key: cord-0828029-wn1gxhk9
authors: Dommett, R. M.; Klein, N; Turner, M. W.
title: Mannose‐binding lectin in innate immunity: past, present and future
date: 2006-09-01
journal: Tissue Antigens
DOI: 10.1111/j.1399-0039.2006.00649.x
sha: 1468d0bcf3c567c4b3a7cb87cea974c8642f9669
doc_id: 828029
cord_uid: wn1gxhk9

The human collectin, mannose‐binding lectin (MBL), is an important protein of the humoral innate immune system. With multiple carbohydrate‐recognition domains, it is able to bind to sugar groups displayed on the surfaces of a wide range of microorganisms and thereby provide first‐line defence. Importantly, it also activates the complement system through a distinctive third pathway, independent of both antibody and the C1 complex. Three single point mutations in exon 1 of the expressed human MBL‐2 gene appear to impair the generation of functional oligomers. Such deficiencies of functional protein are common in certain populations, e.g. in sub‐Saharan Africa, but virtually absent in others, e.g. indigenous Australians. MBL disease association studies have been a fruitful area of research and implicate a role for MBL in infective, inflammatory and autoimmune disease processes. Overall, there appears to be a genetic balance in which individuals generally benefit from high levels of the protein. However, in certain situations, reduced levels of circulating MBL may be beneficial to the host and this may explain the persistence of the deleterious gene polymorphisms in many population groups.

It is now 60 years since the Australian Nobel prize winner Sir Frank Macfarlane Burnet, together with John McCrea, identified three inhibitors in serum (called a, b and g), which were able to inactivate influenza virus (1) . We now know that the b inhibitor was, in fact, a protein called mannosebinding lectin (MBL), a component of the innate immune system (2) . During the past 30 years, our understanding of this protein has steadily increased as a result of extensive research activity in three main areas: (a) bio/immunochemistry (including molecular genetics), (b) microbiology and (c) immunodeficiency. Work in these areas initially proceeded independently as evidence for both an inexplicable biological function and a clinical deficiency state emerged. The isolation and characterization of the protein were necessary in order to illuminate the observations of the so-called RaRf bactericidal activity (3, 4) in the microbiology area and the opsonic deficiency reported in many paediatric populations. Some of the main developments are summarized in Table 1 . This review briefly addresses issues relating to the early history of MBL, its structure, function, genetics and disease associations. Finally, future developments including the potential use of both plasma-derived and recombinant MBL are discussed.

The existence of mammalian serum lectins was first predicted in 1975 by Robinson et al. (5) , and the protein was first isolated in 1978 from cytosolic fractions of rabbit liver by Kawasaki et al. (6) . Subsequently, Wild et al. (7) were able to isolate MBL from both human and rat liver. More recently, extrahepatic transcription of MBL has been reported and this may have implications regarding its role in localized host defence (8) .

MBL belongs to a family of proteins called the collectins, which possess both collagenous regions and lectin domains. The other major human collectins, surfactant protein A and surfactant protein D, possess structural characteristics similar to those of MBL and are found predominantly in the lung and other mucosal sites (9) . Plasma-associated phagocytic defect (28) 1975 Existence of mammalian serum Ôlectin-like proteins specific for mannoseÕ predicted (5) 1976 Association of opsonic defect with frequent infections in infancy, but deficiency also present in 5% of the general population (29) 1978 MBL isolated from rabbit liver (6) 1980 Opsonic deficiency in infants with chronic diarrhoea (30) 1981 Association of yeast opsonization defect with suboptimal C3b deposition (31) 1982 Description of mouse RaRF: a complement-activating bactericidal protein (3) 1983 Human MBL isolated from liver (7); human serum MBL isolated (121) Prospective study of opsonic deficiency in infancy (122) 1984 RaRF activity present in vertebrate classes (4) 1985 Bovine serum MBL described (123) Opsonic defect linked to absence of an unidentified co-factor of the complement system (124) 1987 MBL activation of classical complement pathway (18) 1988 Rat serum MBL A and C described (125) ; description of C-type CRD (126) 1989 Gene for human MBL cloned (33, 34) ; human MBL has bactericidal activity (127) ; opsonic nature of MBL demonstrated (35) MBL inhibits in vitro infection by HIV (85) Correlation of opsonic defect with low serum MBL levels (32) 1990

Bovine and mouse serum b inhibitors of influenza A virus identified as MBL (2) Correlation of MBL levels with classical complement pathway activation at low serum concentrations (128) 1991 Mouse MBL A and C described (129) Opsonic deficiency and low MBL levels linked to single point mutation in codon 54 (variant B) (50) 1992 Human MBL levels in acute-phase responses (59) ; crystallography of MBL CRD (130) ; novel protease (MASP-1) and complement activation by MBL (19) Human RaRF identical to MBL-MASP (131) Low MBL levels in Africans linked to codon 57 (variant C) mutation in the MBL gene (51) 1994

Third MBL mutation in codon 52 (variant D) described (52) 1995 Polymorphisms found in promoter region of MBL gene (55) 1997 Second MASP found to activate complement (20) MBL mutations are an important risk factor for infections in children (132) 1998 Reconstitution of opsonizing activity by infusion of purified MBL into MBL-deficient humans (112) 1999 Truncated form of MASP-2 -MAp19 (21) 2000 Complement-activating complex of ficolins and MASP (133) MBL shown to bind to clinically relevant organisms (15) Structural aspects of MBL

The protein structure of MBL has been studied extensively, and aspects are presented in Figures 1 and 2 . The protein consists of multimers of an identical polypeptide chain of 32 kDa. Each chain comprises four distinct regions encoded by different exons of the MBL-2 gene, as will be discussed in more detail later.

