CPAT 3201 (2020)

Inflammation
Lecture 2: “Neutrophils and
Macrophages”

Professor Georges E. Grau, M.D., Ph.D.

Discipline of Pathology

Vascular
Immunology
Unit
 “INFLAMMATION”
Outline of the 4 lectures

• Lecture 1: Acute inflammation - mediators

• Lecture 2: Neutrophils and macrophages

• Lecture 3: Chronic inflammation

• Lecture 4: Wound healing

Vascular
Immunology
Unit
 Inflammation is important
--- 10 reasons ---
• Pathogenic basis of many diseases
• Roles in triggering IMMUNITY
• A set of processes that keeps us alive!
• Most AI drugs work by preventing formation or actions of mediators
• Works on a daily basis!
• Deficiency in a segment of inflammation can cause severe diseases
• Complex relationship inflammation / cancer
• Dysregulation of inflammation: lethal or chronic, debilitating diseases
• Problems caused by AI drugs (iatrogenic)
• Angiogenesis! (ageing, tissue remodelling, …)
 Today’s lecture (#2)
“Neutrophils and macrophages”
1. Neutrophil emigration
2. Neutrophil movement and chemotaxis.
3. Anti-microbial mechanisms of neutrophils
4. Macrophages.
5. Inflammatory cytokines.
6. Systemic manifestations of inflammation.
7. Recognition of pathogens by phagocytes.
 Today’s key learning issues

• Neutrophil and macrophage properties

• Mechanism of neutrophil emigration

• Neutrophil anti-microbial mechanisms

• Pattern recognition receptors

 Overview: Host defences against micro-organisms

Pre-formed non-specific
effectors: “INNATE IMMUNITY”
bacteria Barriers: • neutrophils
fungi • epithelium (e.g. skin) • plasma proteins
viruses • secretions (e.g. mucous) (e.g. complement system)
• Natural Killer (NK) cells
•  T cells
< 6 hours

Further exposure Antigen drains Activation of

to NK cells and to lymph node macrophages
macrophages

> 120 hours

Humoral and cellular immunity: “ADAPTIVE IMMUNITY”

• B lymphocytes • T lymphocytes
 Roles of neutrophils in inflammation

To kill micro-organisms, particularly bacteria.

To release *chemotactins that attract other neutrophils and

macrophages

*Chemoattractant substances.
 Human peripheral blood leukocytes

basophil monocyte

lymphocyte

neutrophils

eosinophil red blood cells

 Kinetics of human neutrophil
production and loss
• Neutrophils are produced in the bone marrow at ~1.5
million cells per second (= 1 tonne per year).
• They spend about 12 hours in blood and up to 24 hours
in tissues, but they enter tissues only when signalled to
do so (e.g. in inflammation).
• They are terminally differentiated cells and cannot
proliferate under any circumstances.
 Neutrophil events in acute inflammation

• Margination in small vessels.

• Emigration from vessel into tissue.

• Phagocytosis of micro-organisms.

• Killing of micro-organisms.

• Death of neutrophil.
 Tissue accumulation of leukocytes in
inflammation
Oedema Monocytes/
Neutrophils macrophages

• Early (minutes – hours):

➢ maximum rate of migration
of neutrophils.

• Later (hours – days):

➢ maximum rate of migration
Activity

of monocytes.

• Monocytes differentiate into

0 1 2 3 4 macrophages.
Days
 Margination of neutrophils

• Circulating neutrophils touch vessel walls.

• Neutrophils roll slowly along endothelium.

• Neutrophils adhere to endothelium.

• Margination is induced by vasoactive mediators

and cytokines, acting on the endothelium.
Fluid-filled spaces

Congested
vessel

Marginating
neutrophils
 Neutrophil adherence to endothelium
Stage 1: “rolling”
• mediated by proteins called selectins, newly
expressed on the endothelium
Stage 2: “adhesion”
• mediated by integrin proteins, constitutively
expressed on the endothelium and the neutrophil
Stage 3: “emigration”
• also mediated by integrins
 Neutrophil margination and emigration

selectins
integrins
flowing margination

arrest &
rolling activation
spreading
diapedesis
blood (emigration)

tissue
endothelium

Selectins: on neutrophils and endothelial cells

Integrins (on neutrophils) bind to adhesion molecules on endothelial cells
 Neutrophil movement in tissues is
influenced by chemicals
CHEMOTAXIS: increased unidirectional
movement, towards the source of the signal.
TISSUE ACCUMULATION: nett increase in
neutrophils in an anatomical location.
CHEMOTAXIN = CHEMOTACTIN =
CHEMOATTRACTANT: agent that can induce
chemotaxis in target cells.
 Neutrophils move up
chemotactic concentration
gradients Chemoattractant

concentration
gradient of
chemotactin

Neutrophil
margination

Neutrophil Basement membrane Endothelium

From an early edition of Robbins Pathology
 direction of lamellipodium
movement

nucleus

granules

uropod

“Polarised” neutrophil
Structural and biochemical properties of neutrophils
Structure
• multi-lobed nucleus
• two types of cytoplasmic granule
• no endoplasmic reticulum or Golgi apparatus
• few mitochondria
Biochemistry
• protein synthesis capacity impaired
• energy from anaerobic glycolysis (glycogen)
Consequences for function
• short-lived cell
• incapable of proliferation
 Phagocytosis: formation of the phagosome
1 = recognition
granule 2 = engulfment
3 3 = phagosome formation

membrane
invagination
2

nucleus
1
phagosome
particle (e.g. bacterium)
The enzyme NADPH oxidase is located on the outer membrane. Phagocytosis ensures that
it generates reactive oxygen species into the phagosome.
Phagocytosis: formation of the phagolysosome
particle 1
phagosome

phagolysosome
3
nucleus
2 granules fusing
with phagosome
 Neutrophils never give up…

• As they die, neutrophils release “neutrophil

extracellular traps (NETs)”

• Made up of DNA strands with anti-microbial

proteins attached

• Trap and kill bacteria

 Formation of Neutrophil Extracellular Traps

microorganism
granules

nucleus granule proteins neutrophil lyses,

breaks down adhere to DNA NETs released
strands

Modified from Papayannopoulos Trends Immunol 2009:30;513.

