Pulmonary Innate Immune Proteins and Receptors that Interact with Gram-positive Bacterial Ligands

Immunobiol. (2002) 205, pp. 575 – 594 © 2002 Urban & Fischer Verlag http://www.urbanfischer.de/journals/immunobiol

MRC Immunochemistry Unit, Department of Biochemistry, Oxford University, South Parks Road, Oxford, United Kingdom

Pulmonary Innate Immune Proteins and Receptors that Interact with Gram-positive Bacterial Ligands NADES PALANIYAR, JEYA NADESALINGAM, and KENNETH B. M. REID

Abstract The two major gram-positive bacterial (GPB) ligands are peptidoglycan (PGN) and lipoteichoic acid (LTA). These polymeric LTA and highly organized PGN contain repeating carbohydrate moieties, which are potential targets for pattern recognition molecules. The major pattern recognition proteins and receptors, which bind GPB, either have a lectin, PGN recognition, collagen or leucine-rich repeat (LRR) domain. The soluble innate immune proteins (IIPs) that bind to PGN and LTA include pulmonary collectins surfactant-associated proteins (SP-) A and D, lectin-like pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP), and sCD14. Membrane-anchored lectin or lectin-like group members include macrophage mannose receptor (MR), complement receptor 3 (CR3, or Mac-1, or integrin CD11b/CD18), scavenger receptor A (SRCL-1), lectin-like oxidized LDL receptor 1 (LOX-1), and GPI-anchored CD14. Although Toll-like receptor (TLR) 2 and 4, and CD14 contain extracellular LRR domains, only TLRs have a cytoplasmic domain for signal transduction. Three of the four recently discovered human PGN recognition proteins (PGRP) have a transmembrane domain, and hence, considered as true receptors for GPB. Since lysozyme is the only known pulmonary enzyme that can lyse bacterial cell wall PGN, other innate immune molecules appear to be responsible for signalling and enhancing the clearance of GPB infection from the lung. Interestingly, pulmonary collectins bind not only to GPB ligands but also to the receptors, CD14 and TLR, and antigen processing cells such as dentritic cells. These complex interactions appear to play major roles in linking innate and adaptive immunity, and maintaining a pathogen-free lung with minimal, or no inflammation. Abbreviations: AMf = alveolar macrophage; BAL = bronchoalveolar lavage; bOBP = bovine oligosaccharide binding protein (bovine PGRP); C1qRp = C1q receptor; COPD = coronary obstructive pulmonary disease; CR3 = complement receptor 3 (Mac-1, or integrin CD11b/CD18); CRD = carbohydrate recognition domains; CRP = C-reactive protein; DC = dentritic cells; DPPC = dipalmitoylphospatidylcholine; Glc = glucose; GlcNAc = N-acetyl Glucosamine; gp-340 = glycoprotein of 340 kDa, an isoform of salivary agglutinin; GPB = gram-positive bacteria; GNB = gram-negative bacteria; GPI = glycosylphosphatidylinositol; HBD = human b defensins; IIP = innate immune protein; LAM = lipoarabinomannan; LOX-1 = lectin-like oxidized LDL receptor 1; LPS = lipopolysaccharide; LRR = leucine-rich domain; LTA = lipoteichoic acid; Mf = macrophage; MARCO = macrophage receptor with collagenous structure; MBL = mannose-binding lectin; MR = mannose receptor; MurNAc = N-acetyl muramic acid; NF-ÍB = nuclear factor-ÍB; PBD = phospholipid-binding domain; PGN = peptidoglycan; PGRP = peptidoglycan recognition protein; PMN = polymorphonuclear leukocyte; PI = phospatidylinositol; SAP = serum amyloid protein; SP-A = surfactant-associated protein A; SP-D = surfactant-associated protein D; SR = scavenger receptor A (SRCL-1); TA = teichoic acid; TLR = toll-like receptor 0171-2985/02/205/04-05-575 $ 15.00/0

576 · N. PALANIYAR et al. Introduction Although many of the proteins and receptors involved in adaptive immune system have been characterized in great detail, only a few molecules in innate immune system have been thoroughly studied. The adaptive immune system uses antibody for the recognition of non-self molecules whereas the innate immune system relies on a range of molecules that recognize repeated patterns found primarily on the microbial surfaces and debris. Both systems, however, employ phagocytes to clear the foreign molecules or unwanted debris from the host. Since dendritic cells (DC) have both phagocytic and efficient antigen processing capabilities, these phagocytes are considered as important players in connecting the first-line defense with the long-term adaptive immunity. Interestingly, many innate immune proteins (IIPs) and receptors are primarily present in tissues that come in contact with the external environment, and inter link these two immune systems. In this review, we focus our attention to the major IIPs that are related to GPB infection, found in alveolar environment of the lungs. Gram-positive bacterial cell wall Common pulmonary GPB pathogens, Staphylococcus aureus and Streptococcus pneumonia, cause pulmonary inflammation, sepsis and pneumonia, and this group of bacteria do not have an outer membrane. Instead, GBP have a characteristic multi-layered cell wall, which primarily contains peptidoglycan (PGN) and lipoteichoic acid (LTA). PGN is an extensively cross-linked polymer that forms a protective network around the bacteria whereas teichoic acid (TA) is a single chain polymer found either linked to PGN or inserted in the plasma membrane with a terminal glycolipid acyl anchor as LTA. Since PGN is made of repeating disaccharide units, it is one of the major targets for pattern recognition molecules. Although LTA is not organized in a manner similar to that of PGN, its presence on the bacterial surface makes it as another likely candidate for recognitions by IIPs. Both PGN and LTA can activate innate immune system and induce the release of inflammatory molecules such as chemokines and cytokines. PGN has many biological activities including cytotoxicity, adjuvant effect and induction of sleep (1). Presence of PGN together with LTA in the serum causes severe disease and death such as the one caused by GPB-induced sepsis (2). Effective clearance of these bacterial ligands is essential to eliminate the synergic effect of these two molecules. Direct evidence for specific binding of IIPs to these ligands are only beginning to emerge. Structure of PGN

Both gram-negative bacteria (GNB) and GPB contain PGN as a cell wall component. GNB bacteria, however, have only a single layer of PGN below the Lipopolysaccharide (LPS)-containing outer membrane whereas GPB contain multiple layers of PGN above the plasma membrane. In GPB, PGN constitute approximately 90% of the cell wall mass, and is directly exposed to the outer environment. PGN from S.aureus consists of a glycan backbone of alternating units of b1 → 4 glycosidic-bonded N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) which are linked to short peptides (4–5 amino acids) via the lactyl group of MurNAc moieties (Fig. 1). The general struc-

Pulmonary innate immune proteins · 577

Figure 1. Structure of PGN and TA. PGN is an extensively cross-linked muramyl peptide polymer. A b(1 → 4) glycosidic bond forms – [GlcNAc-MurNAc]n – disaccharide polymer which is linked to a short peptide via lactyl bond to the MurNAc. A short poly Gly chain interconnects two adjacent peptide chains via a transpeptide bonds at Lys. TA has a phosphate-linked glycerol or ribitol backbone. Ala, short peptides, or carbohydrate moieties such as GlcNAc or Glc are linked to the backbone via an –OH group of the alcohol.

ture of the short peptide in S.aureus is Ala-Glu-Lys-DAla-DAla. A transpeptide bond between the Lys side chain and polyGly terminal of the adjacent peptide forms the regular cross links seen in PGN. The broad-spectrum b-lactam antibiotics such as penicillin inhibits the formation of this transpeptide bond whereas another antibiotic, vancomycin, binds to the D-Ala:D-Ala dipeptide of GPB and prevent their growth. This general structural feature is also seen in GNB PGN, for example Haemophilus influenza contains similar muramyl peptides (3). Although PGN present in GPB cell wall is an insoluble network, its lysis by pulmonary lysozyme releases soluble form of this b-glycan in to the outer environment (4, 5). Therefore, binding of IIPs to insoluble PGN likely to provide opsonic function whereas their interaction with soluble PGN likely to be important to elicit signalling and receptor-mediated response. Structure of LTA

TA is a single chain polymer made of phosphate-linked repeating units of alcohols such as glycerol or ribitol. Frequently, carbohydrate moieties glucose (Glc) or GlcNAc, or small amino acids Ala, or short peptides are linked to the alcohol backbone, and a typical TA chain contains 5–35 repeating units (Fig. 1). Release of free LTA in to aqueous media results in micelle formation (6), and LTA is a potent elicitor of immune response (7). A