Each chain has a C-terminal, calcium-dependent carbohydrate-recognition domain (CRD); a short, a-helical, hydrophobic neck region (in the so-called coiled-coil configuration); a collagenous region containing 19 Gly-Xaa-Xaa triplets and a cysteine-rich N-terminal region. Three polypeptide chains form a triple helix within the collagenous region, stabilized by hydrophobic interactions and interchain disulphide bonds within the N-terminal cysteine-rich region. This is the basic building block of all circulating molecular forms of MBL. In serum, MBL consists of oligomers ranging from dimers to hexamers, and X-ray crystallographic studies/electron micrographs have revealed that these oligomers have a sertiform or a bouquetlike structure due to an interruption in the collagenous region, giving rise to a kink/hinge. The ability of the protein to bind effectively to microorganisms and activate complement appears to depend on the presence of higher order oligomers (tetramers and above). Work by Drickamer and colleagues (10, 11) and also by Ezekowitz and colleagues (12) has provided an insight into the structure of the CRD. Each CRD binds a calcium ion, enabling it to form co-ordination bonds with the 3-and 4-hydroxyl groups of specific sugars including mannose, N-acetyl-d-glucosamine, N-acetyl-mannosamine, fucose and glucose. The three CRDs in each structural subunit are separated by a constant 45-Å distance (12) . Clustering of the structural subunits provides a flat platform, permitting binding of MBL to the arrays of repeating sugar groups on microbial surfaces. Although the binding affinity of each individual CRD-sugar interaction is relatively low at 10 23 M (13), the formation of higher order oligomers provides multiple CRDs, which are able to bind simultaneously with high avidity.

MBL is a major pattern-recognition molecule of the innate immune system. It primarily recognizes specific sugar groups (as above) on the surface of microorganisms, enabling it to distinguish self from non-self. It can also bind to phospholipids, nucleic acids (14) and non-glycosylated proteins. MBL has been shown to bind promiscuously to a wide range of bacteria, viruses, fungi and protozoa and some selected examples are listed in Table 2 .

Neth et al. used flow cytometry to demonstrate MBL binding to clinically relevant bacterial isolates from immunocompromised children and noted differences in binding within some species such that one isolate might show strong binding, whereas another was much weaker (15) . The role of specific structural features of microorganisms (e.g. the capsule), which permit or prevent binding to MBL, has been explored in several studies. The earliest work was probably by Kawakami et al. on the socalled RaRf complex (which was later identified as MBL) and its interaction with Salmonella enterica serovar Typhimurium (3). This suggested that the structure and composition of lipopolysaccharide play a crucial role in MBL binding and function. Other mechanisms that enable microorganisms to avoid recognition and killing by MBL include lipooligosaccharide sialyation (16, 17) . Despite much progress in this area, many puzzles remain to be addressed, mostly related to the exact disposition of sugars on microbial surfaces.

Our understanding of MBL function has grown rapidly over the past three decades. It is now recognized to have a role in processes as diverse as complement activation, promotion of complement-independent opsonophagocytosis, modulation of inflammation, recognition of altered self-structures and apoptotic cell clearance.

A role for MBL in host defence was first proposed in 1987 when Ikeda et al. observed that the protein was able to activate the classical pathway of complement (18) . However, it is now clear that MBL activates a novel third pathway of complement, often termed the MBL pathway, in an antibody-and C1-independent fashion as illustrated in Figure 3 . This functional activity reflects the fact that MBL circulates in association with a group of MBL-associated serine proteases (the so-called MASPs). In 1992, Matsushita and Fujita demonstrated the presence of a novel complement enzyme in serum, which was thought to generate the C3 convertase (C4bC2a), associated with classical pathway activation (19) . However, this activity was later found to be mediated by MASP-2 (20) , and the original enzyme is now known as MASP-1 and may activate C3 directly. Subsequently, a small separately synthesized fragment of MASP-2 termed sMAP or Map19 was identified (21, 22) and a third MASP (MASP-3) with no known function was also described (23) .

Current understanding suggests that on binding to microorganisms, autoactivation of MASP-2 occurs, permitting cleavage of C4 and C2 to form a C3 convertase, which is indistinguishable in specificity from the convertases found in the other two activation pathways of complement (24) .

It should be noted that the so-called MBL pathway is also activated by another family of proteins called ficolins. The ficolins are structurally similar to collectins, with collagenous domains linked to fibrinogen-like domains having sugar-binding properties. L-and H-ficolins are humoral factors synthesized by hepatocytes, although H-ficolin has also been observed in bronchial/alveolar fluid and in bile (25) . In contrast, M-ficolin is found on peripheral blood mononuclear cells, polymorphonuclear cells and type II lung epithelial cells (26) . Ficolins are also found in complexes with the MASPs and are considered to have different binding specificities compared with MBL (27) .

In 1968, Miller et al. reported a plasma-associated defect of phagocytosis in a child with severe recurrent infections, failure to thrive and diarrhoea (28) . In vitro work revealed a failure of the childÕs plasma to opsonize heat-killed bakers yeast (Saccharomyces cerevisiae). This defect was later detected in the sera of children with recurrent unexplained infections (29) and chronic diarrhoea of infancy (30), but, interestingly, studies in the general population also revealed a relatively high frequency of the defect (5%). In 1981, studies linked this opsonic deficiency to the complement Figure 3 Complement activation pathway. The lectin pathway of complement is activated by MBL and ficolins. On binding to appropriate targets, MBL-MASP-2 complexes cleave C4 and C2 to form C3 convertase (C4bC2a). MBL-MASP-1 complexes may activate C3 directly. Ficolins also work in combination with the MASPs. The classical and alternative pathways also generate C3 convertase enzymes, which cleave C3. The lytic pathway (C5-C9) is common to all three routes of C3 cleavage. MBL, mannose-binding lectin; MASP, MBL-associated serine proteases; MASP-1, MBL-associated serine protease-2; MASP-2, MBLassociated serine protease-2.

system by demonstrating that sera with the deficiency deposited less C3b on yeast surfaces (31) . However, it was not until 1989 that the common opsonic defect was found to be associated with low levels of the mannose-binding protein, which we now refer to as MBL (32) . In that same year, the gene for MBL was cloned (33, 34) (Genetics of Human MBL).

In a study of MBL-coated Salmonella montevideo, Kuhlman et al. reported that MBL was able to interact directly with cell surface receptors and promote opsonophagocytosis (35) . Subsequently, a number of putative MBL-binding proteins/receptors have been proposed including cC1qR/ calreticulin (36), C1qRp (37) and CR1 (38, 39) . However, it is unclear whether MBL is acting as a direct opsonin or is merely enhancing other complement pathways and/or antibody-mediated phagocytosis.

