A receptors on host defense cells. SP-A has been shown to compete with C1q or serum collectins for binding to C1q receptors (C1qR) (165, 166), but has also been shown to interact with distinct receptors on macrophages (and type II cells) (167, 168). Perhaps the best characterized of the non-C1qR receptors is a 210-kD protein that is expressed on alveolar macrophages and type II cells (167). Binding of the 210-kD receptor is calcium-dependent, but is not inhibited by mannan, mannosyl-BSA, and other sugars. Antibodies to this protein were reported to block the human proteinosis SP-A-mediated uptake of Mycobacterium bovis. The specific collectin domains involved in receptor recognition have not been definitively elucidated. C1qR is believed to bind to the collagen domain (169, 170), and binding to macrophages and macrophage chemotaxis are partially blocked by collagenous sequences (171, 172). Human proteinosis SP-A has also been shown to bind to macrophages by a mechanism that appears to involve cellular recognition of the Asn-linked sugar on SP-A (51). The expression of macrophage SP-A receptor(s) on host defense cells is subject to complex regulation. PMA, LPS, and g-interferon increase SP-A binding to marrowderived macrophages, whereas binding is decreased by dexamethasone and granulocyte macrophage colony-stimulating factor (GM-CSF) (173). SP-A can also stimulate the production of GM-CSF by type II cells and macrophages (174). Interestingly, the expression of macrophage SP-A receptor appears inversely related to changes in the

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expression of mannose receptor (173), and SP-A binding can increase mannose receptor expression (51). SP-D receptors. SP-D does not interact with C1q receptors (175). However, SP-D has been shown to be recognized by one or more receptors or binding proteins on alveolar macrophages (175, 176). This binding does not appear to involve the lectin activity of SP-D because binding was performed in the presence of EDTA. It is unclear to what extent the binding moieties may be related to the non-C1qR SP-A receptors. Biotinylated SP-D preferentially binds to alveolar macrophages in frozen sections of rat lung (177). Although labeling of macrophages is markedly reduced in the presence of EDTA or maltose, there is a residual specific binding that is blocked by unlabeled SP-D. Holmskov and coworkers have recently reported the isolation of an SP-D binding protein (GP-340) from human proteinosis lavage (178). Antibodies to the protein show selective binding to alveolar macrophages. The protein apparently binds to the L peptide region. Collectin Ligands (Glycoconjugate Receptors) on Leukocytes Both SP-A and SP-D can interact with leukocytes via lectin-dependent binding to cell surface carbohydrates (177, 179–181). Gold-labeled SP-A shows specific and mannosedependent binding and internalization by human macrophages and monocytes (180) and rat alveolar macrophages (179). Because the mannosyl-albumin inhibitable activity did not correlate with the level of expression of membrane mannose receptors it was concluded that the effects were in part secondary to the lectin activity of SP-A. Interestingly, macrophages can also specifically bind to glycoconjugates expressed on SP-A (51). As indicated in the previous section, biotinylated SP-D shows significant specific maltose-dependent binding to rat lung macrophages

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in frozen tissue sections (177). Although the binding sites have not been elucidated, a glycolipid binding site is suggested (4). Binding of FITC-labeled SP-D to freshly isolated aldehyde-fixed rat alveolar macrophages is eliminated after extraction of the fixed cells with low concentrations of Triton X-100.

Altered Leukocyte Function Mediated by Collectins Collectins can modulate phagocyte function in a variety of ways (Table 4). Some effects involve prior interaction of the collectin with the surface of microorganisms or other particles. Other effects appear to involve direct modulation of leukocyte function through interactions of the collectin with cellular receptors or binding sites. Yet other effects appear to be “indirect” and involve altered presentation of microorganisms as a consequence of collectinmediated agglutination. Opsonization “Opsonization” refers to the process by which particles are modified (usually through binding to a specific protein or opsonin) so that they are more efficiently internalized by phagocytes. There is abundant evidence that lung collectins can bind to microorganisms and enhance their association with leukocytes, and in a few recent studies enhanced internalization and killing of specific organisms has been demonstrated (Figure 8). For example, SP-A has been shown to opsonize and increase the killing of type A, but not type B, H. influenzae by alveolar macrophages (153). Preincubation of specific encapsulated strains of K. pneumoniae with SP-A also results in enhanced internalization and killing of organisms that is inhibited by mannan and partially blocked by EDTA (151). In addition, SP-D can opsonize and enhance the killing of an unencapsulated strain

