Defence against extracellular pathogens
Innate defence molecules
 Various cell types, including leucocytes and epithelial cells, produce antimicrobial peptides called defensins that are directly damaging to certain
microbes.
Antibody interactions with defence components
 Antibodies may have defensive effects simply by binding to antigens, eg.
neutralizing the activities of bacterial toxins or viruses.

Antibodies can also act as intermediaries to activate other defensive
components that interact with the Fc regions of antibodies. Thus, IgM and
IgG antibodies can activate the complement system of proteins; phagocytes
have Fc receptors (FcR) that bind IgG or IgA; natural killer cells similarly bind
IgG; mast cells have FcR specific for IgE; eosinophils have FcR specific for IgG
or IgE.
Complement
 Complement is the name for a collection of about thirty proteins found in the
circulation and in tissue fluids that were initially described by their ability to
‘complement’ the effects of antibodies. Various complement proteins act as
activation enzymes, defence molecules and regulatory proteins. Activation of
complement proteins is triggered by infection and immune activation, and
occurs as a cascade or chain reaction with amplification built in.

Many activation steps involve the splitting (conversion) of complement
proteins into active fragments by enzymes formed from other complement
proteins, eg. C  Ca + Cb. The central event of complement activation is the
conversion reaction C3  C3a + C3b that is catalysed by a C3 convertase
enzyme. There are two C3 convertases that are generated by different
complement activation pathways.

The Classical Pathway of complement activation is initiated by IgM or IgG
antibodies bound to antigens (i.e. free antibodies cannot trigger the
pathway). C1q binds to the Fc regions of these antibodies. This activates C1r
and C1s that are associated with C1q, leading to the conversion of C4 and C2.
The fragments C4b and C2a combine to form C4b2a, which has C3 convertase
activity.

The same C3 convertase can also be generated when mannose binding lectin
(MBL) interacts with microbes. This activates MBL-associated serine
proteases that convert C4 and C2, generating C4b2a.

The Alternative Pathway of complement activation involves the stabilisation of
certain complement protein fragments by their interaction with microbial
surfaces, forming the C3 convertase C3bBbP. Thus, this enzyme contains the
C3b fragment itself, which may be generated as a result of the activity of the
Classical or MBL pathways. It may also be generated by spontaneous
splitting of C3 by tissue proteases that occurs all the time, but in the absence
of microbes that stabilise the C3b it is rapidly degraded.

The C3a and C3b fragments both have immunological activities that are
discussed below. In addition, binding of C3b to the C3 convertase changes its
substrate specificity so that it functions as a C5 convertase that catalyses the
reaction C5  C5a + C5b. This initiates the membrane attack pathway in
which C5b binds to a microbe’s lipid membrane and interacts with C6, C7, C8
and several molecules of C9 to form the membrane attack complex (C5b-9).
The C9 molecules polymerise to form a hollow tube that forms a channel
through the lipid bilayer, thus forming a hole in the membrane. Many such
complexes formed over the surface of a microbe can lead to its death.
Phagocytes
 Macrophages and neutrophils are phagocytes that engulf (phagocytose)
microbes and digest them intracellularly. Macrophages are tissue-resident
cells derived from blood monocytes; neutrophils circulate in the blood and are
recruited into sites of infection and inflammation.

Phagocytes express pattern recognition receptors (eg. mannose receptor and
scavenger receptor) that enable them to bind microbes directly. They also
have receptors for opsonins, i.e. proteins of the immune system that coat
(opsonise) the surface of microbes.

The acute phase proteins C-reactive protein and mannose binding lectin
opsonise bacteria for uptake by phagocytes, and also activate complement.

Antibodies (IgG and IgA) and the complement activation fragment C3b are
potent opsonins. Phagocytes possess receptor proteins that bind to the Fc
regions of IgG or IgA – these are called Fc receptors (FcR). When C3b is
produced by the conversion of the complement protein C3, it can bind
covalently to microbial surfaces, and phagocytes have complement receptors
(CR) that bind to C3b. Thus, microbes coated by antibodies and C3b can be
strongly bound to the surface of phagocytes via FcR and CR.

The interaction of a microbe and opsonins with surface receptors of a
phagocyte triggers phagocytosis, with the microbe being internalised into a
membrane bound vesicle called a phagosome. Cytoplasmic lysosomes that
contain digestive enzymes then fuse with the phagosome, releasing the
digestive enzymes onto the surface of the microbe and bringing about its
degradation. In addition, the phagocyte produces reactive oxygen species
that enter the phagosomes, helping in the degradation and producing a pH
appropriate for the activity of the digestive enzymes.
Eosinophils
 Some infective agents are too large to be engulfed by phagocytes for intracellular digestion – eg. parasitic worms. In this situation, eosinophils can
mediate extra-cellular digestion.

