2. Objective of Lecture
Which cells perform phagocytosis
The physiological location of these cells
The function of phagocytes in the resolution of infection
How phagocytes arrive at the site of infection
The mediators involved in this process and their origin
How phagocytes are activated in response to infection
The mediators involved in this process and their origin
The mechanisms these cells use to kill phagocytosed pathogens
The mediators produced by phagocytes that kill pathogens
The differences in functions of the different subsets of phagocytes
3. The role of phagocytes in innate immunity
The earliest of these mechanisms are preformed and therefore are
constantly available to halt infection.
However, by the time complement activation has taken place, further steps
are usually required to prevent the further development of infection by
removing pathogens, infected cells or cellular debris.
These functions cannot be carried out by preformed mediators and therefore
require the assistance of phagocytes.
Individuals with deficiencies in phagocyte function are highly susceptible to
4. Macrophage/Monocyte Neutrophil
Morphology Large mononuclear cells with
Smaller cells with multi-lobed
nucleus and neutral cytoplasmic
Location Often resident in tissues (remove
routine cell debris)
Blood – requires recruitment to
site of infection
Killing ability Require activation by bacterial
Activated during recruitment,
then able to kill internalised
After killing Migrate to local lymph nodes Die at site by apoptosis (then
taken up by macrophages)
Can present antigen (Class II up-
regulated by IFNg)
Cannot present antigen (don't
normally express Class II)
5. How are Neutrophils recruited to the site of infection?
Neutrophils are the first cells recruited to the
site of an infection and neutrophils carry out
most of the killing at a site of infection.
However, under normal physiological
conditions, neutrophils are not found in
tissues but mainly in the circulation (2-7 X
109/litre of blood).
Neutrophils therefore have to be recruited
from the blood accross the vascular
endothelium into tissue (extravasation).
This occurs at blood vessels close to sites of
inflammation, as a result of production of a
number of proinflammatory signals :
6. How are Neutrophils recruited to the site of infection?
Pro-inflammatory molecules produced in response to
infection/tissue damage include lipid derived
proinflammatory mediators (e.g. prostaglandins (PGs),
leukotrienes (LTs) and platelet activating factor (PAF)) as well as
reactive oxygen and nitrogen species
Plasma enzymes are activated in response tissue damage such as
Plasmin that degrades clots releasing chemotactic and
vasodilating fibrin breakdown products as well as directly
degrading C5 to C5a. Bradykinin is produced via kallekrein and
causes vasodilation and increased vascular permeability.
components are directly
inflammatory (e.g. C5a)
Others (C3a, C4a also
C5a) cause mast cells to
histamine, TNF, IL-1,
LTs and PGs
cytokines produced by
tissue phagocytes. These
include IL-1, IL-6, IL-8,
IL-12 and TNF
9. Production of inflammatory mediators causes blood vessels to dilate and become leaky and this slows the local blood flow, causes
cells to marginate and fluid to accumulate in tissues. This produces the characteristics of inflammation : tumor, rubor, calor
and dolor and they also cause the endothelium to become activated. Activation of the endothelium is the first step towards
neutrophil extravasation Recruitment of neutrophils can be divided in to 4 processes:
1. Endothelial activation and Rolling: Neutrophils constitutively express ligands and
receptors (e.g. sialylated carbohyrates and L-selectin respectively) which interact with reciprocal
receptors and ligands (e.g. P- and E-selectin and GlyCAM-1 respectively) on endothelial cells.
Blood vessels near sites of inflammation dilate and blood flow reduces, this allows these
interactions to take place and the neutrophils marginate and roll along endothelium in these
areas. Inflammatory mediators (esp. IL-1 and TNF) increase expression of E and P-selectin on
endothelial cells which can then bind mucins (sialyl -Lewisx, PGSL) on the neutrophil and this
allows tethering of marginated cells.
2. Activation: activation of the neutrophil is essential for extravasation. Neutrophils are
activated by complement components (iC3b, C5a), inflammatorylipid mediators (eg. leukotrienes
(LTB4), PAF), cytokines (IL-8; MIP1b) and bacterial products (e.g. N-formylated peptides such
as N-formyl-methionyl-leucylphenylalanine (fMLP)). Once activated neutrophils must gain
access to the infected tissue. They do this using adhesion molecules (selectins, integrins,
intercellular adhesion molecules (ICAMs)).
