Humoral Mechanisms Provide An Additional
Defensive Strategy
Microbicidal factors in secretions
Turning now to those defense
systems that are mediated entirely by soluble pattern recognition
molecules (Figure 1.2), we recollect that many microbes activate the
complement system and may be lysed by the insertion of the membrane attack
complex. The spread of infection may be limited by enzymes released through
tissue injury that activate the clotting system. Of the soluble bactericidal
substances elaborated by the body, perhaps the most abundant and widespread is
the enzyme lysozyme, a muramidase that splits the exposed peptidoglycan wall of
susceptible bacteria (see Figure 11.5).
Like the α‐defensins of the
neutrophil granules, the human β‐defensins are peptides derived by proteolytic
cleavage from larger precursors; they have β‐sheet structures, 29–40 amino
acids, and three intramolecular disulfide bonds, although they differ from the
α‐defensins in the placement of their six cysteines. The main human β‐defensin,
hDB‐1, is produced abundantly in the kidney, the female reproductive tract, the
oral gingiva, and especially the lung airways. As the word has it that we are
all infected every day by tens of thousands of airborne bacteria, this must be
an important defense mechanism. This being so, inhibition of hDB‐1 and of a
second pulmonary defensin, hDB‐2, by high ionic strength could account for the
susceptibility of cystic fibrosis patients to infection as they have an ion
channel mutation that results in an elevated chloride concentration in airway
surface fluids. Another airway antimicrobial active against Gram‐negative and Gram‐positive
bacteria is LL‐37, a 37‐residue α‐helical peptide released by proteolysis of a
cathelicidin (cathepsin L‐inhibitor) precursor.
This theme surfaces again in the
stomach where a peptide split from lactoferrin by pepsin could provide the
gastric and intestinal secretions with some antimicrobial policing. A rather
longer two‐domain peptide with 107 residues, termed secretory leukocyte
protease inhibitor (SLPI), is found in many human secretions. The C‐terminal
domain is anti‐protease but the N‐terminal domain is distinctly unpleasant to
metabolically active fungal cells and to various skin‐associated microorganisms,
which makes its production by human keratinocytes particularly appropriate. In
passing, it is worth pointing out that many d‐amino acid analogs of peptide
antibiotics form left‐handed helices that retain the ability to induce membrane
ion channels and hence their antimicrobial powers and, given their resistance
to catabolism within the body, should be attractive candidates for a new breed
of synthetic antibiotics.
Lastly, we may mention the two
lung surfactant proteins SP‐A and SP‐D that, in conjunction with various
lipids, lower the surface tension of the epithelial lining cells of the lung to
keep the airways patent. They belong to a totally different structural group of
molecules termed collectins (Figure 1.35) that contribute to innate immunity
through binding of their lectin‐like domains to carbohydrates on microbes, and
their collagenous stem to cognate receptors on phagocytic cells thereby
facilitating the ingestion and killing of the infectious agents.
Acute phase proteins increase in response to infection
A number of plasma proteins
collectively termed acute phase proteins show a dramatic increase in
concentration in response to early “alarm” mediators such as macrophage‐derived interleukin‐1 (IL‐1) released as a result of
infection or tissue injury. These include C‐reactive protein (CRP), mannose‐
binding lectin (MBL), and serum amyloid P component (Table 1.2). Expression
levels of the latter proteins can increase by as much as 1000‐fold in response
to proinflammatory cytokines such as IL‐1 and IL‐6. Other acute phase proteins
showing a more modest rise in concentration include α1‐ antichymotrypsin, fibrinogen, ceruloplasmin,
C9, and factor B.
The acute phase proteins are a
relatively diverse group of proteins belonging to several different families
(including, but not limited to, the pentraxin, collectin,
and ficolin families) that have a number of functional effects in
common. All of these proteins act as soluble pattern recognition molecules and
are capable of binding directly to infectious agents to function as opsonins
(i.e., “made ready for the table”), thereby enhancing uptake of microorganisms
by macrophages and neutrophils. Many of these proteins also have the ability to
activate complement and the assembly of a membrane attack complex. The ability
to agglutinate microorganisms, thereby impeding their spread within the
infected tissue, is another common theme. Some of these molecules can also form
heterocomplexes, extending the range of PAMPs that can be detected.