The role of MBL as a modulator of inflammation appears to be complex and, accordingly, its mechanism of action remains unexplained. One possible explanation is that MBL is able to trigger proinflammatory cytokine release from monocytes (40, 41) . This concept was addressed in studies by Jack et al. using Neisseria meningitidis incubated with increasing concentrations of MBL before being added to MBL-deficient whole blood. Release of tumour necrosis factor a, interleukin (IL)-1b and IL-6 from monocytes was enhanced at MBL concentrations below 4 mg/ml but suppressed at higher concentrations (42) . Clinical studies in this area are discussed later.

The role of MBL in the recognition of altered self and apoptosis A role for MBL in the clearance of apoptotic cells was first proposed by Ogden et al. in 2001 (43) . MBL was found to bind directly to apoptotic cells that expose terminal sugars of cytoskeletal proteins, thereby permitting their recognition and directly facilitating their phagocytosis by macrophages. Defects in the clearance of apoptotic cells have been implicated in the pathogenesis of certain autoimmune conditions, although the precise role of MBL, if any, remains elusive. For example, in 2005, Stuart et al. reported that although MBL-deficient mice displayed defective apoptotic cell clearance, they did not develop autoimmune diseases (44) .

In animal studies, MBL has been implicated in the pathophysiology of ischaemia reperfusion injury due to its ability to recognize altered self-structures. Stahl and colleagues have proposed the lectin pathway as a mediator of this process in certain organs, and the absence of MBL/MASP pathway activation appears to afford protection in these disease models (45, 46) . However, the relevance of these findings to human health needs to be established.

Changes in cell surface structures during oncogenic transformation appear to promote binding of MBL to cancer cells (47) where the protein can mediate cytotoxic effects including MBL-dependent cell mediated cytotoxicity (48, 49) . The relative importance of such mechanisms in tumour immunology is, at present, unknown.

There are two human MBL genes, but MBL-1 is a pseudogene and only MBL-2 encodes a protein product. The functional MBL-2 gene is located on chromosome 10 (q11.2-q21) and comprises four exons as illustrated in Figure 1 . Exon 1 encodes the signal peptide, a cysteine-rich region and part of the glycine-rich collagenous region. Exon 2 encodes the remainder of the collagenous region and exon 3 encodes an a-helical coiled-coil structure, which is known as the ÔneckÕ region. Exon 4 encodes the CRD, which adopts a globular configuration. The promoter region of the MBL gene contains a number of regulatory elements, which affect transcription of the protein.

In 1991, the complete nucleotide sequence of all four exons of the human MBL-2 gene was determined by Sumiya et al. in two British children with recurrent infections and low MBL levels (50) . In both individuals, a point mutation was observed in codon 54, changing the codon sequence from GGC to GAC and substituting aspartic acid for glycine in the translated protein. Familial studies confirmed that the defect was inherited in an autosomal dominant fashion. In 1992, Lipscombe et al. identified a second exon 1 mutation in codon 57 (Gly / Glu), when studying a sub-Saharan African population (51) , and in 1994, Madsen et al. reported a mutation in codon 52 (Arg / Cys) (52) . These point mutations are now commonly referred to as variants B, C and D respectively, with variant A indicating the wild type. The B variant mutation occurs at a gene frequency of approximately 25% in Eurasian populations. In contrast, the C variant is rare in Eurasians but is commonly seen in sub-Saharan African populations, with frequencies of 50%-60%. Population studies suggest that the B variant mutation may have arisen between 50,000 and 20,000 years ago (53) since no structural gene mutations have been identified in studies of indigenous Australian populations who arrived on the continent approximately 50,000 years ago, whereas the B variant mutation was probably introduced into both North and South America at the time of the last glaciation approximately 20,000 years ago.

The effect of these exon 1 mutations on the protein product continues to be the focus of study. They are believed to impair oligomerization and lead to a functional deficiency. The B and C mutations result in the replacement of critical axial glycines in the triple helix by dicarboxylic acids, resulting in distortion of this important part of the protein (50) . In contrast, the D mutation results in the replacement of arginine with cysteine. This extra cysteine has been proposed to cause formation of adventitious disulphide bonds that hinder higher oligomer formation (54) .

Several polymorphisms have also been reported in the promoter region of the gene. Studies by Madsen et al. investigating the large interindividual variation in serum MBL levels revealed three polymorphisms, H/L, X/Y and P/Q at positions 2550, 2221 and 14 of the MBL gene (55, 56) . Subsequently, four common haplotypes were identified, namely LXP, LYP, LYQ and HYP. Of these, HYP, which is associated with medium to high levels of MBL and LXP, which is associated with low levels of the protein, appear to be most important. These promoter haplotypes are in strong linkage disequilibrium with the exon 1 mutations, resulting in seven common extended haplotypes, namely HYPA, LYPA, LYQA, LXPA, HYPD, LYPB and LYQC. Other rare haplotypes have also been described (57) . Figure 4 illustrates the frequency of these various haplotypes in selected populations and highlights the degree of ethnic variation.

The combination of structural gene and promoter polymorphisms results in a dramatic variation in MBL concentration in apparently healthy individuals of up to 1000-fold (Caucasian: range <20-10,000 ng/ml). In addition, Ezekowitz and colleagues presented evidence in 1988 that MBL was an acute-phase reactant (58) . In these investigations, RNA was isolated from a ÔnormalÕ liver taken as part of a staging biopsy for Hodgkins disease and was compared with RNA isolated from a fresh post-mortem liver of a victim with severe trauma. The authors found that MBL messenger RNA transcripts were barely detectable in normal liver but that induction was seen in liver exposed to acute stress. Subsequent studies have shown that MBL levels can increase between 1.5 and threefold during the acute phase, but this response is variable between individuals (59) . It should also be noted that even during an acutephase response, individuals heterozygous or homozygous for MBL mutations appear unable to achieve the protein levels of those possessing a wild-type genotype. Approximately one-third of the Caucasian population possess genotypes conferring low levels of MBL, with approximately 5% having very low levels. No absolute level of MBL deficiency has been defined. Genotype and phenotype show a relatively strong correlation and studies often use just one measure to infer deficiency. However, there is Ôadded valueÕ in performing both measures and we would strongly advocate this approach whenever possible.