 NETs in action

Bacteria (red) in neutrophil extracellular traps.

From: V Brinkmann, in Medina, J Innate Immunity 2009:1;276

 Formation of the Exudate in Acute Inflammation
Injury, Infection

Formation of Formation of
Vasoactive Mediators Chemotactic Factors

Upregulation of
Vasodilation Endothelial Contraction Adhesion Molecules Neutrophil
(arterioles) (post-capillary venules) on Endothelium Activation
(post-capillary venules)

Hyperaemia
Increased Vascular
and Increased Neutrophil Margination
Permeability
Hydrostatic and Migration
to Proteins
Pressure

Fluid, Salts, Complement, Antibody, Neutrophils

move into affected tissue = EXUDATE

Tissue Damage Sequelae

And now macrophages…..
 Roles of macrophages in inflammation

• Killing of microorganisms
➢bacteria, fungi and parasites

• Phagocytosis and degradation of debris.

• Production of cytokines
➢ regulate the activities of other cells.

Image: Champion, JA (2009)

Pharmaceutical Research 26:244
 Tissue accumulation of leukocytes in
inflammation
Oedema Monocytes/
Neutrophils macrophages

• Early (minutes – hours):

➢ maximum rate of migration
of neutrophils.

• Later (hours – days):

➢ maximum rate of migration
Activity

of monocytes.

• Monocytes differentiate into

0 1 2 3 4 macrophages.
Days
 Monocyte / macrophage kinetics

Bone marrow origin (as

monocytes) at ~100,000 cells
per second.
Monocytes
Circulate (as monocytes) for
~2 – 3 days.

Remain in tissues for months

or years; duration varies from
tissue to tissue
Macrophages
 Human peripheral blood leukocytes

basophil monocyte

lymphocyte

neutrophils

eosinophil red blood cells

Structural and biochemical properties of macrophages
Structure
endoplasmic reticulum • single-lobed nucleus
• no obvious cytoplasmic granules
• prominent endoplasmic reticulum
and Golgi apparatus
• abundant mitochondria

Biochemistry
• large protein synthesis capacity
• energy from aerobic respiration or
anaerobic glycolysis (glycogen)

Consequences for function

• long-lived cell
• can differentiate and proliferate

nucleus mitochondria
Cytokines
 Cytokines

Characteristics Types of cytokine action

• Produced by a cell involved autocrine

in an immune or inflammatory A
reaction paracrine

• Modifies the activity of a

different type of cell involved systemic
in that reaction Blood (endocrine)
vessel
• Stimulates or inhibits
cellular functions
C
Cytokines are proteins
Interleukin-1 (IL-1) and tumour necrosis factor
(TNF) in inflammation
• Produced by several types of cell
➢ macrophages are major source

• Broadly similar activities

➢ Often act synergistically
➢ Products of different genes
➢ Bind to different receptors

• Over-production can contribute to tissue damage

➢ e.g. arthritis
 Synergistic systemic effects of IL-1 and TNF in inflammation

Injury /
Infection T

Stem cells Proliferation

T
Leukocytes Macrophage
T
T
T

TNF
Interleukin-1
Expression

© Nick Hunt 2013

of adhesion Hypothalamus
molecules

Fever
TNF = tumour necrosis factor
 Innate immune cell recognition of
pathogens
• Identification of “non self”

– Evolutionarily conserved microbial components or

products (“pathogen-associated molecular patterns”,
PAMPs)

• Co-evolution of pattern recognition receptors

(PRRs)

– Toll-like receptor system

– Several others
© Nick Hunt 2013
 Pattern recognition receptors:
Interface between the microbial world and immune system

Microbial world

Pattern recognition receptors

Innate immunity

Adaptive immunity
Pattern recognition receptors (PRRs):
Roles

• Detect microbial infections

• Trigger anti-microbial host defences
• Initiate adaptive immune responses
 PRRs recognise
components of pathogens
• Recognise “Pathogen-associated molecular
patterns” (PAMPs)
• PAMPs (microbial origin)
– lipids
– carbohydrates
– nucleic acids
– proteins
• PRRs: Expressed on many cells, including
leukocytes, epithelium, endothelium
 Toll-like receptors (TLRs)

• Most important subfamily of PRRs?

• Molecules equivalent to TLRs found in

mammals, plants and insects
 Toll-like receptors recognise components of pathogens
lipoproteins
LPS
lipoproteins TLR2 TLR6
TLR4 flagellin
TLR2
extracellular
TLR1 TLR5 orientation

single single plasma

stranded stranded membrane
double RNA RNA
stranded
RNA TLR7 TLR8 CpG DNA
TLR9
intracellular
TLR3 orientation

endosomal
membrane
That explains recognition of foreign substances –
but what about sterile inflammation?

Damage-associated molecular patterns

(DAMPs) are released from damaged
cells/tissues and these act through PRRs, like
PAMPs do, to initiate inflammation.

Examples: ATP, RNA, DNA.

 Today’s key learning issues

• Neutrophil and macrophage properties

• Mechanism of neutrophil emigration

• Neutrophil anti-microbial mechanisms

• Pattern recognition receptors