Figure 8. A schematic diagram comparing selected modes of action of SP-A and SP-D in particle opsonization. SP-A can interact directly with encapsulated organisms and enhance interactions with phagocytes through binding to specific receptors. In some cases this may involve cooperation with mannose receptors on alveolar macrophage (151). SP-D can interact directly with unencapsulated or weakly encapsulated organisms and enhance interactions with leukocytes through lectin-dependent and lectin-independent mechanisms.

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of K. pneumoniae by adherent macrophages (Itzhak Ofek, manuscript in preparation). In some case organisms such as pneumocystis may be opsonized by SP-A and internalized, but without associated killing (158). Unfortunately, in most studies, the experimental design does not preclude direct activation of the leukocyte by the collectin. The protein is simply added to the incubation mixture, or the preincubated organisms are not washed prior to their addition to the phagocyte. Chemotaxis/Haptotaxis Natural and recombinant rat SP-D and human proteinosis SP-D are potent chemoattractant and haptotactic agents (maximal chemotactic response at 5 ng/ml or z 10211 M) for both monocytes and neutrophils (163, 181). This effect is mediated by attachment of the CRD to phagocyte surface carbohydrate structures because it is inhibited by competing saccharides and by antibodies directed against the SP-D CRD (181). Interestingly, SP-D concentrations sufficient to cause chemotaxis were not associated with enhanced oxidative metabolism or phagocytic activity. These results suggest that carbohydrate binding properties of SP-D result in distinctive interactions with phagocytes, and that SP-D may play an important role in recruitment or retention of phagocytic cells and modulation of inflammatory or immune responses under certain circumstances in vivo. At much higher concentrations, human SP-A can stimulate chemotaxis of alveolar macrophages (171) and neutrophils (163). The chemotactic activity of SP-A for alveolar macrophages was lectin-independent and appeared to involve interactions with the collagen domain (171). Enhanced Respiratory Activity In several studies the binding of rat SP-A and SP-D have been reported to directly stimulate the respiratory burst response of alveolar macrophages (182–185). However, binding appears to be necessary but not always sufficient for stimulation of respiratory activity. The effects of SP-A on respiration may require its adsorption to a surface (184). In addition, we observed no significant stimulation of the respiratory response of human neutrophils or peripheral blood monocytes by recombinant rat or human SP-D in the absence of a ligand (140, 181). Thus, the state of protein aggregation, method of purification, and various assay conditions may be important variables (185). Non-opsonic Enhancement of Phagocytosis by Lung Collectins Increasingly, there is evidence that collectins can increase the internalization (and sometimes the killing) of a variety of microorganisms by modulating the function of phagocytic cells. The pioneering study by Tenner and coworkers demonstrated that human proteinosis SP-A can enhance FcR- and CR1-mediated phagocytosis of antibody-opsonized sheep erythrocytes by blood monocytes/macrophages (28). Although these effects probably involve “cellular activation” as a consequence of binding to specific “receptors,” similar effects might be observed if particle-collectin interactions are somehow enhanced by prior binding of the collectin to the phagocyte surface.