MBL occurs in two distinct forms in rodents and rhesus monkeys (60), but only one form is found in humans and chickens. As discussed previously, there are two human MBL genes, which are most likely due to a gene duplication event (61) . However, MBL-1 is a pseudogene and the potential mechanisms responsible for silencing the MBL-1 (63). Such substitutions were also found in other higher primates including chimpanzees and gorillas but not in more distant primates such as the rhesus monkey. The authors concluded that both the MBL-1 and the MBL-2 genes have been selectively silenced by the same molecular mechanisms, but skewed in time resulting in overall downregulation of MBL levels in the present human population.

The high frequency of variant alleles observed in certain populations was initially puzzling since it suggests that functional MBL deficiency may well be advantageous. Similarities have been proposed between the MBL genetic system and the role of the sickle cell gene in protection against malaria as occurs in carriers of the sickle cell haemoglobin allele (64) . The argument runs as follows: certain intracellular parasites use C3 opsonization and C3 receptors on monocytes/macrophages to enter their host. Therefore, any reduction in complement-activating function of the host may reduce the probability of parasitization. In support of this notion is a study on patients with visceral leishmaniasis, which revealed that such patients are more likely to have high MBL levels than uninfected controls (65) . A small study of Ethiopian patients with lepromatous or borderline lepromatous leprosy also found that their MBL levels were significantly higher than those of healthy blood donors (66) . An alternative explanation of the unexpectedly high frequency of low MBL phenotype individuals found in many tropical regions is that excessive complement activation can result in immunopathologically mediated host damage; therefore, any mechanism that reduces complement activation may be beneficial (51) .

The identification of MBL deficiency as the cause of the so-called common opsonic defect has been followed by a plethora of disease association studies aimed at defining the precise role of this protein. A number of the early studies concentrated on paediatric populations and MBL was suggested to provide substitute ÔantibodyÕ-like activity during the Ôwindow of vulnerabilityÕ (approximately 6-24 months), when maternal immunoglobulin G (IgG) antibody levels have waned but the infantÕs own adaptive immune response is still immature (32) . Nevertheless, studies in adults suggested that there might be a role for MBL throughout life (67) . Notwithstanding these reports, the majority of individuals possessing a variant MBL allele apparently suffer no ill effects and remain essentially healthy. In a study that apparently confirms this, Dahl et al. monitored 9245 adults in a Danish Caucasian population and found no evidence for significant differences in infectious disease or mortality in MBL-deficient individuals compared with controls (68) . Similar findings were reported by Tacx et al. in unselected adults admitted to hospital with infections (69) . Nevertheless, these studies should not be regarded as proof that MBL levels have no clinical relevance. Many groups have undertaken case-control studies, which do indeed suggest that MBL is an important immunological modulator. In some cases, there is evidence that the significance of MBL deficiency is more readily appreciated when there is another co-existing defect (70), as we first proposed in 1991 (71).

Space does not permit a comprehensive review of all the MBL clinical studies that have been undertaken to date, and the topics covered below have been selected in order to illustrate examples of possible roles for MBL in a variety of clinical situations.

Most studies have explored the role of MBL in relation to the acquisition of an infectious organism (susceptibility) and the nature of the associated clinical course (severity). In clinical practice, this distinction can be difficult. However, for the purposes of this review, we will highlight examples of infections in which MBL appears to have an influence on one or other of these two aspects of infectious diseases.

Hamvas et al. have recently shown a role for MBL in mycoplasma infection (72) . They studied cases of infection in patients with primary antibody deficiencies (PAD) that are known to be particularly susceptible to such organisms and compared them with a control population. More than two-thirds of PAD patients with mycoplasma infections were MBL deficient (in possession of an exon 1 variant allele) compared with one-third of the control group. In the same study, they were able to demonstrate binding of MBL to three strains of Mycoplasma using flow cytometry and proposed a role for MBL in prevention of invasive disease.

In 2003, severe acute respiratory syndrome (SARS) emerged as a highly infectious disease caused by a novel coronavirus (SARS-CoV). It provided a new challenge to previously unexposed individuals predominantly in Asia. Specific antibodies to SARS-CoV could be detected 10 days after the onset of symptoms, making sufferers reliant on innate immune mechanisms during the early phase of infection. Since the structure of the virus was rapidly established (73, 74) , it also became clear that this novel infectious agent was rich in the sugars known to be targeted by MBL and it was hypothesized that this lectin might well be involved in first-line defence against this infection. Subsequent studies found significant differences in the distribution of MBL-deficient genotypes in patients with SARS compared with those in controls (75, 76) . These studies suggested that MBL plays a role in susceptibility to the infection but does not influence subsequent severity. In their investigations, Ip et al. were also able to demonstrate binding of MBL to the virus and its ability to inhibit infection (75) . (77). The B variant allele was found more commonly in patients with symptomatic hepatitis B cirrhosis and in those with spontaneous bacterial peritonitis. It was also noted that MBL levels were lower in this patient cohort with chronic infection. Screening for MBL mutations in such patients was suggested in order to enable identification of those at increased risk of complications who may benefit from prophylactic antibiotic treatment. In 2005, Chong et al. also reported that MBL genotypes correlating with low protein levels were associated with the occurrence of cirrhosis and also hepatocellular carcinoma in hepatitis B carriers (78) . They also demonstrated that MBL is able to bind hepatitis B surface antigen. In the same year, Thio et al. published the results of a nested case-control study of 527 patients who had either naturally recovered from hepatitis B (n ¼ 338) or had persistent infection (n ¼ 189). They found that MBL genotypes correlating with high serum levels were associated with recovery from infection, whereas those correlating with lower levels were associated with persistence of the virus (79) . It should be noted that approximately half of the subjects were also infected with human immunodeficiency virus (HIV), but the authors concluded that this did not influence the results obtained. Matsushita et al. investigated the influence of MBL mutations in hepatitis C infection and found that sufferers who were homozygous for B variant alleles were less likely to respond to interferon treatment (80) . Further work would be warranted in order to define the role of MBL in the pathogenesis of hepatitis infection.