Eosinophils express FcR that bind IgG or IgE and they also express CR. Thus
opsonisation of a parasitic worm by antibodies and C3b facilitates binding of
numerous eosinophils to the parasite surface. Eosinophils have cytoplasmic
granules containing a range of digestive proteins, e.g. major basic protein and
eosinophil cationic protein: these are released onto the surface of the parasite
to bring about extra-cellular digestion.
Mast cells and inflammation
 Mast cells are tissue cells that express high affinity FcR that bind IgE
antibodies from surrounding tissue fluid. Thus, mast cells become coated
with IgE antibodies and therefore acquire the antigenic specificities of these
antibodies.

When an antigen specifically binds simultaneously to two or more IgE
antibodies on a mast cells surface (i.e. the antigen crosslinks or bridges the
IgE), this triggers the release of inflammatory mediators. Release of
inflammatory mediators by mast cells can be similarly triggered by the
complement activation peptides C3a and C5a (these peptides also attract and
activate neutrophils at sites of inflammation).

Mast cells contain numerous granules in which are stored preformed
inflammatory mediators (eg. histamine, heparin, tryptase): these are
released immediately (degranulation) upon mast cell triggering. Mast cell
activation also induces the synthesis of newly formed mediators that are
released over a longer period of time, eg. leukotrienes and prostaglandins
derived from arachidonic acid, and cytokines.

Inflammation is the collection of processes that amplifies the immune
response at sites of infection by facilitating the movement of components of
the immune system to these sites. A whole range of inflammatory mediators
can be involved, including products of the activation of complement, mast
cells, macrophages and T lymphocytes.

Many of the events associated with inflammation involve effects on the walls
of blood vessels, particularly vasodilatation (that increases the volume of
blood reaching the tissues and reduces the rate of blood flow so that
leucocytes move more slowly through the tissues); leucocyte adhesion (see
below); increased vascular permeability (that facilitates the movement of
leucocytes and of plasma, containing antibodies and complement proteins,
into the infected tissues). Movement of leucocytes through the tissue to the
site of infection is achieved by chemotaxis, with immobilisation and activation
when the site of infection is reached.

Within inflamed tissues, the endothelial cells lining blood vessels are activated
by inflammatory mediators to express adhesion molecules that facilitate the
adhesion of leucocytes to the blood vessel walls and their migration across
the walls. The initial interactions (called capture and rolling) are mediated by
selectins (E-selectin and P-selectin expressed by the endothelium, and Lselectin expressed by leucocytes) that bind to highly glycosylated proteins
(mucins). This facilitates the interaction of the leucocytes with chemokines
on the surface of the endothelium that trigger activation of adhesion
molecules called integrins expressed by leucocytes. The integrins form strong
interactions with the endothelial cells by binding to ligands called intercellular
and vascular cell adhesion molecules (ICAM and VCAM): this is called the
activation and flattening stage.

Leucocytes bound to the endothelium pass between adjacent endothelial cells
(facilitated by the loss of tight junctions between endothelial cells), again
using integrin interactions: this is termed extravasation.

Chemotaxis involves the directional movement of cells in response to
chemical signals (chemoattractants). For example, chemokines produced by
activated cells (eg. macrophages) at the site of infection diffuse through the
tissue and bind to receptors where they impinge on the ‘leading edge’ of
leucocytes. This stimulates new integrin expression at the leading edge of
the cell so that it forms new contacts with tissue cells or connective tissue
components. Old integrin contacts at the trailing edge of the cell are
gradually lost, so the cell moves up the concentration gradient of chemokine
towards the site of infection.

In addition to the local inflammatory effects at the site of infection, a body
wide response to infection also occurs, termed the acute phase response.
This is mediated primarily by systemic effects of the cytokines interleukin-1,
interleukin-6 and tumour necrosis factor, that are variously produced by
activated macrophages, lymphocytes, mast cells and other activated cells.
They affect various tissues around the body when released into the blood
stream at significant concentrations. Collectively, these cytokines induce
fever by effects on the hypothalamus, promote increased levels of circulating
leucocytes (leucocytosis) by release from the bone marrow, and stimulate the
liver to produce anti-microbial acute phase proteins like C-reactive protein
and mannan-binding lectin.
Recommended reading:
Todd I, Spickett G (2005) Lecture Notes: Immunology. 5th edition. Blackwell
Publishing. Chapters 5, 6, 7, 8 and 10. OR
Todd I, Spickett G (April 2010) Lecture Notes: Immunology. 6th edition.
Wiley/Blackwell. Chapters 5, 6, 7, 8, and 10.