578 · N. PALANIYAR et al. repeating phosphate-linked backbone provides LTA with a polyanionic property; and hence, it binds to, and acts as a sink for cations such as Ca2+ and Mg2+. Receptors with positively charged collagen-like domains can interact with LTA by polyionic interactions (8, 9). Carbohydrate moieties found in the repeating units and termini vary among bacterial species and strains; and hence, these moieties are potential targets for binding by collectins. Soluble pattern recognition molecules Bronchial secretions and alveolar surfactant/surface fluid contain several soluble IIPs, and some of which can recognize different types of bacteria and microbes (Fig. 2). The major IIPs that are involved in the clearance of GPB infection in the lungs are: Surfactant-associated proteins (SP-) A and D, lysozyme and small-antimicrobial proteins and peptides. The first complement component C1q and some of the acute phase proteins such as mannose-binding lectin (MBL), C-reactive protein (CRP) and serum amyloid component P (SAP) are other group of proteins involved in innate immune clearance of GPB in lung. In addition, the roles of soluble receptors in GPB infection are becoming clear with the recent focus on PGN recognition protein (PGRP). Surfactant-associated Protein A (SP-A)

SP-A is a C-type lectin, and constitutes about 90% of the surfactant associated proteins. This protein interacts with a variety of lipids but has a high affinity for the major pulmonary surfactant phospholipid, dipalmitoylphospatidylcholine (DPPC). SP-A’s ability to modulate surface tension in the lung is controversial, and animal models with SP-A deficiency suggest that this collectin may not have such function in vivo. See other reviews for more discussion (10–12). Many in vitro and in vivo models suggest that SP-A plays a key role(s) in modulating innate immune function (11). Early studies established that the SP-A concentration in bronchoalveolar lavage (BAL) is related to stress and infections including the one caused by GPB (13) (Table 1). Furthermore, SP-A can bind to, and enhance the phagocytosis of Streptococcus pneumoniae (14, 15), group A Streptococcus (16), and S.aureus, (14, 15, 17), in vitro. Recent studies indicate that the absence of SP-A renders mice more susceptible for microbial infection including GPB (e.g., group B Streptococcus) (18, 19). The exact mechanisms involved in SP-A-mediated bacterial clearance in lung, however, are not fully determined. It is envisaged that SP-A is involved in opsonic activity and/or receptor-mediated interactions with phagocytes. The bouquet of flowers-like quaternary structure of native SP-A (18-mer of a 26–30 kDa chain; 540 kDa) presents all the carbohydrate recognition domains (CRD) in a similar orientation so that the protein can interact with lipid membrane (20) or repeating sugars on microbial surfaces. Multivalent binding of an 18-mer array of CRDs to ligands would lead to a higher affinity interaction compared with that of a weak interaction between a single trimeric CRD and the ligand. SP-A also self assembles to form supraquaternary filaments (21), which provides efficient binding to lipid ligands (22). Interestingly, the phospholipid-binding domain (PBD) of SP-A overlaps with that of lectin domain (23). Therefore, it is conceivable that SP-A is anchored on DPPC, and upon binding to bacterial carbohydrate ligands, it is dislodged from the lipid ligand. Detailed

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Figure 2. Soluble (A) and membrane-anchored innate immune proteins or receptors (B). Soluble IIPs. C1q, 18-mer, 460 kDa, 20–25 nm; MBL, 18-mer, 540 kDa, 32 kDa, ∼20–25 nm; SP-A, 18-mer, 540 kDa, ∼20–25 nm; SP-D, 12-mer, 520 kDa, ∼100 nm; CRP or SAP, 5-mer, 120 kDa, ∼10 nm; Lysozyme, 14 kDa; sCD14, soluble form with no GPI anchor, 48 kDa; PRGP, soluble form with no TM domain, 20 kDa; IgG, 2-mer, 150 kDa, ∼5 nm, included for size comparison. B. Membraneanchored IIPs or receptors. C1qRp or CD93, 126 kDa, *, this receptor may interact with IIPs via an accessory component; CR3 or Mac I or CD11b/CD18, 170 kDa/95 kDa; MARCO, 3-mer, 210 kDa, terminal SRCR domain, ∼80 nm; LOX-1, 1-mer, 40 kDa; SR-CL1, 3-mer, 97–110 kDa; ∼44 nm extracellular domain; MR, 180 kDa, 8–10 CRDs, terminal Cys-rich domain and fibronectin type II domains; TLR2 and 4, extracellular LRR domain, cytoplasmic Toll/IL-1 receptor (TIR) domain, 2 and 4 are structurally similar receptors but can interact with different ligands (TLR2 interacts with PGN, LPS, LAM, zymosan, lipoproteins; TLR4 interacts with LTA, LPS, taxol); CD14, 1-mer, 55 kDa, GPIanchor; PGRP, 3 possible configuration, 35–60 kDa, at least one cytoplasmic PGN recognition domain. IIPs are drawn approximately to scale to show the relative dimensions.

studies are required to determine the differential affinity of SP-A towards DPPC and bacterial surface ligands. Although the interaction between the SP-A and GPB had been known for more than a decade, the surface ligands that interact with the protein have not been determined until

580 · N. PALANIYAR et al. Table 1. Concentrations of IIPs in lung and serum, and their changes during certain disease/distress states. Protein

Concentration

Ref.

a

SP-A

SP-D

BAL (mg/mL)

Serum (ng/mL)

Normal Disease

Normal

Disease

3.5 4.7 15

24.9 26.7

74.0, PAP 32.8, PSS

(122, 123) (124, 125) (13)

461, PAP 104, S 339, IPF 472, IPCD 119, TB 59, A 82, P 81, E 152, PSS

(126) (126) (126) (126) (126) (126) (126) (126) (125)

0.88

39.3, PAP (200 mL) 0.88, ARDS (150 mL) 1.93, P-GPB (120 mL) b 5.54, P-GNB (120 mL) b 19.3, PAP (150 mL) 0.97, S (150 mL) 0.58, IPF (150 mL) 0.62 IPCD (150 mL)

(13) 66

46.4 Gp-340 Lysozyme Lactoferrin sCD14

0.5, PAP 0.77 1.9 0.006 0.010

c

12.6, B (100 mL) 23.1, B (100 mL) 0.011, LT-B 0.058, S (100 mL)

900 121-2239 2400 6200 LT-B

(53) (65, 127) (65, 127) (128) (113)

a

, these concentrations are based on the lavage samples, and actual protein concentrations at the bronchial/alveolar lining would expected to be higher. Values in the parenthesis show BAL sample volume; b , only 30-65 mL of 120 mL was recovered; c, sample volume unknown; A, asthma; ARDS, acute respiratory distress syndrome; B, bronchitis; E, emphysema; LT-B, lung transplant-bronchitis; IPCD, interstitial pneumonia with collagen disease; IPF, interstitial pulmonary fibrosis; P, pneumonia with GPB or GNB; PAP, pulmonary alveolar proteinosis; PSS, progressive systemic sclerosis; S, sarcoidosis; TB, pulmonary tuberculosis.

recently. We and others have considered that SP-A may directly interact with PGN and LTA. Our ELISA results suggest that SP-A may interact with soluble PGN in a calciumindependent manner but has very limited or no binding to LTA (unpublished data). VAN DE WETERING et al. (24) recently reported that SP-A may non-specifically bind insoluble form of PGN but not LTA. MURAKAMI et al. (25) detected no direct interaction between SP-A and insoluble PGN. He further showed that SP-A directly interacts with extracellular domain of Toll-like receptor (TLR) 2 and reduces the binding of PGN to the receptor. These differential interactions, thereby, suppress nuclear factor-ÍB (NFÍ-B) activation and cytokine production, and could subsequently limit the inflammation of lung. SP-A also directly interacts with another PGN receptor CD14 (26) and could reduce pulmonary inflammation in a similar manner. Further studies are required to determine the nature of the interaction between PGN and SP-A, and SP-A-mediated signalling pathways.