Gram-negative bacteria. Human proteinosis SP-A has been shown to promote serum-independent phagocytosis of E. coli and P. aeruginosa in parallel with activation of a phosphoinositide/calcium signaling pathway in alveolar macrophages (186). Significantly, pretreatment of bacteria with SP-A enhanced the uptake. In other studies the presence of human proteinosis SP-A was shown to mediate dose-dependent internalization of log-phase, but not stationary, cultures of E. coli (50), and to enhance the phagocytosis of H. influenzae (187). In these studies, enhancement of phagocytosis can be similarly interpreted as resulting from the direct effects of SP-A on macrophage function. The presence of rat or human SP-D increases the binding and internalization of E. coli by neutrophils in the presence of calcium (Hartshorn, manuscript submitted). Significantly, internalization was also somewhat increased to some extent by pre-incubation of the neutrophils with SP-D. As described for IAV, the potency of various molecular forms of SP-D correlated with the degree of subunit multimerization. SP-D-opsonized E. coli also show enhanced binding (Hartshorn, manuscript submitted). Gram-positive bacteria. Proteinosis SP-A and surfactant containing SP-A have been shown to enhance the phagocytosis of serum-opsonized (but not unopsonized) S. aureas by alveolar macrophages (182). This effect was observed following preincubation of SP-A with the macrophage and did not involve opsonization of the organism. In other studies, the presence of human recombinant SP-A during the incubation enhanced the phagocytosis of a non-serumopsonized, log-phase, laboratory strain of S. aureas by alveolar macrophages (50). The presence of human proteinosis similarly enhanced the phagocytosis of S. pneumoniae and Group A streptococcus (186). The effects on S. aureas were shown to be non-opsonic since no enhancement was observed following preincubation of the organism with SP-A. SP-A-stimulated phagocytosis of S. pneumoniae by alveolar macrophages has been reported to involve the stimulation of tyrosine kinase (188). Finally, the presence of proteinosis SP-A can enhance the uptake, but not killing, of S. aureas by blood monocytes, apparently by a mechanism involving C1q receptors (189). As indicated previously, SP-A (2/2) mice show a defective host response to pulmonary challenge by Group B streptococcus and S. aureus (31). However, it has not yet been determined whether this is accompanied by a defect in phagocytic function. Pneumocystis. The attachment and phagocytosis of pneumocystis by rat alveolar macrophages is enhanced in the presence of proteinosis SP-A or by preincubation of the macrophages with the collectin (158). However, enhanced killing has not yet been reported. Although binding of pneumocystis to macrophages is also enhanced by incubation of SP-D-depleted organisms with recombinant SP-D, there is no enhancement of phagocytosis (157, 159). Mycobacteria. SP-A can modulate the interactions of mycobacteria with macrophages with enhanced binding and phagocytosis in vitro (51, 190, 191). The effects of proteinosis SP-A result at least in part from macrophage binding to Asn-linked sugars on the CRD with associated upregulation of the macrophage mannose receptor (51). SP-D enhances the binding but not the uptake of these organisms

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(L. Schlesinger, University of Iowa, Iowa City, IA; personal communication). However, the mechanism of binding has not been elucidated. Altered Secretion of Cytokines and Other Mediators Recent studies have demonstrated direct effects of collectins on other aspects of host defense cell function. Human proteinosis SP-A has been shown to stimulate the production of TNF-a, interleukin (IL)-1a, IL-1b, and IL-6; to decrease the production of g-interferon by human peripheral blood mononuclear cells; to increase TNF-a production by rat alveolar macrophages (10); and to stimulate nitric oxide synthetase expression by macrophages (192, 193). SP-A has also been reported to enhance immunoglobulin production by rat splenocytes (10), and to increase the secretion of colony-stimulating factors by alveolar macrophages (148, 174). On the other hand, proteinosis SP-A was reported to decrease TNF-a activity in the medium of LPSstimulated macrophages, apparently through interactions with the phagocyte (194). The reasons for the discrepant data relating to TNF-a are uncertain but may reflect differences in the methods of isolation of SP-A. Interestingly, the accumulation of TNF-a in cultures of alveolar macrophages exposed to pneumocystis is decreased in the presence of SP-D (157). Effects on T-lymphocyte Proliferation SP-A has been found to increase the proliferation of concanavalin A (Con A)-stimulated rat splenic T-lymphocytes, and counteract the inhibitory effects of surfactant lipids (11). In other studies, SP-A inhibited the proliferation of phytohemagglutinin and anti-CD3 stimulated T cells, while slightly increasing the proliferation of Con A-stimulated cells (9, 195). The inhibitory effects were accompanied by decreased production of IL-2. More recently, recombinant rat and human SP-D were found to potently inhibit proliferation under all three conditions of lymphocyte stimulation (196). This effect did not require longrange bridging interactions because trimeric subunits of rat SP-D were more potent than dodecamers. It is not yet known whether these effects involve direct interactions of the collectin with binding sites on the lymphocyte or are mediated through interactions with a subpopulation of mononuclear phagocytes. “Indirect Effects” on Leukocyte Function In addition to binding to and activating phagocytes, collectins can indirectly influence phagocyte function by altering the presentation of pathogens or microbial ligands to the host defense cells. The best-characterized example is the effect of aggregation on the interactions of influenza A with neutrophils. Effects of SP-D on the response of neutrophils to IAV. Lectin-dependent binding of SP-D to IAV enhances the binding of virus to neutrophils, augments the respiratory burst response to bound virus, and enhances viral uptake (140, 145, 146). In these assays SP-D is considerably more potent than human SP-A, MBP, or various anti-HA monoclonal antibodies. Significantly, neuraminidase treatment of the neutrophil abrogates all of these effects, indicating that SP-D enhances the binding of virus to sialic acid

