Phagocyte function and Macrophages

Question 1

Why is phagocytosis important?

Answer 1

*Protects against invading organisms
*Processes foreign antigens
*Presents parts of foreign antigens activating adaptive immune system
(links innate and adaptive immunity)
*Purges debris and damaged/dying host cells

Question 2

Which cells are phagocytic?

Answer 2

-Macrophage
-Neutrophil (Polymorphonuclear leukocyte, PMNs)
-Dendritic cell
-eosinophil
-basophil

Question 3

Where do phagocytes act ?

Answer 3

• Sites of infection (attracted by inflammatory response)
• Mucosal lymphoid tissue (patrolling of Respiratory & GI tracts, Liver)
• Lymph nodes (filtering from Lymph fluid)
• Spleen (filtering from Blood)

Question 4

List the process of phagocytosis in simple terms

Answer 4

• Activation of resting phagocytes
• Chemotaxis
• Attachment and signalling
• Phagosome maturation
• Killing and antigen presentation

Question 5

Describe the Activation of resting phagocytes

Answer 5

Question 6

Describe the Chemotaxis of phagocytes (Step 2)

Answer 6

Movement towards an increasing concentration of attractant bacterial protein, capsule, cell wall, complement (C5a), chemokine (CXCL-8)

Question 7

Compared the enhanced and unenhanced attachment of phagocytes (step 3)

Answer 7

Unenhanced
Pattern Recognition Receptors (PPRs) for
Pathogen-Associated Molecular Patterns (PAMPs)
(eg. Microbe cell wall components, viral DNA)

Enhanced
Opsonin Recognition receptors for Antibody (Fc) or Complement (C5a) fractions previously fixed to microbe components

Question 8

Describe unehanced attachment of phagocytes, giving examples (step 3)

Answer 8

pattern recognition receptors (PRRs) are designed to recognise a few highly conserved structures Pathogen-Associated Molecular Patterns (PAMPs) (examples in image)

PAMPs can be hidden/absent by pathogen diversity in cell wall structure

Unenhanced - Pattern recognition receptors (PRRs) can be one of two:
 Endocytic pattern recognition receptors
 Signalling pattern recognition receptors

Question 9

Describe Unenhanced - Endocytic pattern recognition receptors

Answer 9

Mannose receptors
bind to terminal mannose and fucose on microbial glycoproteins/lipids

Scavenger receptors
bind to other bacterial cell wall components
e.g. Lipopolysaccharide (LPS), peptidoglycan

Question 10

Describe Unenhanced - Signalling pattern recognition receptors

Answer 10

Toll-like receptors
different TLRs bind different microbial molecules but TLR’s do NOT induce phagocytosis

 Binding to bacterial and fungal components:
transmits a signal to the nucleus inducing cytokine production e.g. IL-12, TNF-a, IL-6
leads to innate immune defences such as inflammation, activation of other phagocytes

 Binding to viral components:
triggers anti-viral interferons (IFN)

Question 11

List the types of TLRs, their ligands and cytokines

Answer 11

TLR4 detects Gram-negative bacteria because of its ability to recognize endotoxin. It can also activate the macrophage by a second pathway that is initiated by Trif, which leads to a secondary production of IFNbeta and
autocrine activation of additional macrophage genes.

CD14 is a GPI-linked membrane protein that facilitates the recognition of LPS by TLR4 so that it increases LPS sensitivity. Recently CD14 has also been shown to facilitate recognition of ligands by TLR2 and TLR3.

TLR4 also recognizes degraded extracellular matrix and the nuclear protein high mobility group 1 protein (HMGB1) which can be released by necrotic cells, an example
of damage-associated molecules.

Question 12

Describe enhanced attachment

Answer 12

More specific and efficient
Mediated through opsonins
C3b + C5a promote enhanced attachment
Fc fragments of IgA and IgG

Macrophages use both enhanced and unenhanced attachment
Neutrophils use mainly enhanced attachment

Opsonic receptors require antibody or complement to recognize the target

Bacteria opsonized by C3 fragments or antibody engage complement receptors (CR) or Fc receptors (FcR).

Monocytes and macrophages express a range of receptors (CR1, CR3, CR4) for C3 cleavage products that may become
bound to pathogens, immune complexes or other
complement activators.

Question 13

Describe phagosome formation

Answer 13

1. The microbe attaches to macrophage receptors on its cell surface
2. This causes actin cytoskeleton rearrangement, whereby the Actin filaments align in pseudopodiaaround the microbe
3. The microbe is slowly engulfed by the cell membrane which invaginates to form a vesicle on its inner side
4. the vesicle then separates from the cell membrane forming a phagosome.