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Surfactant-associated protein D (SP-D)

SP-D is the other major collectin found in alveolar environment. Recent reports suggest that SP-D is not only present in the lung but also in many other tissues, particularly, on mucosal surfaces (27–29). This collectin binds phospatidylinositol (PI), a minor phospholipid component of pulmonary surfactant, and hence, SP-D can also be anchored in the alveolus. SP-D also binds pulmonary pathogens and enhances their clearance both in vitro and in vivo (30). This hydrophilic protein can efficiently present the bacterial antigens to DCs, hence links innate immunity with adaptive immunity (31). The mechanism by which SP-D exerts its effect is not entirely similar to that of SP-A, in vivo. SP-D deficiency leads to multiple abnormalities: (i) Alveolar macrophages (AMf) are found in increased numbers, become large and foamy, and generate more reactive oxygen species, and (ii) lung become emphysemic and fibrotic with alveolar lipidosis (32, 33). GPB infection causes high level of pulmonary inflammation in SP-D knockout mice suggesting that SP-D regulates generation of reactive oxygen species, in vivo (34). Although SP-D agglutinates GPB, in vitro, the SP-D knockout mice are capable of effectively clearing pulmonary GPB infection, in vivo. These phenotypes suggest that although SP-A and SP-D have some overlapping functions, they are also involved in significantly different pathways/functions. Our results indicate that SP-D interacts with both of the GPB surface ligands, LTA and soluble form of PGN, isolated from S.aureus (unpublished data). This binding activity is completely abolished in the absence of calcium suggesting that the lectin activity of SP-D is involved in this interaction. VAN DE WETERING et al. (24) recently reported a similar lectin type interaction between SP-D and bacterial ligands LTA and insoluble form of PGN. These new findings suggest that SP-D can efficiently interact with both of the major cell wall components of GPB. The oligomeric structure of native SP-D resembles that of an X (12-mer of a 43 kDa chain; 520 kDa) or asterisks (>12-mer) (35–37). This organization presents the trimeric CRDs (38) of an oligomeric SP-D in different directions. The oligomeric structure of native SP-D is more favorable for agglutination/aggregation of microbes, and this ability of the protein has been well documented. For example, a single trimeric subunit (3-mer of 43 kDa chain, 129 kDa) of SP-D (39) representing a quarter of the whole molecule shown in Figure 2, or a truncated protein with only neck + CRD (3-mer of 22 kDa chain, 66 kDa) has reduced agglutination ability (40). Since SP-D can bind receptors such as CD14 (26) and ligands such as PGN and LTA via its lectin activity, this collectin could effectively cross link GPB and its receptors, in vivo. Mannose-binding lectin (MBL)

MBL is an acute phase response serum collectin secreted primarily by the liver. The oligomeric structure of MBL, from several animals, resembles that of SP-A and C1q, but MBL exhibits varying degrees of oligomerization (ranging from 6-mer to 18-mer of a 32 kDa chain, 540 kDa) and affinity for ligands (35, 41, 42). Unlike SP-A (43) and SP-D, MBL can bring about complement activation. It has been well established that MBL binds many microbes and activates the complement system via the lectin pathway (42, 44). Like lung collectin SP-A, MBL can bind to a PGN receptor CD14 by protein-protein interactions (45). Although MBL binds to GPB and activates complement cascade (46), the ligands on

582 · N. PALANIYAR et al. the GPB have not been fully characterized. MBL binds efficiently to the terminal manosyl moiety found on Micrococcus luteus LTA, but binds weakly to LTA that lack terminal sugars such as the one from S.aureus (47). Hence, MBL appear to interact with the GPB primarily via mannose sugar moieties. Although MBL is usually not detected in normal BAL, influenza viral infection leads to the detection of MBL in the lung (48). This serum collectin may only play a limited role in pulmonary innate immunity. Gp-340

Gp-340 (native Mr ∼1000 kDa) is the pulmonary isoform of salivary agglutinin, and contains multiple scavenger receptor cysteine-rich (SRCR) domains (49–51). Since SRCR domains have no clearly defined functions, biological function of gp-340 is not completely understood. However, gp-340 is present not only in lung but also in many tissues including trachea, salivary glands, small intestine and stomach (28). Several other tissues also show low level of expression. In the human tracheal tissue, gp-340 is localized to Serous cells and submucosal glands, and found as part of respiratory mucus gel-forming mucin MUC5AC or MUC5B (52). Although the genomic sequence of gp-340 contains a transmembrane domain, expression of this domain has not yet been detected. This molecule, however, is detected as a soluble molecule in BAL and found in association with AMf (53). This tissue distribution is reminiscent of a typical innate immune protein. Salivary agglutinin binds to, and agglutinates Streptococcus mutant and several other GPB (51, 54). Like agglutinin, the mouse homolog of gp-340, CRP-ductin, also agglutinates a variety of bacteria including GPB (55); however, the surface ligands for gp-340 are not clearly defined. Interestingly, the globular heads of C1q directly bind to salivary agglutinin and activate complement pathway (56). Since gp-340 interacts with SP-D (53), more complex interactions with pathogens and phagocytes are also possible. Although the function of gp-340 is not clearly defined in the lung, in view of its ability to interact with C1q and SP-D, and what is known about the properties of salivary agglutinin, gp-340 is likely involved in innate immunity. Peptidoglycan recognition protein (PGRP)

PGRP is evolutionarily conserved from fly to humans and show sequence similarity to lysozyme (57); however, PRGP does not have any PGN-lytic activity. Twelve PGRP genes that could express soluble and transmembrane domain-containing proteins, are present in fruit fly, and at least five of them are expressed in different compartments of the body (58). The molecular mass of the protein varies between 19-60 kDa, and the smaller soluble form appears to exist as monomers (59). All of the known isoforms bind PGN and activate cytokine response. In fruit fly, PGRP-LC isoform specifically recognizes the GNB (Escherichia coli) but not GPB suggesting that this protein may also bind to other ligands such as LPS (60). Interestingly, bovine PGPR also kills a fungal pathogen, Cryptococcus neoformans, which dose not contain PGN; hence, this newly identified member is referred to as bovine oligosaccharide binding protein (bOBP) (61). In mammals, PGRP is expressed in circulating polymorphonuclear leukocytes (PMNs) and eosinophils, and other tissues but not in monocytes or Mf (61, 62). Since PMNs and eosinophils contain PGRP, it is likely that the protein is released in to the lung during GPB infection. Our ELISA analysis of the BAL fluid from alveolar proteinosis patients suggests that PGRP is

Pulmonary innate immune proteins · 583

present in alveolar environment (unpublished data). Presence of PGRP mRNA in the lung (63) indicates that this protein is expressed in situ. Since phagocytes can express PGRP, whether the PGRP detected in lung was secreted by those cells or pulmonary type II cells are unknown. However, the presence of PGRP in the lung suggests that this protein is likely involved in the clearance of pulmonary infection of GPB. Lysozyme

Lysozyme is a 14 kDa protein and expressed and secreted by type II cells, and its tissue distribution is similar to that of SP-A (64). It is one of the predominant soluble proteins present in the BAL (65). Lysozyme binds to PGN and digests glycosidic bonds rendering the bacterial cell wall soluble. This process together with the action of other small bacteriostatic IIPs helps to kill GPB (see section below on antimicrobial proteins and factors). The smallest disaccharide unit of PGN, the muramyl peptide, can act as adjuvant suggesting that the soluble form of PGN is sufficient to assist to elicit a high level of adaptive immune response. Soluble PGN generated by lysozyme digestion is involved in signalling pathways that are mediated through TLRs (66). Bacteriocidal activity of lysozyme shows synergistic effect in the presence of lactoferrin, another bacteriostatic protein present in the lung. This effect is attributed to the binding of lactoferrin to LTA while lysozyme digest PGN (4). However, functions of other IIPs and receptors involved in the signalling process are not clearly defined. Hence, lysozyme appears to be the major enzyme directly responsible for solublizing the PGN wall of the GPB whereas the other proteins participate in different aspects of clearing the GPB infection. C1q and complement proteins

The first complement component C1q, and anaphylatoxins C3a (9 kDa) and C5a (9 kDa) have been detected in the lung (67). C1q (18-mer, 460 kDa) is structurally similar to that of SP-A and MBL but consist of 6 copies each of polypeptide chains A (26.5 kDa), B (26.5 kDa) and C (24 kDa). Pulmonary type II cell synthesizes complement proteins C2, C3, C4, C5 and Factor B whereas AMf expresses C2, C3 and Factor B (68). Furthermore, bacterial pneumonia significantly increase concentrations of C1q (from 0.02 mg/mL (almost undetectable) to 0.4–10 mg/mL) and C3 in BAL, with minimal changes in their serum concentrations, which suggests that complement proteins are particularly secreted in the lung during infection (69). However, the relative contributions of these adaptive immune response-related proteins in pulmonary innate immunity are not completely understood. Some reports suggest that C1q can bind GPB in the absence of antibodies (70, 71) and activate complement cascade (72). C1q appear to binds LTA (73), and capsular polysaccharide (70); however, whether C1q binds to PGN is unknown. The iC3b deposited on microbial surfaces can be recognized by innate immune system (see section on CR3); therefore, complement components could communicate with innate immune system via complement receptors in the lung. Interestingly, SP-A can bind C1q and SP-D (74). WATFORD et al., (43) suggest that the weak binding between SP-A and C1q is sufficient to prevent complement activation, and thereby, minimizes the inflammation in lung. Therefore, these protein-protein interactions may regulate the balance between the innate immune systems-based clearance of microbes and complement activation.