Secondary immunodeficiencies due to disease or treatment have provided interesting patient populations within which to study the role of MBL. One such group comprises those receiving chemotherapy for malignancy. These patients are rendered neutropenic by their treatment (or underlying disease process) and are subsequently at increased risk of infectious complications. In 2001, two studies were published reporting an effect of MBL deficiency in such patients. Neth et al. studied 100 children and measured MBL levels and genotype. Children in possession of MBL variant alleles spent twice as many days in hospital with febrile neutropenia during the first 6 months of their treatment compared with wild-type individuals (81) . In the other study, Peterslund et al. followed 54 adults undergoing chemotherapy for various haematological malignancies and found that those who developed ÔsignificantÕ infections (bacteraemia, pneumonia or both) in the 3-week periods post-treatment had significantly lower levels of MBL compared with those without significant infections (82) . Subsequent studies have shown differing results, but drawing comparisons between them is inherently difficult. These patients are a highly heterogeneous population, with different underlying disease processes, undergoing treatment regimens of differing intensity, resulting in various degrees of immunosuppression. In one contrasting study, Bergmann et al. followed 80 adults undergoing therapy for acute myeloid leukaemia, which involves intense highly myelosuppressive treatment. They found no effect of MBL deficiency on frequency, severity or duration of fever and suggested that the nature of the treatment overwhelmed any potential influence of MBL (83) . Further clinical studies in such patients are required in order to delineate the exact role of MBL.

An MBL double-knockout mouse model has been used to explore the above clinical conundrum. In 2004, Shi et al. demonstrated that MBL null mice were highly susceptible to intravenous inoculation with Staphylococcus aureus, all dying within 48 h, compared with 55% survival of MBL wild-type mice. However, when the mice were inoculated via the intraperitoneal route and rendered neutropenic (using cyclophosphamide), neutropenic MBL null mice were found to have higher accumulations of bacteria in the blood and organs compared with neutropenic wild-type mice. By day 8 post-infection, the neutropenic wild-type mice had cleared their blood, but the neutropenic MBL null mice had persistent bacteraemia. The authors were able to reverse the phenotype by treating the MBL null mice with recombinant MBL (84) .

To date, nearly 40 million humans have been infected with HIV. The clinical consequences of viral exposure are variable. Some individuals can be repeatedly exposed to the virus but remain free from infection. Others can be infected but remain free from clinical disease. While numerous viral and host factors will determine the fate of an individual exposed to HIV, there are data to indicate that MBL can influence both susceptibility and severity of HIV infection.

The likely target for HIV binding is the heavily glycosylated glycoprotein, gp120. While MBL can be readily demonstrated to bind to purified gp120 (85) , the capacity of MBL to neutralize primary HIV isolates is less convincing. Recent data indicate the MBL can opsonize HIV but does not induce neutralization at the levels at which it is normally present in serum. However, binding and opsonization of HIV by MBL may alter virus trafficking and viral antigen presentation during HIV infection. MBL may influence uptake by dendritic cells (DC), which express a cell surface lectin called ÔDC-specific intracellular adhesion molecule 3-grabbing non-integrinÕ (DC-SIGN). DC-SIGN has been shown to mediate a type of infection called ÔtransÕ-infection, where DC bind HIV and efficiently transfer the virus to T cells. Preincubation of HIV strains with MBL prevents DC-SIGN-mediated trans-infection of T cells and indicates that at least in vitro, MBL may inhibit DC-SIGN-mediated uptake and spread of HIV (86) .

Whatever the mechanism of MBL interactions with HIV, a number of clinical studies have suggested that deficiency of MBL is a risk factor for acquiring HIV infection.

MBL deficiency appears to increase the acquisition of HIV infection by between three-and eightfold (87) (88) (89) (90) . There is also an increased risk of vertical transmission from infected mothers to their offspring (91) . However, these findings have not been replicated in all populations, with some studies failing to demonstrate a role for MBL in HIV infection (92) (93) (94) . There is even less clarity with regard to the role of MBL in HIV disease progression. Garred et al. (87) demonstrated that men with MBL variant alleles had a shorter survival time following the onset of acquired immune deficiency syndrome (AIDS) than did patients with wild-type MBL alleles. However, in a well-characterized cohort of homosexual men, variant MBL alleles had an insignificant effect on survival following the diagnosis of AIDS (95) . In this latter study, there appeared to be a protective effect of MBL variant alleles, with a delay in the development of AIDS from the time of HIV seroconversion. Patients with MBL variant alleles had lower CD4 counts at the time of developing AIDS, indicating that MBL deficiency may influence the onset of AIDS for any given CD4 count. Furthermore, MBL mutations appeared to protect against the development of Kaposi sarcoma, a finding that was difficult to explain (95) . In another study, Prohaszka et al. (90) found that MBL levels were lower in asymptomatic HIV-positive individuals compared with HIV-negative controls. However, the protective effect of MBL was lost in patients with an AIDS diagnosis; patients with high MBL levels had significantly lower numbers of CD4 cells. A possible explanation is that enhanced proinflammatory cytokine production in advanced HIV disease acts to increase MBL synthesis (96) , elevating levels in patients with late-stage disease. Indeed, a recent study has shown in vitro that MBL can enhance proinflammatory cytokine production and viral replication (97) . In the light of studies indicating a role for MBL in inflammatory modulation, it is tempting to suggest that under some circumstances, MBL may act to promote inflammatory cell activation, thereby accelerating the rate of CD41 T-cell depletion.

Few studies have assessed the impact of MBL in the context of effective antiviral therapy. However, one study has attempted to relate MBL status and HIV-infected longterm non-progressors (LTNPs) (98) . MBL levels were consistent with a wild-type genotype in the six LTNPs studied. Amoroso and colleagues had also suggested such an effect in a study showing that children with rapidly progressing disease were more likely to have MBL variant alleles (codon 54) than slower progressors (99) .