3. Arrest: activation of neutrophil causes conformational change in integrin (LFA-1), which
allows it to bind to ICAM. Local inflammatory mediators also cause upregulation of integrins
(e.g. LFA-1, Mac-1) on neutrophils which interact with receptors (e.g. ICAM-1, ICAM-2)
upregulated on endothelium. These stronger interactions allow the neutrophil to stop rolling and
cross the endothelium into tissue in a process known as diapedesis
4. Migration: Next, the Neutrophil responds to a group of molecules called chemoattractants
to make its way between the endothelial cells of the blood vessel by a process called diapedesis.
Chemoattractants include complement protein C5a, bacterial products (e.g. fMLP), lipid
mediators (LTB4, PAF) and cytokines (in particular IL-8, also MIP1b). These chemoattractants
also form a chemical gradient in the tissue, and the neutrophil migrates up this gradient (a
process called chemotaxis) in order to find the site of infection. During this process the
neutrophil is activated and ready to kill on its arrival.
Normal tissues harbour low numbers of resident macrophages that are long lived cells (months compared
with neutrophils (6h in circulation, 2-3 days in tissue). They come under a number of tissue specific names
Kupffer cells (liver), Microglia (brain), Histiocytes (skin), Mesangial cells (kidney). These cells are not
activated and would not automatically be able to kill a pathogen – these cells require to undergo
activation in order to get their pathogen killing processes going. This is important, as most of the time
these macrophages are digesting the normal cell debris or dead cells that result from normal wear and tear
in tissues. Classically activated macrophages can be found in 2 activation states:
i. Primed – this happens in response to IFNg, which is produced mainly by T cells. This causes
macrophages to increase the expression of Class II molecules on their surface, and upregulate
phagocytosis and some killing mechanisms. Natural Killer (NK) cells can act as an innate source of IFNg,
allowing macrophage activation to occur in advance of adaptive immune responses (see Figure).
ii. Hyperactivated – this requires the presence of pathogen derived molecules, the best studied is
lipopolysaccharide (LPS), which is abundant on the surface of gram negative bacteria. LPS causes
macrophages to secrete inflammatory mediators such as IL-1, IL-6 and TNF, and also fully upregulate
their killing machinery
However, macrophages can also be alternatively activated through engagement of different cellular
receptors, or mediators of the innate and adaptive immune responses (see Figure 1 from Gordon (2003)
Nat. Rev. Immunol.)
Macrophage numbers in tissues are topped up by migrating monocytes from the blood and approximately
24 hours after infection, large numbers of monocytes are actively recruited from the blood to the site of
infection. IL-1 induces expression of ICAM on endothelial cells which interacts with beta integrins (e.g.
Mac1(CD11b:CD18)) to induce rolling and adhesion. Like Neutrophils, Monocytes follow gradients of
chemokines to the site of infection of which, IL-8 and MCP (monocyte chemoattractant protein) are
11. Once formed, the pathogen-containing phagosome can then fuse with other
cellular compartments containing microbiocidal products.
Neutrophils have 3 main microbiocidal compartments:
Primary granules - contain serine proteases, lysozyme and
phospholipase A2, highly acidic.
Secondary granules - similar to primary, but also include lactoferrin,
elastase and collagenase
Tertiary granules - at leading edge of migrating neurophil and contains
gelatinases capable of degrading basement membranes
Macrophages kill pathogens by fusing phagosomes with lysosomes which
have similar constituents to the neutrophil primary granule.
In addition to these mediators, phagocytes also posess a variety of oxygen
dependent killing mechanisms. Both phagocytes produce a respiratory
burst which produces superoxides and hydrogen peroxide. Neutrophils
contain an enzyme, myeloperoxidase, which can convert superoxide into
hypochlorite (very effective bleach). Macrophages contain an enzyme called
nitric oxide synthase which is activated by IFNg and TNF (or LPS)
treatment which produced nitric oxide, which is very effective at killing.