584 · N. PALANIYAR et al. Pentraxins

CRP and SAP are acute phase lectin-like pentraxins (non-covalent 5-mer of 24 kDa chains, 120 kDa) (75, 76), and can bind many bacteria and their surface ligands including altered lipids and DNA (77, 78). Like MBL, pentraxins are secreted by liver and released in to serum, however, CRP and SAP are detected in BAL fluid during acute pulmonary infections (79). CRP is also expressed in AMf (80). For example, CRP binds group A Streptococcus pyogenes (81, 82), and its concentration is increased 15 fold during Streptococcus pneumoniae infection in coronary obstructive pulmonary disease (COPD) (83). These homopentameric ring proteins act as “ancient antibodies” that could be recognized by C1q and cleared by classical complement pathway (77, 84). Therefore, CRP and SAP act as innate immune molecules, and exert their function via the well-established complement pathway. Other antimicrobial proteins and factors

Proteins such as lactoferrin, secretory leukoproteinase inhibitor (SLPI), secretory phospholipase A2, human b defensins (HBD) 1 and 2, human neutrophil peptides (HNPs), anionic peptides and cathelicidin LL-37 are found in the lung and other surfaces exposed to external environment. For detailed information on these proteins see other reviews (85, 86). Many of these molecules act both on GPB and GNB, which suggest the existence of multiple modes for ligand binding/interaction with bacteria. Receptors for GPB Two major types of receptors are primarily involved in signalling GPB infections (Fig. 2). The first group of receptors have lectin activity, hence, can directly bind saccharide ligands found on bacterial surfaces. This group of receptors includes mannose receptor (MR), complement receptor 3 (CR3 or Mac-1 or CD11b/CD18 integrin) and some members of the SR family. The second group of receptors contains tandem arrays of leucine-rich repeat (LRR) domains, and this group includes CD14 and TLRs. Presence of a cytoplasmic Toll/Interleukin 1 receptor (TIR) domain suggests that TLR is a classical receptor for signal transduction. Some reports suggest that CD14 also contains lectin-like activity (87, 88), hence, this receptor may exert its function by multiple mechanisms. Macrophage mannose receptor (MR)

MR is a 180 kDa type I transmembrane protein with extracellular multiple (8–10 tandem CRDs) lectin domains (89, 90), and a high affinity saccharide ligand binding of MR is achieved via CRD clustering (90). MR is expressed in Mf, DC and a population of endothelial cells, and this receptor can internalize both soluble and insoluble ligands. MR transduces the signal by inducing the release of leukocyte lysosomal enzymes and cytokines including TNF-a, GM-CSF, IL1, IL6 and IL12, and upregulating FcgR activity. MR not only interacts with mannan but also binds efficiently to GlcNAc (91), hence, this receptor is a potential candidate to interact with PGN.

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Phagocytic complement C1q receptor (C1qRp or CD93)

Human C1qRp is a type I tramsmembrane glycoprotein (126 kDa) that has a C-type CRD, five EGF domains and a short cytoplasmic domain (92). C1qRp is predominantly expressed in human endothelial cells and appear to play a direct role in leukocyte-endothelial cell interaction, which can facilitate the phagocyte migration (93). Although early studies suggested that C1qRp can directly bind collagen-like domains of C1q, MBL and SP-A (92, 94) recent studies suggest that these interactions may be weak or indirect (95). It is now known that the leukocyte antigen CD93 is C1qRp, and is also expressed in monocytes and immature DC (96). A44 (110-130 kDa glycoprotein), the murine homolog of C1qRp, is highly expressed in endothelial and alveolar type II cells but not in Mf (97). Since type II cells are efficient phagocytes (98), C1qRp likely assist in the uptake of particles from the lung. Interestingly, recent studies show that C1qRp directly interacts with S.aureus protein A and enhance its phagocytosis (99). These studies suggest that C1qRp plays a role in innate immunity both via direct interaction with pathogens and by binding with C1q and collectins. Complement receptor 3 (CR3 or CD11b/CD18)

CR3, or Mac-1, is a type III transmembrane integrin composed of heterodimers of CD11b (170 kDa) and CD18 (95 kDa). CR3 is present on monocytes , Mf, PMNs, and DC (100). In addition to its ability to bind extracellular matrix proteins and assist in leukocyte migration, CR3 can also enhance the phagocytosis of iC3b-coated pathogens (101). Interestingly, CR3 also contains lectin activity and binds b-glycans, the long chain b-linked glucose oligosaccharides (102). Upon simultaneous binding to iC3b and b-glycans, CR3 found on leukocytes activates the phagocytosis and induces their degranulation (103). This effect was not found when CR3 binds to iC3b-opsonized targets that does not contain b-glucan. CR3 is essential for phagocytosis of group B streptococcus by human Mf (101, 104), which suggests that this receptor plays key roles in innate immunity. Noticeably, binding of CR3 to Zymosan can be competed with GlcNAc (102), suggesting that CR3 likely to bind PGN, which contains repeating units of GlcNAcMurNAc disaccharides. Scavenger receptor (SR)

Several SRs, with different domain organization, are present on Mf (105). Many SR contain multiple SRCR domains that have no known functions. Typical class A SR (SR-A) contains a transmembrane segment, coiled-coil, collagen, and an optional CRD but has no SRCR domain. SR-A (now known as SR-CL1) has the C-terminal domain organization (CRD:collagen:coiled-coil) opposite to that of typical collectins but it binds to LTA and elicit a cytokine response (106, 107), which eventually lead to the clearance of bacteria in an opsonin-independent manner (9). Another SR-A member, macrophage receptor with collagenous structure (MARCO) (70 kDa), is an 80 nm-long trimeric collagen-like protein expressed in AMf. This receptor has no coiled-coil or CRD but has carbohydrate attachment region (spacer domain III) and SRCR domains (108). MARCO can bind S.aureus and enhances its phagocytosis (109), and its bacterial binding region is located in the vicinity of SRCR domains

586 · N. PALANIYAR et al. (108). MARCO participates in clearing pathogens including S.aureus by AMf (8, 110), indicating its role in pulmonary innate immunity. Lectin-like oxidized LDL receptor-1 (LOX-1) is a member of class E SR (SR-E), and is expressed in human lung, particularly in vascular epithelium and mature AMf. It is a type II transmembrane, or soluble, protein and contains a CRD (111). LOX-1 (40 kDa) can bind S.aureus and enhance their adhesion to leukocytes (112). Since LOX-1 can interact with pathogens in an antibody-independent manner, this protein may also play innate immune function. Taken together, SRs are an important group of innate immune proteins in the alveolar environment. CD14

CD14 is a soluble (48 kDa) or glycosylphosphatidylinositol (GPI)-anchored (55 kDa) glycoprotein. Since CD14 does not contain a cytoplasmic domain, the presence of other receptors is envisaged for classical signal transduction process (see below). GPI-anchored CD14 is expressed and displayed on the surface of monocytes, AMf, DC and PMNs whereas the soluble form is detected both in serum and BAL (113). Both forms of the protein can interact not only with LPS but also with glycan moiety of PGN (87, 114), and induce cell signalling via AP-1 pathway (115, 116). Interestingly, SP-A, SP-D and MBL can bind CD14 (26, 45). SP-A and MBL interact with CD14 via protein-protein interactions but SP-D binds to the carbohydrate moiety of CD14. Since SP-A interacts with CD14 via its hydrophobic neck domain (26), the CRD of the protein is available to bind bacterial ligands. Conversely, since CD14 binds many carbohydrate-based microbial polymers, the protein itself is considered as a lectin (87, 88). Since CD14 can bind bacterial ligands, soluble IIPs and receptors on phagocytes, CD14 plays an important role in innate immune system-mediated clearance of GPB infection. Toll-like receptors (TLRs)

TLRs are type I transmembrane proteins that contain extra cellular LRR domain, and participate in signalling variety of microbial infections, including gram-negative and positive bacterial infection (117). Many ligands including PGN, LTA, LPS, LAM, lipoproteins, and unmethylated DNA with CpG motif activate TLRs. At least 10 TLRs are present in humans, and they appear to control immune and inflammatory response (117). Particularly, TLR 2 and 4 are related to GPB infection signalling/clearing. Interestingly, TLR2 is constitutively expressed by lung epithelial (A549) and monocytic (THP-1) cell lines (118). HARJU et al. (119) recently showed that expression of both TLR2 and 4, in human lung, are developmentally regulated, and TLR mRNA concentrations reaches a maximum after the birth of an infant. This development-dependent expression of TLR suggests that this group of receptors is important in infant life, when the adaptive immune system is not fully developed. TLR2 binds primarily to PGN, LTA, LPS and bacterial lipoproteins (66, 120) whereas TLR4 interacts with LTA and LPS (117). Furthermore, TLR2 upregulates the expression of HBD-2 in response to bacterial lipoproteins (118). Typically, extracellular domain of TLRs bind various bacterial ligands, in association with other binding proteins, transmit signal via the cytoplasmic TIR domain and results in the activation of NF-ÍB pathway (121). A recent report suggests that SP-A can directly interact with TLR2 and reduce the