Cystic fibrosis provides an example of a clinical condition where MBL appears to be exerting its role as an infection susceptibility gene and inflammatory modulator. Garred et al. were the first group to report that patients with MBL variant alleles have significantly impaired lung function and decreased life expectancy in comparison with wild-type individuals (100) . The effect of MBL deficiency on the severity of lung disease was most apparent in patients with chronic Pseudomonas aeruginosa infection and it was also found that Burkholderia cepacia infection was more common in patients with MBL deficiency. In 2004, Davies et al. reported that an effect of MBL was only seen in adults homozygous for MBL mutations. These patients had significantly reduced lung function, more frequent hospital admissions and raised systemic inflammatory markers. However, there was no evidence of increased susceptibility to Burkholderia cepacia and Pseudomonas aeruginosa (101) . Whether MBL has an effect on early colonization with Burkholderia cepacia and Pseudomonas aeruginosa or subsequent secondary viral infections or whether there is an (anti)inflammatory effect on subsequent lung damage remains unclear.

Clinical studies of critically ill patients requiring intensive care management have shown that individuals who are MBL deficient are more likely to develop the systemic inflammatory response syndrome (SIRS) ( Figure 5 ) and progress to septic shock and death (102, 103) , findings which may well relate to the proinflammatory cytokine response.

It should also be noted that chronic inflammation is now increasingly accepted to be a risk factor for myocardial infarction (MI), and a recent study by Saevarsdottir et al. has found that patients with high MBL levels have a decreased likelihood of suffering a MI -again suggesting a potential role for MBL in modulating the inflammatory response (104).

As a component of the complement system with similarities to C1q, but also as a player in infectious and inflammatory processes, the structure and function of MBL have prompted studies exploring a possible role in autoimmune conditions. Systemic lupus erythematosus (SLE) has been the focus of a number of MBL genotyping studies, but the results have been somewhat inconsistent. Nevertheless, a recent meta-analysis has reviewed studies in this area and found that MBL variant alleles are indeed SLE risk factors (105) . As with infectious disease, there is some evidence that the risk of pathology increases if there is another co-existing immune defect. For example, in a cohort of Spanish patients, the odds ratio for developing SLE was 2.4 for individuals with MBL deficiency, but this increased to 3.2 when there was also a co-existing partial C4 deficiency (106) . Studies in patients with SLE have reported that MBL deficiency also influences their risk of developing certain complications, which include arterial thromboses (107) and respiratory tract infections (108, 109) .

A role for MBL in the pathogenesis of rheumatoid arthritis has also been suggested. Malhotra et al. reported that changes in IgG glycosylation secondary to the underlying disease results in MBL-associated complement activation (110) . Such complement activation then contributes to chronic inflammation of the synovial membrane. However, Graudal et al. found that patients with lower MBL levels experienced earlier, more severe, symptoms and had more rapid joint destruction as visualized radiologically (111) .

Several recent research publications suggest the directions in which future work on this collectin and its associated molecules may proceed. These include therapeutic interventions, functional assays and the evaluation of the importance of MBL in disease. These are considered briefly below.

Therapeutic potential of MBL MBL replacement was first attempted (without any knowledge of the deficiency) when fresh frozen plasma was given to patients and found to correct the opsonic defect (28, 29) . Since then, affinity-purified, plasma-derived MBL has been safely given to many patients, resulting in normalization of enzyme-linked immunosorbent assay detectable MBL and complement-mediated opsonic activity (112) . A phase 1 study showed the half-life of the protein to range between 18 and 115 h (113) . The development of recombinant MBL is also at the phase 1 trial stage and such developments provide exciting prospects for the future exploration of the therapeutic potential of MBL. Exactly who would benefit from replacement therapy is under debate and the importance of targeting well-defined patient groups will be vital to its success.

The discovery of other components of the lectin pathway including the ficolins and the MASPs indicates that this limb of the immune system is complex and extends beyond MBL and MASP-2 alone. This knowledge enables us to question the impact of these molecules either in isolation or in combination. Functional assessment of the lectin pathway may be a far more accurate and clinically relevant measurement than MBL level and/or genotype alone. A number of different assays have been reported, which assess activity at different stages of the functional pathway; therefore, the results must be interpreted accordingly (114, 115) .

The impact of deficiencies of the various adjunctive components is also the subject of much current research. In 2003, Stengaard-Pedersen et al. reported the first identified case of MASP-2 deficiency (116) . Functional analysis of the ability of MBL to activate the lectin pathway, estimating C4b deposition on a mannan surface, was performed on a group of patients with suspected immunodeficiency. One patient was found to have deficient pathway activity despite having sufficient MBL. No MASP-2 or Map19 was found in the plasma, and genetic analysis indicated that the patient was homozygous for a point mutation in exon 3 of the gene (D105G). Clinically, the patient suffered from recurrent infections and autoimmune symptoms. Subsequently, the frequency of this mutation has been assessed in a small number of populations and values range from 1.3% to 6.3% (117) . As discussed previously, the contribution of MASP-1 and MASP-3 in the pathway remains unexplained.

The role of ficolins is now beginning to be addressed in clinical studies. Like MBL, no absolute levels of deficiency have yet been defined. Atkinson et al. studied more than 300 children with recurrent respiratory tract infections and measured L-ficolin levels (118) . An association with MBL deficiency in the same patient cohort had already been reported (119) . In this study, low levels of L-ficolin were more common in patients than in controls and most common in patients with co-existing atopic disorders, suggesting a role for L-ficolin in protection from microorganisms complicating allergic disease. Polymorphisms in the ficolins have been identified, although their clinical significance is as yet unknown.