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“ligands” on the leukocyte surface; i.e., enhances binding to the natural receptor for IAV on the neutrophil. The concentrations of collectins required to elicit these effects closely correlate with those required for IAV aggregation (44, 53). Furthermore, multimers of SP-D dodecamers are significantly more effective than dodecamers on a protein-concentration basis at enhancing IAV binding. Single arms and isolated trimeric CRDs do not increase IAV binding, and actually decrease IAV binding at relatively high concentrations. It is possible that the altered cellular response to bound virus results from bridging or clustering of the “receptors” by viral aggregates, or enhanced viral internalization. The propensity of IAV infection to promote bacterial superinfection contributes heavily to the morbidity and mortality associated with IAV epidemics, and correlates temporally with a marked depression of phagocyte functional responses (197). Pre-incubation of IAV with collectins protects neutrophils against the deactivating effects of the virus as measured by O22 responses of the cells to formylmethionylleucylphenylalanine after virus treatment (44, 53, 145, 146). SP-D is the most potent of the collectins in protecting against deactivation, and 10- to 40-fold more potent than antibodies to HA on a protein-concentration basis. Again, the protective effects directly correlate with their capacity to induce viral aggregation, and enhance binding of the virus to neuraminidase-sensitive sites. Effects on LPS signaling pathways. Interactions between collectins and LPS suggest that under appropriate conditions these molecules could modulate cellular activation and signaling pathways involving LPS. Such effects could theoretically involve completion for binding by other LPS binding proteins or altered presentation of LPS to inflammatory cells. SP-D has been reported to be recovered as a complex with LPS in LPS-induced acute lung injury in rats (198). Leukocyte apoptosis. Interestingly, Hartshorn and coworkers have observed that pretreatment of E. coli with SP-D can enhance E. coli or E. coli 1 influenza-mediated apoptosis of neutrophils in vitro (Hartshorn, manuscript submitted). Presumably this effect results as a consequence of increased or altered binding of E. coli. Summary Both SP-A and SP-D can specifically interact with a wide variety of respiratory pathogens and modulate the leukocyte response to these organisms. The specific mechanism of these effects, in most cases, has not been fully elucidated. At least in the case of SP-D some effects appear to result as a consequence of microbial aggregation with enhanced binding of the agglutinated organism to their natural “receptors.” Although SP-D is generally more potent as an agglutinin than is SP-A under the usual assay conditions, both collectins can elicit aggregation and this is likely to be an important mechanism. There is evidence that both SP-A and SP-D can function as true opsonins for certain organisms under specific assay conditions in vitro. However, enhanced internalization is not an invariant consequence of enhanced binding. Furthermore, pretreatment of the phagocyte with the collectin can sometimes lead to enhanced internalization, and many of the published experiments have not excluded direct, non-opsonic effects re-

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sulting as a consequence of the presence of the collectin in the incubation mixture.