Pulmonary innate immune proteins · 587

interaction between PGN and the receptor, which results in the inhibition of PGN-induced TNF-a production by U937 monocytic cell line (25). Therefore, SP-A may be one of the important molecules that maintain low levels of inflammation during bacterial infection in the lung. Conclusion Several IIPs present in the lung and serum bind primarily to PGN and LTA on GPB cell wall. The concentration and the acute response vary among these IIPs. It is logical to think that the lysozyme, a PGN-lytic enzyme, is responsible for the digestion of insoluble PGN found on GPB (also on GNB). Since lysozyme is an enzyme, reasonably low concentrations of it would be sufficient to exert its function. On the contrary, opsonic or pattern recognition proteins would expected to be at a higher concentrations to effectively signals the clearance of whole or fragment of a pathogen. Typically, a protein that has a CRD with a relatively low affinity for a given saccharide would require an oligomeric state to bind with high affinity so that it could act as a pattern recognition molecule. Lung collectins are oligomeric proteins, and would satisfy this requirement. SP-A is found at higher concentration than most other proteins in the lung, and can self assemble to form supraquaternary structures, which could further enhance the affinity of this protein for arrays of repeating ligands. Conversely, SP-D is present at low concentrations but appears to increase under certain infections or allergic antigen insult. The concentration of wellstudied acute phase proteins, MBL, CRP and SAP are increased upon infection, and therefore, could be present in sufficient quantities to act as pattern recognition molecules at high microbial load situations. Most of the IIPs are present as soluble proteins but could interact with phagocytes, thereby; could exert its innate immune function. TLR2 is anchored on the membrane of type II cells and Mf so that they could initiate the signalling process using conventional pathways. Since gp-340 (pulmonary isoform of salivary agglutinin) is present in the lung, and interacts with AMf membrane, C1q and SP-D, this protein may also be involved in innate immunity. Many of the pathways involved in these interactions are not clearly understood and therefore require detailed experimentations to dissect the signalling mechanisms and clearance of GPB in lung. Acknowledgements

This work was supported by EU/MRC-UK research grant QLK2 1999 00325 (K. B. M.R and J.N) and Postdoctoral fellowships from the Wellcome Trust-UK/CIHR-Canada (N.P).

References 1. JOHANNSEN, L. 1993. Biological properties of bacterial peptidoglycan. Apmis 101: 337. 2. DE KIMPE, S. J., M. KENGATHARAN, C. THIEMERMANN, and J. R. VANE. 1995. The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure. Proc. Natl. Acad. Sci. USA 92: 10359. 3. BURROUGHS, M., E. ROZDZINSKI, S. GEELEN, and E. TUOMANEN. 1993. A structure-activity relationship for induction of meningeal inflammation by muramyl peptides. J. Clin. Invest. 92: 297.

588 · N. PALANIYAR et al. 4. LEITCH, E. C., and M. D. WILLCOX. 1999. Elucidation of the antistaphylococcal action of lactoferrin and lysozyme. J. Med. Microbiol. 48: 867. 5. AKINBI, H. T., R. EPAUD, H. BHATT, and T. E. WEAVER 2000. Bacterial killing is enhanced by expression of lysozyme in the lungs of transgenic mice. J. Immunol. 165: 5760. 6. FISCHER, W., S. MARKWITZ, and H. LABISCHINSKI. 1997. Small-angle X-ray scattering analysis of pneumococcal lipoteichoic acid phase structure. Eur. J. Biochem. 244: 913. 7. MORATH, S., A. GEYER, and T. HARTUNG. 2001. Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus. J. Exp. Med. 193: 393. 8. PALECANDA, A., J. PAULAUSKIS, E. AL-MUTAIRI, A. IMRICH, G. QIN, H. SUZUKI, T. KODAMA, K. TRYGGVASON, H. KOZIEL, and L. KOBZIK. 1999. Role of the scavenger receptor MARCO in alveolar macrophage binding of unopsonized environmental particles. J. Exp. Med. 189: 1497. 9. THOMAS, C. A., Y. LI, T. KODAMA, H. SUZUKI, S. C. SILVERSTEIN, and J. EL KHOURY. 2000. Protection from lethal gram-positive infection by macrophage scavenger receptor-dependent phagocytosis. J. Exp. Med. 191: 147. 10. PALANIYAR, N., M. IKEGAMI, T. KORFHAGEN, J. WHITSETT, and F. X. MCCORMACK. 2001. Domains of surfactant protein A that affect protein oligomerization, lipid structure and surface tension. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 129: 109. 11. LEVINE, A. M., and J. A. WHITSETT. 2001. Pulmonary collectins and innate host defense of the lung. Microbes Infect. 3: 161. 12. MCCORMACK, F. X., and J. A. WHITSETT. 2002. The pulmonary collectins, SP-A and SP-D, orchestrate innate immunity in the lung. J. Clin. Invest. 109: 707. 13. BAUGHMAN, R. P., R. I. STERNBERG, W. HULL, J. A. BUCHSBAUM, and J. WHITSETT. 1993. Decreased surfactant protein A in patients with bacterial pneumonia. Am. Rev. Respir. Dis. 147: 653. 14. MCNEELY, T. B., and J. D. COONROD. 1993. Comparison of the opsonic activity of human surfactant protein A for Staphylococcus aureus and Streptococcus pneumoniae with rabbit and human macrophages. J. Infect. Dis. 167: 91. 15. HARTSHORN, K. L., E. CROUCH, M. R. WHITE, M. L. COLAMUSSI, A. KAKKANATT, B. TAUBER, V. SHEPHERD, and K. N. SASTRY. 1998. Pulmonary surfactant proteins A and D enhance neutrophil uptake of bacteria. Am. J. Physiol. 274: L958. 16. TINO, M. J., and J. R. WRIGHT. 1996. Surfactant protein A stimulates phagocytosis of specific pulmonary pathogens by alveolar macrophages. Am. J. Physiol. 270: L677. 17. VAN IWAARDEN, F., B. WELMERS, J. VERHOEF, H. P. HAAGSMAN, and L. M. VAN GOLDE. 1990. Pulmonary surfactant protein A enhances the host-defense mechanism of rat alveolar macrophages. Am. J. Respir. Cell. Mol. Biol. 2: 91. 18. LEVINE, A. M., M. D. BRUNO, K. M. HUELSMAN, G. F. ROSS, J. A. WHITSETT, and T. R. KORFHAGEN. 1997. Surfactant protein A-deficient mice are susceptible to group B streptococcal infection. J. Immunol. 158: 4336. 19. LEVINE, A. M., K. E. KURAK, J. R. WRIGHT, W. T. WATFORD, M. D. BRUNO, G. F. ROSS, J. A. WHITSETT, and T. R. KORFHAGEN. 1999. Surfactant protein-A binds group B streptococcus enhancing phagocytosis and clearance from lungs of surfactant protein-A-deficient mice. Am. J. Respir. Cell. Mol. Biol. 20: 279. 20. PALANIYAR, N., R. A. RIDSDALE, C. E. HOLTERMAN, K. INCHLEY, F. POSSMAYER, and G. HARAUZ. 1998. Structural changes of surfactant protein A induced by cations reorient the protein on lipid bilayers. J. Struct. Biol. 122: 297. 21. PALANIYAR, N., R. A. RIDSDALE, F. POSSMAYER, and G. HARAUZ. 1998. Surfactant protein A (SP-A) forms a novel supraquaternary structure in the form of fibers. Biochem. Biophys. Res. Commun. 250: 131. 22. PALANIYAR, N., R. A. RIDSDALE, S. A. HEARN, Y. M. HENG, F. P. OTTENSMEYER, F. POSSMAYER, and G. HARAUZ. 1999. Filaments of surfactant protein A specifically interact with corrugated surfaces of phospholipid membranes. Am. J. Physiol. 276: L631. 23. PALANIYAR, N., F. X. MCCORMACK, F. POSSMAYER, and G. HARAUZ. 2000. Three-dimensional structure of rat surfactant protein A trimers in association with phospholipid monolayers. Biochemistry 39: 6310.