MBL is an ancient molecule, which has probably been subject to a large number of evolutionary pressures. The last 50,000 years of human evolution have been associated with major changes as hominids moved from an essentially nomadic lifestyle to increasingly crowded living arrangements in large settled communities. Associated with these changes, the spectrum of common infectious diseases would also have changed. More recently, the introduction of antibiotics, the emergence of novel infections and increasing use of immunosuppressive therapies have provided new challenges to our innate host defence system. Despite all these changing evolutionary pressures, MBL gene polymorphisms persist at high frequencies, suggesting that they offer potential advantages to the host. Thus, there exists a balance in which certain individuals benefit from the expression of high levels of the protein, whereas others (living in differing environments, eg. the tropics) may benefit from reduced levels of circulating MBL ( Figure 6 ).

MBL status may also be either advantageous or disadvantageous when considered from the viewpoint of the severity of a particular illness. Thus, it is known that those with higher levels of MBL are better able to modulate inflammation, probably through an effect on cytokine responses. In contrast, those deficient in MBL appear to be at risk of sepsis and SIRS. For these reasons, we believe that analyses of the relevance of MBL (120) should be extended beyond its role in infectious disease and include clinical areas such as autoimmunity and inflammatory disorders. 

Inhibitory and inactivating action of normal ferret sera against an influenza virus strain

Bovine and mouse serum beta inhibitors of influenza A viruses are mannose-binding lectins

Properties of a new complement-dependent bactericidal factor specific for Ra chemotype salmonella in sera of conventional and germ-free mice

A group of bactericidal factors conserved by vertebrates for more than 300 million years

Affinity chromatography of human liver alpha-D-mannosidase

Isolation and characterization of a mannan-binding protein from rabbit liver

Isolation of mannosebinding proteins from human and rat liver

Extra-hepatic transcription of the human mannose-binding lectin gene (mbl2) and the MBL-associated serine protease 1-3 genes

Collections and ficolins: humoral lectins of the innate immune defense

Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing

Trimeric structure of a C-type mannose-binding protein

Human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil

Binding of sugar ligands to Ca(21)-dependent animal lectins

Analysis of mannose binding by site-directed mutagenesis and NMR

Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin

Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition

Activation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B

The lipopolysaccharide structures of Salmonella enterica serovar Typhimurium and Neisseria gonorrhoeae determine the attachment of human mannose-binding lectin to intact organisms

Serum lectin with known structure activates complement through the classical pathway

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease

A second serine protease associated with mannan-binding lectin that activates complement

A truncated form of mannose-binding lectin-associated serine protease (MASP)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway

Two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene

MASP-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway

Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated serine protease-2

Hakata antigen, a new member of the ficolin/opsonin p35 family, is a novel human lectin secreted into bronchus/alveolus and bile

Human M-ficolin is a secretory protein that activates the lectin complement pathway

L-ficolin specifically binds to lipoteichoic acid, a cell wall constituent of gram-positive bacteria, and activates the lectin pathway of complement

A familial plasma-associated defect of phagocytosis

Defective opsonization. A common immunity deficiency

Yeast opsonisation in children with chronic diarrhoeal states

A study of C3b deposition on yeast surfaces by sera of known opsonic potential

Association of low levels of mannan-binding protein with a common defect of opsonisation

Exon structure reveals its evolutionary relationship to a human pulmonary surfactant gene and localization to chromosome 10

Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein

The human mannose-binding protein functions as an opsonin

Human leukocyte C1q receptor binds other soluble proteins with collagen domains

Mannose binding protein (MBP) enhances mononuclear phagocyte function via a receptor that contains the 126,000 M(r) component of the C1q receptor

Complement receptor 1/CD35 is a receptor for mannan-binding lectin

Complement receptor type 1 (CR1, CD35) is a receptor for C1q

Activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by CD14 antigen, and mannan binding protein inhibits TNF-alpha release

Induction of TNF-alpha in human peripheral blood mononuclear cells by the mannoprotein of Cryptococcus neoformans involves human mannose binding protein

Mannose-binding lectin regulates the inflammatory response of human professional phagocytes to Neisseria meningitidis serogroup B

C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells

Mannose-binding lectin-deficient mice display defective apoptotic cell clearance but no autoimmune phenotype

Gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving C1q

Mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury

Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines

Antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding proteindependent cell-mediated cytotoxicity

Antitumor activity of mannan-binding protein

Molecular basis of opsonic defect in immunodeficient children

High frequencies in African and non-African populations of independent mutations in the mannose binding protein gene

A new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein

Restricted polymorphism of the mannose-binding lectin gene of indigenous Australians

Molecular determinants of oligomer formation and complement fixation in mannose-binding proteins

Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein

Different molecular events result in low protein levels of mannan-binding lectin in populations from southeast Africa and South America

A new strategy for mannosebinding lectin gene haplotyping

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

The concentration of the C-type lectin, mannan-binding protein, in human plasma increases during an acute phase response

Characterization of two mannose-binding protein cDNAs from rhesus monkey (Macaca mulatta): structure and evolutionary implications

Characterization of murine mannose-binding protein genes Mbl1 and Mbl2 reveals features common to other collectin genes

The human ortholog of rhesus mannose-binding protein-A gene is an expressed pseudogene that localizes to chromosome 10

The ÔinvolutionÕ of mannose-binding lectin

Protection afforded by sickle-cell trait against subtertian malareal infection

Mannan-binding lectin enhances susceptibility to visceral leishmaniasis

Dual role of mannan-binding protein in infections: another case of heterosis?