Complexities Relating to the Study of Lung Collectin Function Source and Preparation of Collectins As emphasized throughout this review, the activity of the collectins can be influenced by the number and spatial distribution of CRDs within a molecular complex. Thus, any factor that influences the state of aggregation may alter the biologic properties of the protein. For example, exposure to oxidizing agents during the chemical radioiodination of SP-A was shown to lead to a loss of oligomerization, with an associated loss of lectin activity (199). For studies examining interactions with leukocytes, contamination with endotoxin can become a significant issue. As is usually the case, the best strategy is to avoid contamination from the outset and to include appropriate controls. In our own laboratory all water is ultrafiltered to remove endotoxins, and “pyrogen-free” glass- and plasticware are used wherever possible. The measured contamination is usually low, much less than required to stimulate leukocytes by purified LPS, even in the presence of serum. Nevertheless, given the LPS binding properties of these proteins, it is conceivable that they might be able to present even small amounts of endotoxin to cells in such a way that they influence the biologic behavior. In the case of SP-D, binding to LPS is calcium-dependent and final purification by gel filtration in the presence of EDTA may help minimize the introduction of LPS/SP-D complexes. Microbial Binding to Collectins Microorganisms possess a wide range of specialized cellsurface attachment molecules, including various carbohydrate binding proteins (155). Examples of viral and mycobacterial binding to sugars on SP-A and certain SP-D mutants have been discussed in this review. This possibility must always be considered and can be approached experimentally by characterizing profiles of saccharide inhibition in the presence and absence of calcium, and by examining interactions with enzymatically deglycosylated proteins or mutant collectins lacking specific sugar attachments. Some interactions with microorganisms may eventually be found to involve hydrophobic interactions, or specific binding to the collagen domains. Influence of Microbial Growth Conditions Environmental or growth conditions (aeration, mechanical agitation, pH, CO2 concentrations, medium composition, growth substrate) can influence the amount, structure, and maturation of LPS displayed on the bacterial cell surface (200, 201). The amount and composition of the capsular polysaccharides expressed by encapsulated strains may also depend on the growth conditions. Phase variations within a given bacterial strain may dramatically alter the structure of the cell wall, and thereby bacterial adhesion, as well as influencing interactions with antibodies or other host defense molecules (155, 202). Many organisms release components of their cell wall (e.g., LPS, lipotechoic acids, or capsular

polysaccharides), which may actively compete for binding to microbial cell-surface ligands (155). There are two important corollaries. First, the phenotype of organisms may change under the influence of changing environmental conditions in vivo and influence the binding of host defense proteins. For example, changes in O-antigen and mucoid exopolysaccharide expression, as observed for virulent strains of P. aeruginosa in lungs of cystic fibrosis patients, can influence the accessibility of cell-wall antigens to specific antibodies (203). Second, interactions with an organism in vivo may not parallel their interactions with the organism following isolation and culture in vitro. For example, rough and unencapsulated forms of bacteria may be critical during certain stages of bacterial colonization and invasion by strains that show a smooth and encapsulated phenotype in the laboratory. Similar considerations may also apply to experiments with viruses. Patterns of glycosylation may be influenced by the cell type used for viral propagation (e.g., eggs versus mammalian cells). Cooperative/Competitive Collectin Interactions The potential for cooperative or competitive interactions of SP-A and SP-D in their interactions with particulate ligands and cells seems extraordinarily high but has not yet been explored. A major problem rests with defining an appropriate assay system. Purified SP-A can also specifically bind to surface adsorbed SP-D (204); the mechanism of this apparent interaction has not been determined, but may involve binding to hydroxylysyl glycosides in the collagen domain of SP-D. The functional consequences of such an interaction have not been defined. Potential Competition or Cooperation with Other Lectins It must be emphasized that the collectins are not the only C-type lectins expressed in the normal lung. The mannose receptor, which is expressed on alveolar macrophages, is an integral membrane protein with multiple tandem repeats of a C-type lectin CRD that preferentially interacts with mannose-rich glycoconjugates (205, 206). This protein has been implicated in modulating the interactions of various pathogens with macrophages, and the expression or activity of the receptor can be influenced by collectin binding (51, 151). The lung also contains a number of other carbohydrate binding proteins with a variety of saccharide specificities that may have access to the airspace. For example, a-ficolin, which may be preferentially expressed in lung and placenta, is a secreted protein that contains collagenous and fibrinogen-like domains (207). An apparently identical serum protein shows specific binding of the fibrinogen-like domain to N-acetylglucosamine, and can enhance the phagocytosis of Salmonella typhimurium by neutrophils (208). Interactions with Serum Proteins MBP and conglutinin can exert antimicrobial effects through interactions with the classical or alternative pathway of the complementary system, respectively. However, such interactions have not been demonstrated for SP-A or SP-D. Although SP-D shows some activity in conglutination assays consistent with an interaction with C3bi, it is