Pulmonary innate immune proteins · 589 24. VAN DE WETERING, J. K., M. VAN EIJK, L. M. VAN GOLDE, T. HARTUNG, J. A. VAN STRIJP, and J. J. BATENBURG. 2001. Characteristics of surfactant protein A and D binding to lipoteichoic acid and peptidoglycan, 2 major cell wall components of gram-positive bacteria. J. Infect. Dis. 184: 1143. 25. MURAKAMI, S., D. IWAKI, H. MITSUZAWA, H. SANO, H. TAKAHASHI, D. R. VOELKER, T. AKINO, and Y. KUROKI. 2002. Surfactant protein A inhibits peptidoglycan-induced tumor necrosis factoralpha secretion in U937 cells and alveolar macrophages by direct interaction with toll-like receptor 2. J. Biol. Chem. 277: 6830. 26. SANO, H., H. CHIBA, D. IWAKI, H. SOHMA, D. R. VOELKER, and Y. KUROKI. 2000. Surfactant proteins A and D bind CD14 by different mechanisms. J. Biol. Chem. 275: 22442. 27. MIYAMURA, K., R. MALHOTRA, H. J. HOPPE, K. B. REID, P. J. PHIZACKERLEY, P. MACPHERSON, and A. LOPEZ BERNAL. 1994. Surfactant proteins A (SP-A) and D (SP-D): levels in human amniotic fluid and localization in the fetal membranes. Biochim. Biophys. Acta 1210: 303. 28. MADSEN, J., A. KLIEM, I. TORNOE, K. SKJODT, C. KOCH, and U. HOLMSKOV. 2000. Localization of lung surfactant protein D on mucosal surfaces in human tissues. J. Immunol. 164: 5866. 29. VAN ROZENDAAL, B. A., L. M. VAN GOLDE, and H. P. HAAGSMAN. 2001. Localization and functions of SP-A and SP-D at mucosal surfaces. Pediatr. Pathol. Mol. Med. 20: 319. 30. REID, K. B. 1998. Interactions of surfactant protein D with pathogens, allergens and phagocytes. Biochim. Biophys. Acta 1408: 290. 31. BRINKER, K. G., E. MARTIN, P. BORRON, E. MOSTAGHEL, C. DOYLE, C. V. HARDING, and J. R. WRIGHT. 2001. Surfactant protein D enhances bacterial antigen presentation by bone marrowderived dendritic cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 281: L1453. 32. BOTAS, C., F. POULAIN, J. AKIYAMA, C. BROWN, L. ALLEN, J. GOERKE, J. CLEMENTS, E. CARLSON, A. M. GILLESPIE, C. EPSTEIN, and S. HAWGOOD. 1998. Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D. Proc. Natl. Acad. Sci. USA 95: 11869. 33. KORFHAGEN, T. R., V. SHEFTELYEVICH, M. S. BURHANS, M. D. BRUNO, G. F. ROSS, S. E. WERT, M. T. STAHLMAN, A. H. JOBE, M. IKEGAMI, J. A. WHITSETT, and J. H. FISHER. 1998. Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo. J. Biol. Chem. 273: 28438. 34. LEVINE, A. M., J. A. WHITSETT, J. A. GWOZDZ, T. R. RICHARDSON, J. H. FISHER, M. S. BURHANS, and T. R. KORFHAGEN. 2000. Distinct effects of surfactant protein A or D deficiency during bacterial infection on the lung. J. Immunol. 165: 3934. 35. LU, J., H. WIEDEMANN, R. TIMPL, and K. B. REID. 1993. Similarity in structure between C1q and the collectins as judged by electron microscopy. Behring Inst. Mitt. 6: 16. 36. CROUCH, E., D. CHANG, K. RUST, A. PERSSON, and J. HEUSER. 1994. Recombinant pulmonary surfactant protein D. Post-translational modification and molecular assembly. J. Biol. Chem. 269: 15808. 37. HOLMSKOV, U., S. B. LAURSEN, R. MALHOTRA, H. WIEDEMANN, R. TIMPL, G. R. STUART, I. TORNOE, P. S. MADSEN, K. B. REID, and J. C. JENSENIUS. 1995. Comparative study of the structural and functional properties of a bovine plasma C-type lectin, collectin-43, with other collectins. Biochem. J. 305 (Pt 3): 889. 38. HAKANSSON, K., N. K. LIM, H. J. HOPPE, and K. B. REID. 1999. Crystal structure of the trimeric alpha-helical coiled-coil and the three lectin domains of human lung surfactant protein D. Structure Fold. Des. 7: 255. 39. HARTSHORN, K., D. CHANG, K. RUST, M. WHITE, J. HEUSER, and E. CROUCH. 1996. Interactions of recombinant human pulmonary surfactant protein D and SP-D multimers with influenza A. Am. J. Physiol. 271: L753. 40. EDA, S., Y. SUZUKI, T. KAWAI, K. OHTANI, T. KASE, Y. FUJINAGA, T. SAKAMOTO, T. KURIMURA, and N. WAKAMIYA. 1997. Structure of a truncated human surfactant protein D is less effective in agglutinating bacteria than the native structure and fails to inhibit haemagglutination by influenza A virus. Biochem. J. 323 (Pt 2): 393. 41. WALLIS, R., and J. Y. CHENG. 1999. Molecular defects in variant forms of mannose-binding protein associated with immunodeficiency. J. Immunol. 163: 4953.

590 · N. PALANIYAR et al. 42. LU, J. H., S. THIEL, H. WIEDEMANN, R. TIMPL, and K. B. REID. 1990. Binding of the pentamer/ hexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2C1s2 complex, of the classical pathway of complement, without involvement of C1q. J. Immunol. 144: 2287. 43. WATFORD, W. T., J. R. WRIGHT, C. G. HESTER, H. JIANG, and M. M. FRANK. 2001. Surfactant protein A regulates complement activation. J. Immunol. 167: 6593. 44. WALLIS, R., and K. DRICKAMER. 1999. Molecular determinants of oligomer formation and complement fixation in mannose-binding proteins. J. Biol. Chem. 274: 3580. 45. CHIBA, H., H. SANO, D. IWAKI, S. MURAKAMI, H. MITSUZAWA, T. TAKAHASHI, M. KONISHI, H. TAKAHASHI, and Y. KUROKI. 2001. Rat mannose-binding protein a binds CD14. Infect. Immun. 69: 1587. 46. NETH, O., D. L. JACK, A. W. DODDS, H. HOLZEL, N. J. KLEIN, and M. W. TURNER. 2000. Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect. Immun. 68: 688. 47. POLOTSKY, V. Y., W. FISCHER, R. A. EZEKOWITZ, and K. A. JOINER. 1996. Interactions of human mannose-binding protein with lipoteichoic acids. Infect. Immun. 64: 380. 48. READING, P. C., L. S. MOREY, E. C. CROUCH, and E. M. ANDERS. 1997. Collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice. J. Virol. 71: 8204. 49. HOLMSKOV, U. L. 2000. Collectins and collectin receptors in innate immunity. APMIS Suppl. 100: 1. 50. HOLMSKOV, U., J. MOLLENHAUER, J. MADSEN, L. VITVED, J. GRONLUND, I. TORNOE, A. KLIEM, K. B. REID, A. POUSTKA, and K. SKJODT. 1999. Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D. Proc. Natl. Acad. Sci. USA 96: 10794. 51. LIGTENBERG, T. J., F. J. BIKKER, J. GROENINK, I. TORNOE, R. LETH-LARSEN, E. C. VEERMAN, A. V. NIEUW AMERONGEN, and U. HOLMSKOV. 2001. Human salivary agglutinin binds to lung surfactant protein-D and is identical with scavenger receptor protein gp-340. Biochem. J. 359: 243. 52. THORNTON, D. J., J. R. DAVIES, S. KIRKHAM, A. GAUTREY, N. KHAN, P. S. RICHARDSON, and J. K. SHEEHAN. 2001. Identification of a nonmucin glycoprotein (gp-340) from a purified respiratory mucin preparation: evidence for an association involving the MUC5B mucin. Glycobiology 11: 969. 53. HOLMSKOV, U., P. LAWSON, B. TEISNER, I. TORNOE, A. C. WILLIS, C. MORGAN, C. KOCH, and K. B. REID. 1997. Isolation and characterization of a new member of the scavenger receptor superfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-D binding molecule. J. Biol. Chem. 272: 13743. 54. PRAKOBPHOL, A., F. XU, V. M. HOANG, T. LARSSON, J. BERGSTROM, I. JOHANSSON, L. FRANGSMYR, U. HOLMSKOV, H. LEFFLER, C. NILSSON, T. BOREN, J. R. WRIGHT, N. STROMBERG, and S. J. FISHER. 2000. Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340. J. Biol. Chem. 275: 39860. 55. MADSEN, J., I. TORNOE, O. NIELSEN, I. KREBS, J. MOLLENHAUER, A. POUSTKS, K. SKODT, and U. HOLMSKOV. 2001. CRP-ductin, the mouse homolog of gp-340/DMBT1 binds specifically to lung surfactant protein D (SP-D), Haemophilus influenzae and Klebsiella oxytoca. Intl. Workshop on C1 and Collectins Mainz 4: 13. 56. BOACKLE, R. J., M. H. CONNOR, and J. VESELY. 1993. High molecular weight non-immunoglobulin salivary agglutinins (NIA) bind C1Q globular heads and have the potential to activate the first complement component. Mol. Immunol. 30: 309. 57. KANG, D., G. LIU, A. LUNDSTROM, E. GELIUS, and H. STEINER. 1998. A peptidoglycan recognition protein in innate immunity conserved from insects to humans. Proc. Natl. Acad. Sci. USA 95: 10078. 58. WERNER, T., G. LIU, D. KANG, S. EKENGREN, H. STEINER, and D. HULTMARK. 2000. A family of peptidoglycan recognition proteins in the fruit fly Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 97: 13772. 59. YOSHIDA, H., K. KINOSHITA, and M. ASHIDA. 1996. Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori. J. Biol. Chem. 271: 13854.

