MBL in Innate Immunity JID 2003:187 (Suppl 2) S335

S U P P L E M E N T A R T I C L E
Role of the Mannose-Binding Lectin
in Innate Immunity
R. Alan Ezekowitz
Laboratory of Developmental Immunology, Massachusetts General Hospital for Children, and Department of Pediatrics, Harvard Medical School,
Boston, Massachusetts
The ability to recognize infectious agents from self is intrinsic to innate immunity. One basic tenet of innate
immunity is the evolution of classes of molecules that are termed pattern-recognition receptors and mol-
ecules. Many pattern-recognition molecules conspire together to protect the host in the rst minutes and
hours after exposure to an infectious challenge. The mannose-binding lectin (MBL; also termed mannose-
binding protein) is a prototypic pattern-recognition molecule that appears to play a role as an ante-antibody
in rst line host defense. The serum levels of the human MBL are regulated in serum so that any one person
will display a phenotype of low, intermediate, or high levels. There appears to be a relationship between
circulating MBL and susceptibility and resistance to infection. MBL levels also appear to be regulated by
distinct haplotypes. Thus, the question to be answered is what constitutes the innate immunity haplotype in
any individual and how does this impact on the relationship between the host and infectious agents?
The basic templates of innate immunity were laid down
hundreds of millions of years ago and conserved from
primitive life forms to humans. From insects, we have
learned that there are 3 basic responses to septic injury:
the induction of proteolytic cascades that are aimed at
walling off the infection (melanization), recognition
and engulfment of the infectious agent by phagocytic
cells, and the induction of a humoral response in the
guise of antimicrobial peptide release [1]. These 3 phys-
iologic processes are conserved in higher mammals. In
humans, the proteolytic cascades are represented by
complement and the kallikrein and clotting systems.
The basic cellular processes of phagocytosis are similar
Financial support: National Institutes of Health (grant R01 AI-42788).
R.A.E. has a commercial interest in a privately held company, Natimmune, which
seeks to explore the therapeutic potential of a recombinant form of human
mannose-binding lectin.
Reprints or correspondence: Dr. R. Alan Ezekowitz, Laboratory of Developmental
Immunology, Massachusetts General Hospital for Children, Dept. of Pediatrics,
Harvard Medical School, 5 Parkman St., Boston, MA 02114 (ezekowitz.alan@mgh
.harvard.edu).
The Journal of Infectious Diseases 2003; 187(Suppl 2):S3359
2003 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2003/18712S-0006$15.00
in ies and humans. The components of humoral re-
sponses in humans encompass a much broader rep-
ertoire of antimicrobial and antiviral molecules that
include natural antibodies, the complement system,
collectins, and a host of other circulating pattern-rec-
ognition molecules. In higher animals, effector cells
such as macrophages, gdT cells, NK T cells, and gran-
ulocytes guard the portals of potential microbial entry.
In our laboratory, we abide by 4 basic principles of
innate immunity: defense of the host in the rst
minutes, hours, or days of exposure to an infectious
agent during the lag period of 2436 h required to
generate a specic adaptive immune response; limit the
infection; induce a heightened sense of awareness in
the organism; and act as necessary antecedent to the
development of an adaptive immune response.
ALLELIC VARIATION IN GENES
INVOLVED IN INNATE IMMUNITY:
AN INFECTION HAPLOTYPE?
It is important to realize that there is a constant tussle
between the host and the potential pathogen and that
the innate immune system is critical to the maintenance

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S336 JID 2003:187 (Suppl 2) Ezekowitz
Figure 1. Schematic diagram of human mannose-binding lectin (MBL)
trimer and the following domains: amino terminal cysteine-rich, collagen,
a-helical coiled coil, and carbohydrate recognition domain (CRD) with
sugar ligand model.
of the balance in favor of the host. It is my contention that
subtle variations in components of the innate immune system
may be critical in determining the balance between host and
pathogen. When one extends the ideas of allelic variation to
host defense, at least 2 models emerge [2]. One model proposes
a critical subset of !100 genes that are involved in innate im-
munity. The other model contends that many genes (100)
play this role in dening susceptibility or resistance to infection.
What adds to the complexity is that the complement of any
of these haplotypes in the aggregate, under certain circum-
stances, may predispose to infection with particular agents,
whereas, under other circumstances, these same genes may pro-
tect against infection with a different potential pathogen. Ac-
cumulating evidence indicates that the human mannose-bind-
ing lectin (MBL) is a gene product that plays a role in rst-line
host defense and is illustrative of this paradigm.