These soluble pattern recognition
molecules are frequently synthesized by activated macrophages upon stimulation
of their pattern recognition receptors, or are
stored within neutrophil granules available for immediate release via
degranulation in response to infection. The liver is another major source of
many acute phase proteins that are released into the circulation as a result of
the systemic effects of the major proinflammatory cytokines IL‐1 and IL‐6. Let
us look at some examples further.
Pentraxins
Pentraxins, so‐called because
these agents are made up of five identical subunits, constitute a superfamily
of conserved proteins typified by a cyclic multimeric structure and a
C‐terminal 200‐amino‐acid‐long pentraxin domain. CRP, serum amyloid P component
(SAP), and pentraxin 3 are members of this family (Figure 1.36). Human CRP is
composed of five identical polypeptide units noncovalently arranged as a cyclic
pen- tamer around a calcium (Ca)‐binding cavity, was the first pentraxin to be
described, and is the prototypic acute phase response protein. Pentraxins have
been around in the animal kingdom for some time, as a closely related homolog,
limulin, is present in the hemolymph of the horseshoe crab, not exactly a close
relative of Homo sapiens. A major property of CRP is its ability to bind
in a Ca‐dependent fashion, as a pattern recognition molecule, to a number of microorganisms
that contain phosphorylcholine in their membranes, the complex having the
useful property of activating complement (by the classical and not the
alternative pathway with which we are at present familiar). This results in the
deposition of C3b on the surface of the microbe that thus becomes opsonized for
adherence to
phagocytes.
SAP can complex with chondroitin
sulfate, a cell matrix glycosaminoglycan, and subsequently bind lysosomal
enzymes such as cathepsin B released within a focus of inflammation. The
degraded SAP becomes a component of the amyloid fibrillar deposits that
accompany chronic infections – it might even be a key initiator of amyloid
deposition. SAP also binds several bacterial species via LPS and, similar to
CRP, can also activate the classical complement pathway. CRP and SAP represent
the main acute phase reactants in human and mouse, respectively.
Collectins
Nine members of the collectin
family have been described in vertebrates to date, the most intensively studied
of which is mannose‐binding lectin (MBL). MBL can react not only
with mannose but several other sugars, so enabling it to bind with an
exceptionally wide variety of Gram‐negative and Gram‐ positive bacteria,
yeasts, viruses, and parasites; its subsequent ability to trigger the classical
C3 convertase through two novel associated serine proteases (MASP‐1 and MASP‐2)
is the basis of what is known as the lectin pathway of complement
activation. (Please relax, we unravel the secrets of the classical and lectin
pathways in the next chapter.)
MBL is a multiple of trimeric
complexes, each unit of which contains a collagen‐like region joined to a
globular lectin‐binding domain (Figure 1.37). This structure places it in the
family of collectins (collagen + lectin) that have
the ability
to recognize “foreign”
carbohydrate patterns differing from “self ” surface polysaccharides, normally
terminal galactose and sialic acid groups, whereas the collagen region can bind
to and activate phagocytic cells through complementary receptors on their
surface. The collectins, especially MBL and the alveolar surfactant molecules
SP‐A and SP‐D mentioned earlier (Figure 1.35), have many attributes that
qualify them for a first‐line role in innate immunity as soluble PRRs. These
include the ability to differentiate self from nonself, to bind to a variety of
microbes, to generate secondary effector mechanisms, and to be widely
distributed throughout the body including mucosal secretions. They are of
course the soluble counterparts to the cell surface C‐type lectin PRRs
described earlier.
Interest in the collectin
conglutinin has intensified with the demonstration, first, that it is found in
humans and not just in cows, and second, that it can bind to N‐acetylglucosamine;
being polyvalent, this implies an ability to coat bacteria with C3b by
cross‐linking the available sugar residue in the complement fragment with the
bacterial proteoglycan. Although it is not clear whether conglutinin is a
member of the acute phase protein family, we mention it here because it
embellishes the general idea that the
evolution of lectin‐like molecules that bind to microbial rather than self
polysaccharides, and which can then hitch themselves to the complement system
or to phagocytic cells, has proved to be such a useful form of protection for
the host.