Pulmonary innate immune proteins · 591 60. RAMET, M., P. MANFRUELLI, A. PEARSON, B. MATHEY-PREVOT, and R. A. EZEKOWITZ. 2002. Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416: 644. 61. TYDELL, C. C., N. Y. YOUNT, D. TRAN, J. YUAN, and M. E. SELSTED. 2002. Isolation, characterization, and antimicrobial properties of bovine oligosaccharide binding protein: a microbicidal granule protein of eosinophils and neutrophils. J. Biol. Chem. (in press). 62. LIU, C., E. GELIUS, G. LIU, H. STEINER, and R. DZIARSKI. 2000. Mammalian peptidoglycan recognition protein binds peptidoglycan with high affinity, is expressed in neutrophils, and inhibits bacterial growth. J. Biol. Chem. 275: 24490. 63. KISELEV, S. L., O. S. KUSTIKOVA, E. V. KOROBKO, E. B. PROKHORTCHOUK, A. A. KABISHEV, E. M. LUKANIDIN, and G. P. GEORGIEV. 1998. Molecular cloning and characterization of the mouse tag7 gene encoding a novel cytokine. J. Biol. Chem. 273: 18633. 64. GIBSON, K. F., and S. PHADKE. 1994. Intracellular distribution of lysozyme in rat alveolar type II epithelial cells. Exp. Lung. Res. 20: 595. 65. THOMPSON, A. B., T. BOHLING, F. PAYVANDI, and S. I. RENNARD. 1990. Lower respiratory tract lactoferrin and lysozyme arise primarily in the airways and are elevated in association with chronic bronchitis. J. Lab. Clin. Med. 115: 148. 66. SCHWANDNER, R., R. DZIARSKI, H. WESCHE, M. ROTHE, and C. J. KIRSCHNING. 1999. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J. Biol. Chem. 274: 17406. 67. WATFORD, W. T., A. J. GHIO, and J. R. WRIGHT. 2000. Complement-mediated host defense in the lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 279: L790. 68. STRUNK, R. C., D. M. EIDLEN, and R. J. MASON. 1988. Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways. J. Clin. Invest. 81: 1419. 69. SODA, K., M. ANDO, T. SAKATA, M. SUGIMOTO, H. NAKASHIMA, and S. ARAKI. 1988. C1q and C3 in bronchoalveolar lavage fluid from patients with summer-type hypersensitivity pneumonitis. Chest 93: 76. 70. LEVY, N. J., and D. L. KASPER. 1985. Antibody-independent and -dependent opsonization of group B Streptococcus requires the first component of complement C1. Infect. Immun. 49: 19. 71. KOROLEVA, I. V., A. G. SJOHOLM, and C. SCHALEN. 1998. Binding of complement subcomponent C1q to Streptococcus pyogenes: evidence for interactions with the M5 and FcRA76 proteins. FEMS Immunol. Med. Microbiol. 20: 11. 72. LEVY, N. J., and D. L. KASPER. 1986. Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway. J. Immunol. 136: 4157. 73. LOOS, M., F. CLAS, and W. FISCHER. 1986. Interaction of purified lipoteichoic acid with the classical complement pathway. Infect. Immun. 53: 595. 74. OOSTING, R. S., and J. R. WRIGHT. 1994. Characterization of the surfactant protein A receptor: cell and ligand specificity. Am. J. Physiol. 267: L165. 75. SRINIVASAN, N., H. E. WHITE, J. EMSLEY, S. P. WOOD, M. B. PEPYS, and T. L. BLUNDELL. 1994. Comparative analyses of pentraxins: implications for protomer assembly and ligand binding. Structure 2: 1017. 76. EMSLEY, J., H. E. WHITE, B. P. O’HARA, G. OLIVA, N. SRINIVASAN, I. J. TICKLE, T. L. BLUNDELL, M. B. PEPYS, and S. P. WOOD. 1994. Structure of pentameric human serum amyloid P component. Nature 367: 338. 77. HICKS, P. S., L. SAUNERO-NAVA, T. W. DU CLOS, and C. MOLD. 1992. Serum amyloid P component binds to histones and activates the classical complement pathway. J. Immunol. 149: 3689. 78. ROBEY, F. A., K. D. JONES, T. TANAKA, and T. Y. LIU. 1984. Binding of C-reactive protein to chromatin and nucleosome core particles. A possible physiological role of C-reactive protein. J. Biol. Chem. 259: 7311. 79. HOHENADEL, I. A., M. KIWORR, R. GENITSARIOTIS, D. ZEIDLER, and J. LORENZ. 2001. Role of bronchoalveolar lavage in immunocompromised patients with pneumonia treated with a broad spectrum antibiotic and antifungal regimen. Thorax 56: 115.

592 · N. PALANIYAR et al. 80. DONG, Q., and J. R. WRIGHT. 1996. Expression of C-reactive protein by alveolar macrophages. J. Immunol. 156: 4815. 81. HIND, C. R., P. M. COLLINS, M. L. BALTZ, and M. B. PEPYS. 1985. Human serum amyloid P component, a circulating lectin with specificity for the cyclic 4,6-pyruvate acetal of galactose. Interactions with various bacteria. Biochem. J. 225: 107. 82. NOURSADEGHI, M., M. C. BICKERSTAFF, J. R. GALLIMORE, J. HERBERT, J. COHEN, and M. B. PEPYS. 2000. Role of serum amyloid P component in bacterial infection: protection of the host or protection of the pathogen. Proc. Natl. Acad. Sci. USA 97: 14584. 83. DEV, D., E. WALLACE, R. SANKARAN, J. CUNNIFFE, J. R. GOVAN, C. G. WATHEN, and F. X. EMMANUEL. 1998. Value of C-reactive protein measurements in exacerbations of chronic obstructive pulmonary disease. Respir. Med. 92: 664. 84. DU CLOS, T. W. 2000. Function of C-reactive protein. Ann. Med. 32: 274. 85. TRAVIS, S. M., P. K. SINGH, and M. J. WELSH. 2001. Antimicrobial peptides and proteins in the innate defense of the airway surface. Curr. Opin. Immunol. 13: 89. 86. GANZ, T. 2002. Antimicrobial polypeptides in host defense of the respiratory tract. J. Clin. Invest. 109: 693. 87. WEIDEMANN, B., J. SCHLETTER, R. DZIARSKI, S. KUSUMOTO, F. STELTER, E. T. RIETSCHEL, H. D. FLAD, and A. J. ULMER. 1997. Specific binding of soluble peptidoglycan and muramyldipeptide to CD14 on human monocytes. Infect. Immun. 65: 858. 88. RIETSCHEL, E. T., J. SCHLETTER, B. WEIDEMANN, V. EL-SAMALOUTI, T. MATTERN, U. ZAHRINGER, U. SEYDEL, H. BRADE, H. D. FLAD, S. KUSUMOTO, D. GUPTA, R. DZIARSKI, and A. J. ULMER. 1998. Lipopolysaccharide and peptidoglycan: CD14-dependent bacterial inducers of inflammation. Microb. Drug. Resist. 4: 37. 89. TAYLOR, M. E., J. T. CONARY, M. R. LENNARTZ, P. D. STAHL, and K. DRICKAMER. 1990. Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. J. Biol. Chem. 265: 12156. 90. TAYLOR, M. E., K. BEZOUSKA, and K. DRICKAMER. 1992. Contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor. J. Biol. Chem. 267: 1719. 91. MULLIN, N. P., P. G. HITCHEN, and M. E. TAYLOR. 1997. Mechanism of Ca2+ and monosaccharide binding to a C-type carbohydrate-recognition domain of the macrophage mannose receptor. J. Biol. Chem. 272: 5668. 92. NEPOMUCENO, R. R., A. H. HENSCHEN-EDMAN, W. H. BURGESS, and A. J. TENNER. 1997. cDNA cloning and primary structure analysis of C1qR(P), the human C1q/MBL/SPA receptor that mediates enhanced phagocytosis in vitro. Immunity 6: 119. 93. FONSECA, M. I., P. M. CARPENTER, M. PARK, G. PALMARINI, E. L. NELSON, and A. J. TENNER. 2001. C1qR(P), a myeloid cell receptor in blood, is predominantly expressed on endothelial cells in human tissue. J. Leukoc. Biol. 70: 793. 94. GEERTSMA, M. F., P. H. NIBBERING, H. P. HAAGSMAN, M. R. DAHA, and R. VAN FURTH. 1994. Binding of surfactant protein A to C1q receptors mediates phagocytosis of Staphylococcus aureus by monocytes. Am. J. Physiol. 267: L578. 95. REID, K. B., M. COLOMB, F. PETRY, and M. LOOS. 2002. Complement component C1 and the collectins – first-line defense molecules in innate and acquired immunity. Trends Immunol. 23: 115. 96. STEINBERGER, P., A. SZEKERES, S. WILLE, J. STOCKL, N. SELENKO, E. PRAGER, G. STAFFLER, O. MADIC, H. STOCKINGER, and W. KNAPP. 2002. Identification of human CD93 as the phagocytic C1q receptor (C1qRp) by expression cloning. J. Leukoc. Biol. 71: 133. 97. DEAN, Y. D., E. P. MCGREAL, and P. GASQUE. 2001. Endothelial cells, megakaryoblasts, platelets and alveolar epithelial cells express abundant levels of the mouse AA4 antigen, a C-type lectin-like receptor involved in homing activities and innate immune host defense. Eur J. Immunol. 31: 1370. 98. FEHRENBACH, H. 2001. Alveolar epithelial type II cell: defender of the alveolus revisited. Respir. Res. 2: 33. 99. NGUYEN, T., B. GHEBREHIWET, and E. I. PEERSCHKE. 2000. Staphylococcus aureus protein A recognizes platelet gC1qR/p33: a novel mechanism for staphylococcal interactions with platelets. Infect. Immun. 68: 2061.