MBL AS PARADIGM OF INNATE IMMUNITY
MBL (also called mannose-binding protein) was rst isolated
from rabbit serum in the late 1970s [3]. The characterization
of cDNA that encoded for rat MBL-A revealed homologies to
surfactant apoprotein A in that both molecules had C-terminal
lectinlike domains and collagen tails that form trimers and
then multimers of trimers up to hexamers of trimers (gure
1) [4]. The overall similarity in the organization of MBL as a
collagen containing protein with 16 head domains with C1q,
the rst component of the classical complement pathway, led
to the idea that MBL could substitute for C1q and activate the
classical pathway of complement [5].
In a series of papers, my colleagues and I dened cDNAs that
encode for the human MBL [6]; dened the role of MBL as an
opsonin in vitro and demonstrated that it was capable of binding
gram-negative bacteria, not just mannan [7], and the outer en-
velope glycoproteins of viruses, such as gp120 of human im-
munodeciency virus [8]; determined that human MBL is en-
coded by a single gene located at 10q21.1 [9]; and characterized
the 2 murine MBL cDNAs, MBL-A and -C [10]. Later the same
year we dened a functional role for MBL in vitro.
Super et al. [11] determined that a relative lack of MBL
accounted for a defect in opsonization of yeast in persons who
appeared to be prone to recurrent infections, despite apparent
adequate adaptive immunity. It was subsequently shown that
amino acid substitutions that disrupt the collagen helix at co-
don 54 (B allele) account for low serum levels of MBL [12].
B and C (codon 52) alleles are structural variants of MBL that
appear to affect the stability of MBL oligomers. In addition to
these structural variations, several single-nucleotide polymor-
phisms (SNPs) have been identied in the MBL promoter. Of
these, LXP, LYP, LYQ, and HYP are the most common. HYP
is associated with the highest MBL levels, and LXP is associated
with low levels when associated with the wild-type MBL coding
region. The 3 structural gene mutations are in linkage dis-
equilibrium with the promoter polymorphisms so that each
individual will express 2 of 8 possible haplotypes: HYPA, LYQA,
LYPA, LXPA, LYPB, PYPB, LYQC, and HYPD [13, 14].
MBL acts like an ante-antibody in that it has a broad
MBL in Innate Immunity JID 2003:187 (Suppl 2) S337
Figure 2. Proposed model for carbohydrate pattern recognition by human mannose-binding lectin (carbohydrate binding to carbohydrate recognition
domain [CRD]).
spectrum of binding to bacterial and viral infectious agents and
is constitutively present in serum before the development of a
specic antibody response [15]. Of importance, in 1992, Mat-
sushita and Fujita [16] described a serine protease associated
with MBL that bears 50% homology to C1r and C1s, key
components in the initiation of the classical pathway conver-
tase. Subsequently, it was determined that there is a homologue
of this MBL-associated serine protease (MASP) 1, termed
MASP2, and an alternatively spliced form of MASP1, termed
MASP3 [17, 18]. It now appears that MASP2 is the enzyme
responsible for lectin pathway activation; the precise role for
MASP1 is not clear. The so-called lectin pathway of comple-
ment activation originated in Ascidians, which have collectin-
like proteins, MASP, and C3, whereas sea urchins have C3 and
factor B-like molecules. Thus, MBL has the right pedigree to
play a key role as a pattern-recognition molecule in rst-line
host defense. It acts as an opsonin either directly or by virtue
of its ability to bind complement and is present in serum.
MBL AS PARADIGM FOR PATTERN
RECOGNITION
An essential feature of innate immunity is the ability to dis-
tinguish infectious agents from self. Janeway [19] proposed that
pathogen-associated molecule patterns are invariant structures
that are common and immutable components of infectious
agents and are recognized by putative pattern-recognition re-
ceptors. This concept was subsequently rened to include com-
mensals, as well as pathogens and circulating pattern-recog-
nition molecules. A key principle of pattern recognition is the
ability to distinguish species self from non-self. MBL binds a
wide range of infectious agents and apoptotic cells but appears
not to recognize self-glycoproteins.
Structural studies rst dened the binding site of rat MBL
carbohydrate recognition domain ligated to a mannose terminal
oligosaccharide and dened what we term the micropattern.
This is dictated by the orientation of C3 and C4 hydroxyl
groups of the ligand [20]. This kind of ligand geometry is found
in mannose, fucose, N-acetylglucosamine, glucose, and deriv-
atives of these sugars but not in galactose and sialic acid, which
are the ultimate and penultimate sugars of most mammalian
glycoproteins. This interaction is lowafnity (10
5
M), yet MBL
binds to yeast mannan with an afnity of M [21].
10
10 10
The high-afnity binding requires that ligands span 45 A

in 3
dimensions to allow for high-afnity binding. It appears that
this geometry is predominantly in infectious agents and not
self-glycoproteins [22]. Therefore, pattern recognition requires
both a micro- and macropattern (gure 2) [1]. This concept
appears to extend to other collectins such as surfactant pro-
teinD [23].