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considerably less potent than conglutinin. Adherence of blood monocytes/macrophages to SP-A can enhance CR1mediated phagocytosis of antibody opsonized red cells. However, there is no evidence to suggest that this effect involved any direct interaction with components of the complementary system. Because C1q can enhance the binding of radiolabeled SP-D to macrophages (175), it is possible that the interactions of SP-D to C1q involve the binding of the SP-D CRD to glucose-containing hydroxylysine-derived glycosides in the collagen domain of C1q. Interactions with Matrix Proteins SP-D shows lectin-dependent binding to type IV but not to interstitial collagens, in various solid-phase binding assays (Crouch, unpublished data). In fact SP-D is apparently identical to a 43-kD type IV collagen binding protein previously identified in cultures of rat type II cells (209). We speculate that SP-D binds to glucose-containing hydroxylysyl glycosides, which are abundant in type IV collagen. Soluble versus Insoluble Phases The distribution of SP-A and SP-D collectins among soluble and insoluble compartments could influence the outcome of collectin phagocyte interactions. In this regard, the activity of SP-A can be influenced by its mode of presentation. For example, highly oligomerized preparations of proteinosis SP-A are much more effective in enhancing the adherence of mycobacteria to macrophages than natural or recombinant SP-As in solution, even though all three preparations show similar activity when adsorbed to a substratum (51). Likewise, rat and canine SP-A were found to enhance oxygen radical production only when adsorbed to a surface (184). Factors that determine or alter the distribution of SP-A and SP-D between soluble and insoluble compartments have not been elucidated. Potential interactions with mucus glycoproteins or other components of the mucus layer have yet to be explored.

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What Determines Specificity for Foreign Glycoconjugates? Given that complex carbohydrates are important components of the mammalian plasma membrane, the normal function of these proteins clearly requires selectivity for foreign glycoconjugates. Preferential recognition of specific saccharides that are not present or abundant on mature mammalian oligosaccharides (e.g., glucose, heptose, terminal N-acetylglucosamine) is probably an important mechanism. Terminal sialylation, a common modification on host oligosaccharides, may also limit interactions with host glycoconjugates because none of the known collectins recognize sialic acid residues. Perhaps more importantly, the density of glycoproteins in the eukaryotic plasma membrane is comparatively low. Given that the spacing of individual carbohydrate ligands within a single complex oligosaccharide is insufficient to allow efficient binding of all sites of a trimeric CRD, the very high density of glycoconjugates associated with the microbial cell wall may confer relative specificity for microorganisms versus host cells (210). Normal blood-sugar concentrations may also help limit all but very high affinity lectin-mediated interactions.

Why Do We Need More than One Pulmonary Collectin?

Why Haven’t Microorganisms Developed Effective Ways to Avoid “Static” Collectin Defenses? Collectin-mediated defenses are inherently less able to adapt to structural changes in the surface structure of microorganisms than specific immune mechanisms. However, they may be highly effective given their propensity to interact with required, integral structural components of the microbial cell wall (e.g., the LPS of gram-negative bacteria) or molecules required for infectivity. This likely accounts for the widespread utilization of lectins by invertebrates and plants, not only in the defense against infection (211), but in the neutralization or clearance of biologically active molecules released by microorganisms, such as LPS. Of course, another answer to this question is “They have.” It seems likely that some organisms have developed ways to utilize these interactions to facilitate attachment, colonization, or invasion to the detriment of the host. Knowledge of the outcomes from various microbial-collectin interactions will be needed before embarking on strategies designed to augment collectin defenses.

There are several biochemical differences between SP-A and SP-D that likely confer distinctive functional properties in vivo. The most important of these include (1) differences in solubility at physiologic ionic strength, which may influence compartmentalization and availability in the airspace lining material; (2) differences in the length of the collagen domain, which determine their capacities to participate in long-range bridging interactions; (3) differences in CRD specificity or valency; and (4) differences in the localization of Asn-linked sugars. Differences in ligand specificity may allow these proteins to complement one another in vivo (Figure 8) (150). For example, colonization and initial invasion by gramnegative bacteria is probably favored by a capsule-deficient phenotype that is expected to facilitate SP-D binding. By contrast, evasion of leukocyte host defenses may depend on an encapsulation, which could favor interactions with SP-A.