Pulmonary innate immune proteins · 593 100. VASTA, G. R., M. QUESENBERRY, H. AHMED, and N. O’LEARY. 1999. C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway. Dev. Comp. Immunol. 23: 401. 101. LE CABEC, V., C. COLS, and I. MARIDONNEAU-PARINI. 2000. Nonopsonic phagocytosis of zymosan and Mycobacterium kansasii by CR3 (CD11b/CD18) involves distinct molecular determinants and is or is not coupled with NADPH oxidase activation. Infect. Immun. 68: 4736. 102. THORNTON, B. P., V. VETVICKA, M. PITMAN, R. C. GOLDMAN, and G. D. ROSS. 1996. Analysis of the sugar specificity and molecular location of the beta-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18). J. Immunol. 156: 1235. 103. XIA, Y., V. VETVICKA, J. YAN, M. HANIKYROVA, T. MAYADAS, and G. D. ROSS. 1999. The betaglucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells. J. Immunol. 162: 2281. 104. GOODRUM, K. J., L. L. MCCORMICK, and B. SCHNEIDER. 1994. Group B streptococcus-induced nitric oxide production in murine macrophages is CR3 (CD11b/CD18) dependent. Infect. Immun. 62: 3102. 105. PEISER, L., S. MUKHOPADHYAY, and S. GORDON. 2002. Scavenger receptors in innate immunity. Curr. Opin. Immunol. 14: 123. 106. DUNNE, D. W., D. RESNICK, J. GREENBERG, M. KRIEGER, and K. A. JOINER. 1994. The type I macrophage scavenger receptor binds to gram-positive bacteria and recognizes lipoteichoic acid. Proc. Natl. Acad. Sci. USA 91: 1863. 107. GREENBERG, J. W., W. FISCHER, and K. A. JOINER. 1996. Influence of lipoteichoic acid structure on recognition by the macrophage scavenger receptor. Infect. Immun. 64: 3318. 108. ELOMAA, O., M. SANKALA, T. PIKKARAINEN, U. BERGMANN, A. TUUTTILA, A. RAATIKAINENAHOKAS, H. SARIOLA, and K. TRYGGVASON. 1998. Structure of the human macrophage MARCO receptor and characterization of its bacteria-binding region. J. Biol. Chem. 273: 4530. 109. ELSHOURBAGY, N. A., X. LI, J. TERRETT, S. VANHORN, M. S. GROSS, J. E. ADAMOU, K. M. ANDERSON, C. L. WEBB, and P. G. LYSKO. 2000. Molecular characterization of a human scavenger receptor, human MARCO. Eur. J. Biochem. 267: 919. 110. PALECANDA, A., and L. KOBZIK. 2001. Receptors for unopsonized particles: the role of alveolar macrophage scavenger receptors. Curr. Mol. Med. 1: 589. 111. MURASE, T., N. KUME, H. KATAOKA, M. MINAMI, T. SAWAMURA, T. MASAKI, and T. KITA. 2000. Identification of soluble forms of lectin-like oxidized LDL receptor-1. Arterioscler. Thromb. Vasc. Biol. 20: 715. 112. SHIMAOKA, T., N. KUME, M. MINAMI, K. HAYASHIDA, T. SAWAMURA, T. KITA, and S. YONEHARA. 2001. LOX-1 supports adhesion of Gram-positive and Gram-negative bacteria. J. Immunol. 166: 5108. 113. STRIZ, I., L. ZHENG, Y. M. WANG, H. POKORNA, P. C. BAUER, and U. COSTABEL. 1995. Soluble CD14 is increased in bronchoalveolar lavage of active sarcoidosis and correlates with alveolar macrophage membrane-bound CD14. Am. J. Respir. Crit. Care Med. 151: 544. 114. DZIARSKI, R., R. I. TAPPING, and P. S. TOBIAS. 1998. Binding of bacterial peptidoglycan to CD14. J. Biol. Chem. 273: 8680. 115. GUPTA, D., Q. WANG, C. VINSON, and R. DZIARSKI. 1999. Bacterial peptidoglycan induces CD14-dependent activation of transcription factors CREB/ATF and AP-1. J. Biol. Chem. 274: 14012. 116. JIN, Y., D. GUPTA, and R. DZIARSKI. 1998. Endothelial and epithelial cells do not respond to complexes of peptidoglycan with soluble CD14 but are activated indirectly by peptidoglycaninduced tumor necrosis factor-alpha and interleukin-1 from monocytes. J. Infect. Dis. 177: 1629. 117. AKIRA, S., K. TAKEDA, and T. KAISHO. 2001. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2: 675. 118. BIRCHLER, T., R. SEIBL, K. BUCHNER, S. LOELIGER, R. SEGER, J. P. HOSSLE, A. AGUZZI, and R. P. LAUENER. 2001. Human Toll-like receptor 2 mediates induction of the antimicrobial peptide human beta-defensin 2 in response to bacterial lipoprotein. Eur. J. Immunol. 31: 3131.

594 · N. PALANIYAR et al. 119. HARJU, K., V. GLUMOFF, and M. HALLMAN. 2001. Ontogeny of Toll-like receptors Tlr2 and Tlr4 in mice. Pediatr. Res. 49: 81. 120. YOSHIMURA, A., E. LIEN, R. R. INGALLS, E. TUOMANEN, R. DZIARSKI, and D. GOLENBOCK. 1999. Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J. Immunol. 163: 1. 121. MUSHEGIAN, A., and R. MEDZHITOV. 2001. Evolutionary perspective on innate immune recognition. J. Cell. Biol. 155: 705. 122. HONDA, Y., H. TAKAHASHI, N. SHIJUBO, Y. KUROKI, and T. AKINO. 1993. Surfactant proteinA concentration in bronchoalveolar lavage fluids of patients with pulmonary alveolar proteinosis. Chest 103: 496. 123. KUROKI, Y., H. TAKAHASHI, H. CHIBA, and T. AKINO. 1998. Surfactant proteins A and D: disease markers. Biochim. Biophys. Acta 1408: 334. 124. GREENE, K. E., J. R. WRIGHT, K. P. STEINBERG, J. T. RUZINSKI, E. CALDWELL, W. B. WONG, W. HULL, J. A. WHITSETT, T. AKINO, Y. KUROKI, H. NAGAE, L. D. HUDSON, and T. R. MARTIN. 1999. Serial changes in surfactant-associated proteins in lung and serum before and after onset of ARDS. Am. J. Respir. Crit. Care Med. 160: 1843. 125. TAKAHASHI, H., Y. KUROKI, H. TANAKA, T. SAITO, K. KUROKAWA, H. CHIBA, A. SAGAWA, H. NAGAE, and S. ABE. 2000. Serum levels of surfactant proteins A and D are useful biomarkers for interstitial lung disease in patients with progressive systemic sclerosis. Am. J. Respir. Crit. Care Med. 162: 258. 126. HONDA, Y., Y. KUROKI, E. MATSUURA, H. NAGAE, H. TAKAHASHI, T. AKINO, and S. ABE. 1995. Pulmonary surfactant protein D in sera and bronchoalveolar lavage fluids. Am. J. Respir. Crit. Care Med. 152: 1860. 127. TORSTEINSDOTTIR, I., L. HAKANSSON, R. HALLGREN, B. GUDBJORNSSON, N. G. ARVIDSON, and P. VENGE. 1999. Serum lysozyme: a potential marker of monocyte/macrophage activity in rheumatoid arthritis. Rheumatology (Oxford) 38: 1249. 128. WARD, C., E. H. WALTERS, L. ZHENG, H. WHITFORD, T. J. WILLIAMS, and G. I. SNELL. 2002. Increased soluble CD14 in bronchoalveolar lavage fluid of stable lung transplant recipients. Eur. Respir. J. 19: 472. Professor K. B. M. REID, MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK. Tel: xx44-(0)-1865-275353, Fax: xx44-(0)1865-275729; e-mail: [email protected]