Mannose binding protein gene mutations associated with unusual and severe infections in adults

A population-based study of morbidity and mortality in mannose-binding lectin deficiency

Mannan binding lectin in febrile adults: no correlation with microbial infection and complement activation

Mannan binding lectin deficiency and concomitant immunodefects

The molecular basis of a common defect of opsonization

Role for mannose binding lectin in the prevention of Mycoplasma infection

Characterization of a novel coronavirus associated with severe acute respiratory syndrome

The genome sequence of the SARS-associated coronavirus

Mannose-binding lectin in severe acute respiratory syndrome coronavirus infection

Association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection

Mannose binding lectin gene mutations are associated with progression of liver disease in chronic hepatitis B infection

Mannose-binding lectin in chronic hepatitis B virus infection

Mannose binding lectin genotypes influence recovery from hepatitis B virus infection

Hepatitis C virus infection and mutations of mannose-binding lectin gene MBL

Deficiency of mannose-binding lectin and burden of infection in children with malignancy: a prospective study

Association between deficiency of mannose-binding lectin and severe infections after chemotherapy

Low levels of mannose-binding lectin do not affect occurrence of severe infections or duration of fever in acute myeloid leukaemia during remission induction therapy

Mannose-binding lectin-deficient mice are susceptible to infection with Staphylococcus aureus

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

Interaction of mannose-binding lectin with HIV type 1 is sufficient for virus opsonization but not neutralization

Susceptibility to HIV infection and progression of AIDS in relation to variant alleles of mannose-binding lectin

Mannan-binding lectin in the sub-Saharan HIV and tuberculosis epidemics

The level of the serum opsonin, mannan-binding protein in HIV-1 antibody-positive patients

Mannan-binding lectin serum concentrations in HIV-infected patients are influenced by the stage of disease

Polymorphisms in the MBL2 promoter correlated with risk of HIV-1 vertical transmission and AIDS progression

Absence of association between mannose-binding lectin gene polymorphisms and HIV-1 infection in a Colombian population

Mannose-binding protein in HIV-seropositive patients does not contribute to disease progression or bacterial infections

Circulating levels of mannose binding protein in human immunodeficiency virus infection

Presence of the variant mannose-binding lectin alleles associated with slower progression to AIDS

Human mannose-binding protein gene is regulated by interleukins, dexamethasone and heat shock

Modulatory effect of mannose-binding lectin on cytokine responses: possible roles in HIV infection

Low mannose-binding lectin serum concentrations in HIV long-term nonprogressors?

Polymorphism at codon 54 of mannose-binding protein gene influences AIDS progression but not HIV infection in exposed children

Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis

Impaired pulmonary status in cystic fibrosis adults with two mutated MBL-2 alleles

Association of mannose-binding lectin polymorphisms with sepsis and fatal outcome, in patients with systemic inflammatory response syndrome

Increased incidence and severity of the systemic inflammatory response syndrome in patients deficient in mannose-binding lectin

Mannan binding lectin as an adjunct to risk assessment for myocardial infarction in individuals with enhanced risk

The mannose-binding lectin gene polymorphisms and systemic lupus erythematosus: two case-control studies and a meta-analysis

A dysfunctional allele of the mannose binding protein gene associates with systemic lupus erythematosus in a Spanish population

Mannose-binding lectin variant alleles and the risk of arterial thrombosis in systemic lupus erythematosus

Association of mannose-binding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients

Association of mannose binding lectin (MBL) gene polymorphism and serum MBL concentration with characteristics and progression of systemic lupus erythematosus

Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein

Mannan binding lectin in rheumatoid arthritis. A longitudinal study

Reconstitution of opsonizing activity by infusion of mannan-binding lectin (MBL) to MBL-deficient humans

Human plasma-derived mannose-binding lectin: a phase I safety and pharmacokinetic study

An assay for the mannan-binding lectin pathway of complement activation

Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA

Inherited deficiency of mannan-binding lectin-associated serine protease 2

Deficiency of the mannan-binding lectin pathway of complement and poor outcome in cystic fibrosis: bacterial colonization may be decisive for a relationship

L-ficolin in children with recurrent respiratory infections

Mannan-binding lectin insufficiency in children with recurrent infections of the respiratory system

Human mannose-binding lectin in immunity: friend, foe, or both?

Isolation and characterization of a mannan-binding protein from human serum

A common congenital immunodeficiency predisposing to infection and atopy in infancy

Mannan-binding protein and conglutinin in bovine serum

Suboptimal C3b/C3bi deposition and defective yeast opsonization. I. Evidence for the absence of essential co-factor activity

Isolation and characterization of two distinct mannan-binding proteins from rat serum

Two distinct classes of carbohydrate-recognition domains in animal lectins

A serum lectin (mannan-binding protein) has complement-dependent bactericidal activity

The level of mannan-binding protein regulates the binding of complement-derived opsonins to mannan and zymosan at low serum concentrations

Molecular characterization of the mouse mannose-binding proteins. The mannose-binding protein A but not C is an acute phase reactant

Structure of a C-type mannose-binding protein complexed with an oligosaccharide

Human mannose-binding protein is identical to a component of Ra-reactive factor

Association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series

Cutting edge: complementactivating complex of ficolin and mannose-binding lectin-associated serine protease

Mannose-binding lectin accelerates complement activation and increases serum killing of Neisseria meningitidis serogroup C

Activation of the lectin complement pathway by H-ficolin (Hakata antigen)

Differential recognition of obligate anaerobic bacteria by human mannose-binding lectin

Differential binding of mannose-binding lectin to respiratory pathogens in cystic fibrosis

Human mannose-binding protein inhibits infection of HeLa cells by Chlamydia trachomatis

Binding of mannan-binding protein to various bacterial pathogens of meningitis

Interaction of human mannose-binding protein with Mycobacterium avium

Interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1

High mannose glycans and sialic acid on gp120 regulate binding of mannose-binding lectin (MBL) to HIV type 1

Mannose binding lectin (MBL) and HIV

Mannan-binding protein and bovine conglutinin mediate enhancement of herpes simplex virus type 2 infection in mice

Mannan-binding lectin modulates the response to HSV-2 infection

Mannose binding protein is involved in first-line host defence: evidence from transgenic mice

Binding of host collectins to the pathogenic yeast Cryptococcus neoformans: human surfactant protein D acts as an agglutinin for acapsular yeast cells

Mannose-binding lectin is a component of innate mucosal defense against Cryptosporidium parvum in AIDS

Recognition of plasmodium falciparum proteins by mannan-binding lectin, a component of the human innate immune system

The major surface glycoprotein of Trypanosoma cruzi amastigotes are ligands of the human serum mannose-binding protein

Novel MASP2 variants detected among North African and Sub-Saharan individuals

Analysis of mannose-binding lectin 2 (MBL2) genotype and the serum protein levels in the Korean population

Association of mannose-binding lectin gene haplotype LXPA and LYPB with interferon-resistant hepatitis C virus infection in Japanese patients