MBL IS A HIGH-PREVALENCE DISEASE ALLELE
THAT REFLECTS THE DIVERSE ROLE PATTERN-
RECOGNITION MOLECULES PLAY IN
RESISTANCE AND SUSCEPTIBILITY TO
INFECTION
Humans have 7 common haplotypes that set the MBL serum
levels in each person. Studies of different ethnic groups indicate
that the prevalent haplotypes that specify high, intermediate, low,
or undetectable levels of MBL in serum vary considerably [13,
14, 24, 25]. For instance, 10% of white persons have haplotypes
S338 JID 2003:187 (Suppl 2) Ezekowitz
that result in MBL levels of 50 ng/mL, whereas up to 40% of
Xhozas in South Africa have low-secretor haplotypes [26]. The
question thus becomes, what is the selection advantage for each
ethnic group? The answer appears to be in part that MBL has a
Jekyll and Hyde role in innate immunity [27].
MBL and Dr. Jekyll. The hypothesis would predict that
low or undetectable levels of MBL predispose the host to in-
fections. Super et al. [11] rst proposed that an opsonic defect
in serum that correlated with a broad phenotype of recurrent
infection was due to a lack of MBL in young children. Later,
Turner [28] modied this idea and suggested that the phe-
notype of susceptibility to infection was more obvious if there
was an associated defect in adaptive or innate immunity. An
example is that an antibody isotype deciency in conjunction
with low MBL levels would collectively present the host with
an increased risk to infection. Garred et al. [29] provided an-
other excellent example: SNPs in the MBL gene modify sus-
ceptibility to infection. They demonstrated that patients with
cystic brosis who also inherit low-secretor MBL haplotypes
have a reduction in life expectancy of 58 years. Although the
exact mechanism is not clear, one plausible explanation is that
the lungs of these patients are colonized earlier with Burkhold-
eria cepacia and Pseudomonas aeruginosa. Once this occurs, a
refractory cycle of lung injury begins that ultimately results in
the patients death.
The importance of the innate immune system as an ante-
cedent to adaptive immunity is well illustrated when innate
immunity is disrupted as a side effect of cancer chemotherapy.
An association between febrile neutropenia and infection in
patients with cancer undergoing chemotherapy has been rec-
ognized since 1966 [30]. It is also clear that febrile neutropenic
patients are heterogeneous. Many thoughtful studies have strat-
ied patients according to risk [31]. However, until recently
there has been little attention to assessing patients premorbid
genetic susceptibility to infection. Two recent studies addressed
this question and, once again, MBL presents a paradigm. Neth
et al. [32] found that children with febrile neutropenia after
chemotherapy who had low secretor haplotypes had hospital
stays that were, on average, 2 days longer than those of children
who were MBL sufcient. Peterslund et al. [33] found that, of
54 adults with cancer, 16 with clinically signicant events (e.g.,
bacteremia and pneumonia) had low levels of MBL. Taken
together, these preliminary studies indicate that a threshold
MBL level correlates with increased risk of infection.
MBL and Mr. Hyde. The nding that 50%60% of sub-
Saharan African populations express the C allele that correlates
with low MBL levels suggests that under certain circumstance
MBL acts as a disease resistance gene [26]. This supposition
gains support from the observations that low MBL alleles ap-
pear to be protective against infections with Plasmodium fal-
ciparum [34], Cryptosporidium parvum [35], mycobacterial tu-
berculous meningitis, and Leishmania chagasi [36]. This raises
the idea that MBL provides intracellular pathogens with a se-
lective advantage. However, it is likely to be more complex,
because it appears that MBL might also modulate inamma-
tion. MBL appears to trigger proinammatory cytokine release
in the human system in vitro and in vivo [26]. This idea is
conrmed by recent observations from our laboratory that in-
dicate that MBL-A null mice appear to be protected from death
in a model of acute septic peritonitis and that this phenotype
appears to correlate with a muted, but not absent, proinam-
matory response [37].
CONCLUSIONS
It is likely that allelic variations in genes whose products play
a role in innate immunity may be considered in the collective
as infection haplotypes. These infection haplotypes may con-
tribute to susceptibility to infection under certain circum-
stances. However, under other circumstances, the same infec-
tion haplotypes may protect the host against an overly rigorous
inammatory response. MBL is clearly an important gene in
these infection haplotypes, but a more-complete analysis will
include cytokine genes, chemokine genes, genes that regulate
nonoxidative and oxidative killing, and pattern-recognition
molecules (e.g., Toll-like receptors and peptidoglycan recep-
tors). The challenge is to identify the subtle phenotypes and
apply SNP analysis to informative patient populations.
Acknowledgments
I thank all members of my laboratory for stimulating dis-
cussions and hard work and, in particular, Thilo Stehle for
assistance in preparation of the gures and Kazue Takahashi
for leading the mannose-binding lectin group.
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