Who’s Scratching Whom? And Why? Some interactions between collectins and microorganisms may involve the specific recognition of Asn-linked oligosaccharides on the collectin by lectin-like proteins displayed on the microbial surface. Although these two modes of interaction may yield similar results in various in vitro assays, their relative biologic importance remains to be elucidated. Cooperativity of such reciprocal interactions could conceivably occur. In some cases (e.g., with pneumocystis) it is unclear whether collectin binding favors the host, or provides an advantageous niche for microbial colonization of the airspace in immunosuppressed patients. In this regard, human alveolar proteinosis, which is associated with large accumulations of SP-A and SP-D in the alveoli, is associated with an increased incidence of infection by a variety of microorganisms. Finally, it is unclear whether the enhanced internalization of mycobacte-

Important Questions and Directions for Future Research

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ria or pneumocystis by SP-A results to the benefit or detriment of the host, since the internalized organisms are not effectively killed. What Is the Significance of Microbial Aggregation? Studies with influenza A virus strongly suggest that aggregation can enhance leukocyte responses and potentially facilitate clearance by phagocytic mechanisms. Aggregation may also facilitate clearance by mucociliary mechanisms or inhibit microbial colonization and invasion (Table 4). Highly multimeric proteins—like collectins and IgM—may be particularly well suited to mediate host defense during the early phases of an infectious process by virtue of their ability to aggregate the larger numbers of organisms. In any case, in designing experiments it is important to assess the contributions of prior particle aggregations to their effects on host defense cells. What Is the Functional Significance of Lipid Binding? Although the collectins may be found to participate in important physical transformations of surfactant lipids or in some way contribute to surfactant metabolism, it seems equally plausible that interactions with surfactant lipids are primarily required to optimally orient the CRDs in relation to the air/lipid or lipid/hypophase interface or facilitate uptake of foreign materials embedded in the surfactant layer. Thus, tubular myelin may simply represent an organization of surfactant lipids resulting from the insertion of SP-A, rather than a necessary intermediate in the formation of the surface active layer. Exposed hydrophobic sites could also mediate interactions with hydrophobic domains (e.g., hydrophobins) displayed on various microorganisms, and perhaps stabilize relatively weak lectin-dependent interactions. Finally, the binding to various glycolipids in solid-phase binding assays suggests possible regulatory functions involving interactions with glycolipids appropriately displayed on the surfaces of epithelial or host defense cells. It is possible that this binding activity can compete for microbial binding to glycolipid receptors recognized by various bacteria and viruses or bacterial toxins (131). How Do Collectins Modulate the Function of Host Defense Cells? Virtually nothing is now known about the mechanisms by which lung collectins directly modulate the function of host defense cells. In future studies it will be important to more systematically examine the effects of pre-incubating phagocytes with collectins (and washing) prior to exposure to microorganisms or other target particles. Further definition of the relevant receptors and glycoconjugate binding sites, and their associated mechanisms of signal transduction, should prove to be a fruitful area of investigation. Are There Acquired Deficiency States of Lung Collectins? Acute lung injuries of various types could theoretically lead to collectin inactivation as a consequence of protein modification (e.g., oxidation) or proteolytic degradation. Proteolytic cleavage at sites amino-terminal to the coiledcoil domain or disruption of intersubunit disulfide bonds