Ficolins
These proteins are structurally
and functionally related to col- lectins (Figure 1.38), and can also recognize
carbohydrate‐ based PAMPs on microorganisms to activate the lectin pathway of
complement activation. Ficolins typically recognize N‐acetylglucosamine
residues in complex‐type carbohydrates in addition to other ligands. Three
ficolins have been identified in humans, ficolin‐1, ‐2, and ‐3 (also known as
M‐, L‐, and H‐ficolin, respectively), and a role as opsonins for the enhancement
of phagocytosis has also been demonstrated for these proteins. Ficolins can
also interact with CRP to widen the range of bacteria recognized by the latter
and also to enhance complement‐mediated killing. The range of bacterial structures
recognized by ficolins and MBL are complementary and recognize different but
overlapping bacterial species.
Interferons inhibit viral replication
Recall from our earlier discussion
of pattern recognition receptors (PRRs) that engagement of many of these
receptors by PAMPs results in the production of cytokines and chemokines that act to amplify immune responses by
binding to cells in the vicinity. An important class of cytokines induced by
viral as well as bacterial infection is the type I interferons (IFNα
and IFNβ). These are a family of broad‐spectrum antiviral agents present in
birds, reptiles, and fish as well as the higher animals, and first recognized
by the phenomenon of viral interference in which an animal infected with one
virus resists superinfection by a second unrelated virus. Different molecular
forms of interferon have been identified, the genes for all of which have been
isolated. There are at least 14 different α‐interferons (IFNα) produced by leukocytes,
while fibroblasts, and probably all cell types, synthesize IFNβ. We will keep a
third type (IFNγ), which is not directly induced by viruses, up our sleeves for
the moment.
Cells synthesize interferon when
infected by a virus and secrete it into the extracellular fluid, where it binds
to specific receptors on uninfected neighboring cells. As we saw earlier,
engagement of several members of the TLR family, as well as the RIG‐like
helicase receptors and the cytoplasmic DNA sensors, with their cognate PAMPs
results in the induction of members of the interferon‐regulated factor (IRF)
family of transcription factors (Figure 1.22 and Figure 1.23). In combination
with NFkB, another transcription factor activated by engagement of several of
the PRRs, IRFs induce expression of type I interferons that are secreted and
bind to cells in the vicinity. Long double‐stranded RNA molecules, which are
produced during the life cycle of most viruses, are particularly good inducers
of interferons. The bound interferon now exerts its antiviral effect in the
following way. At least two genes are thought to be derepressed in the
interferon‐binding cell, allowing the synthesis of two new enzymes. The first,
a protein kinase called protein kinase R (PKR), catalyzes the
phosphorylation of a ribosomal protein and an initiation factor (eIF‐2)
necessary for protein synthesis. The net effect of this is to dramatically
reduce protein translation as a means of reducing the efficiency of virus
production. Another gene product induced by interferons, oligoadenylate
synthetase, catalyzes the formation of a short polymer of adenylic acid
which activates a latent endoribonuclease; this in turn degrades both viral and
host mRNA. This is another clever adaptation that is designed to reduce the
production of viral products. Another consequence of the downturn in protein
synthesis is a reduction in the expression of major histocompatibility complex
(MHC) proteins, making cells susceptible to the effects of natural killer
cells.
The net result is to establish a
cordon of uninfectable cells around
the site of virus infection, so restraining its spread. The effectiveness of
interferon in vivo may be inferred from experiments in which mice
injected with an antiserum to murine interferons could be killed by several
hundred times less virus than was needed to kill the controls. However, it must
be presumed that interferon plays a significant role in the recovery from, as
distinct from the prevention of, viral infections.
As a group, the interferons may prove
to have a wider bio- logical role than the control of viral infection. It will
be clear, for example, that the induced enzymes
described above would act to inhibit host cell division just as effectively as
viral replication.