could liberate trimeric carbohydrate binding fragments similar to RrSP-Dser15/20 or recombinant trimeric CRDs. Such molecules are defective in mediating bridging interactions, and may also function as competitive inhibitors of collectin binding. The capacity of SP-D to bind with high affinity to type IV collagen in vitro suggests that collectin–matrix interactions could also occur in the setting of lung injury. Disorders associated with prominent epithelial injury and alveolar fibrosis (e.g., organizing diffuse alveolar damage) might be associated with sequestration of collectins by binding to matrix or serum components. Disorders associated with pulmonary edema, such as mitral valve disease and diseases characterized by impaired clearance (e.g., chronic bronchitis, cystic fibrosis), could conceivably alter the compartmentalization of collectins or permit inhibitory interactions with serum proteins that might compromise their function. The recoveries of lung collectins are reduced by 2- to 3-fold in the lavage from smokers as compared to nonsmokers (104). Thus, the increased risk of respiratory infection associated with smoking and smoking-related disorders such as emphysema and chronic bronchitis might result in part from an acquired collectin deficiency. SP-A and SP-D levels were reported to be decreased in lavage from cystic fibrosis patients (212). However, higher levels of SP-A were observed in another study (213). Diabetes mellitus is associated with an increased risk of respiratory infection by a wide variety of microorganisms (including S. aureas), a number of gram-negative bacteria, Mycobacterium tuberculosis, and various fungi (214). Infections by influenza and streptococci (including Group B streptococcus) have also been associated with increased morbidity or mortality in this setting. It is conceivable that functional impairment of host defense lectins secondary to elevated glucose concentrations or the accumulation of non-enzymatically glycosylated proteins contributes to this increased susceptibility in patients with poorly controlled diabetes. Are There Genetic Deficiency States Involving Lung Collectins? Although no genetic deficiency states of the lung collectins have been described in the literature, it seems likely that they exist. Allelic variants of the human MBP gene have been identified that are associated with an increased risk of infection (215, 216). These “mutations” appear to influence the level of circulating protein as a consequence of interfering with normal folding of the collagen domain and/or oligomerization, and/or by altering promoter gene activity (217, 218). At least one of the allelic forms of MBP is defective in its association with the MBP-associated serine protease (219). It is possible that various alleles of SP-A and SP-D are associated with analogous differences in collectin accumulation or function. Do Collectins Participate in the Pathogenesis of Certain Immune-mediated Lung Disorders? Human SP-A and SP-D have been shown to inhibit allergen-specific IgE binding to mite extracts through lectindependent binding to glycoconjugates on mite allergens

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(13). Given that SP-A also shows lectin-dependent binding to pollens, collectins may influence the host response to a variety of inhaled organic particulate antigens. It seems reasonable to suggest that such interactions could decrease IgE-mediated mast cell activation or modulate allergen sensitization and thereby play some role in the pathogenesis of allergic asthma (13) or other immune-mediated disorders, such as hypersensitivity pneumonitis. Interestingly, the recovery of SP-A from lavage was increased by . 2-fold relative to controls in patients with hypersensitivity pneumonitis or sarcoidosis (220).

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5. 6. 7. 8. 9.

10.

Summary There is growing evidence that the lung collectins SP-A and SP-D modulate important interactions between the host and inhaled microorganisms. There is also considerable circumstantial evidence that these molecules participate in aspects of pulmonary immune and inflammatory regulation. The collectins show calcium-dependent, lectin-mediated interactions with glycoconjugates expressed on a wide variety of microorganisms (and certain complex organic particulates) in vitro. In addition, the collectins can influence the activity of phagocytes through lectin-dependent and -independent interactions. Although both SP-A and SP-D can enhance the internalization and killing of some organisms, in some cases the proteins mediate enhanced binding and/or internalization without associated microbial killing. Trimeric subunits of collectin molecules show specific and high-affinity binding to various saccharide ligands. However, the oligomerization of trimeric subunits is essential for agglutination and other long-range bridging interactions. The actions of SP-A and SP-D may be complementary in vivo. The molecules appear to occupy different airspace compartments, and recognize different ligands or ligand domains. The biologic importance of binding to surfactant lipids is unknown; however, these interactions could primarily serve to orient the molecules in relation to the alveolar lining material or to modify the presentation of other bound materials to pulmonary cells. Much has been learned but many more questions remain.

11. 12. 13.

14.

15. 16. 17. 18. 19. 20. 21.

22.

23. Acknowledgments: The author thanks Dr. Itzhak Ofek for many stimulating discussions during his time in her laboratory as a Visiting Professor and for making available some of his unpublished data regarding SP-D interactions with K. pneumoniae. The author is grateful for long and productive collaborations with Dr. Kevin Hartshorn, which have led to a detailed understanding of the interactions of SP-D and other collectins with influenza A; and with Margot Anders, University of Melbourne, Melbourne, Australia, for collaborative studies correlating interactions of SP-D with influenza A and viral multiplication in vivo. Finally, the author thanks Drs. T. R. Korfhagen (Childrens Hospital Medical Center, Cincinnati, OH) and Larry Schlesinger (University of Iowa, Iowa City, IA); and Janet North for excellent secretarial assistance. The personal studies cited in the review were supported by NIH grants HL44015, and HL29594.

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