Question 14

Describe phagosome maturation

Answer 14

• electron pump uses ATP to pump protons into the phagosome, lowering the pH inside - has a direct effect on microbial growth

Destruction
-Phagosome fusion with lysosome form phagolysosome
-Oxygen-dependent – ROS, NO : Oxygen-independent – enzymes, Proteases, Nucleases

Processing
-MHC-II presentation of antigens, TLR signalling inducing pro-inflammatory cytokine release

Question 15

How do lysosomes contribute to Oxygen-independent killing?

Answer 15

Lysosomes also contain

Lysozyme – breaks down proteoglycans in bacterial cell walls
Cathepsins – proteolytic enzymes that degrade bacteria
Lactoferrin – deprives bacteria of iron
Defensins – small peptides that form channels in lipid bilayers

Question 16

Describe Oxygen-dependent killing

Answer 16

the generation of reactive oxygen and nitrogen species.

Oxidation of proteins, carbohydrates, DNA destroys function
Oxidation of lipids can destabilise cytoplasmic membrane structure

Question 17

How does Streptococcus pneumoniae evade phagocytosis?

Answer 17

Streptococcus pneumoniae – has a capsule
Resists unenhanced attachment by preventing binding ofpattern recognition receptors
Resists C3b opsonisation

Question 18

How does Yersinia evade phagocytosis?

Answer 18

Yersinia – depolymerise actin preventing engulfment

Question 19

How do Salmonella and Mycobacterium Block phagolysosome killing

Answer 19

Salmonella – more resistant to toxic ROS and defensins
Mycobacterium – blocks phagosome fusing with lysosome

Question 20

How does Staphylococcus aureus Kill phagocytes

Answer 20

Staphylococcus aureus – produces leukocidin,
a toxin which damages membranes

Question 21

Why is Phagocytosis of apoptotic cells important?

Q. Give an example in the immune system where macrophages phagocytose apoptotic cells.

Answer 21

 Important for regulation of immune responses
 Prevents leakage of cytotoxic or antigenic contents
 Rapid, efficient and ‘silent’ (no inflammation)

A. Thymocytes that fail the processes of positive and negative selection die by apoptosis and are phagocytosed by thymic macrophages. Also B cells that die within lymphoid follicles are taken up by tingible body macrophages

Question 22

Which phagocytes eat apoptotic cells?

Answer 22

Professional phagocytes – macrophages etc.
mobile and can infiltrate tissues

‘Amateur’ phagocytes – essentially any cell can take up a neighbouring dying cell
slower kinetics than the professional phagocytes

Question 23

What are the signals from apoptotic cells?

Answer 23

‘find-me’ signals attracting phagocytes
soluble and secreted by apoptotic cells

‘eat-me’ signals activating phagocytes
expressed on the apoptotic cell surface

‘don’t eat-me’ signals preventing phagocytosis
NOT expressed by dying cells

Question 24

Describe the Host “Find me” signals

Answer 24

Chemoattractants
-Lysophosphatidylcholine (LPC)
-ATP
-Chemokines e.g. CXCL3 and CXCL5 (neutrophil
chemoattractants)

Apoptotic neutrophils produce ‘find me’ signals (e.g. lysophosphatidylcholine) that attract macrophages

Question 25

Describe the Host “Eat me” signals

Answer 25

Normally expressed on the inner leaflet of the plasma membrane. In apoptosis PtdSer is translocated to the outer part of the lipid
bilayer. Involves a flipase and scramblase
enzyme

The appearance of phosphatidylserine (PS) in the outer leaflet of the plasma membrane is characteristic of apoptotic cells. PS recognition by PS-binding proteins stimulates the uptake of apoptotic cells and the production of antiinflammatory mediators, especially TGFb, which inhibit production of proinflammatory chemokines and cytokines.

Question 26

Describe scenarios where there is no full phagocytosis but just host cell housekeeping

Answer 26

a) RPE (retinal epithelial cells) in the eye remove
fragments of photoreceptors that die by apoptosis in response to light – essential for
maintaining vision

b) Microglia in the brain help trim and drive neuronal structuring by “nibbling” them (trogocytosis)

Question 27

Describe don’t eat me signals

Answer 27

Host cells control the phagocytic activity of macrophages by displaying the ‘don’t eat me’ signal CD47. CD47 engages a receptor in macrophages called SIRPalpha that through
its immunoreceptor tyrosine-based inhibitory motif (ITIM) motif inhibits the uptake process. CD47 expression by tumor cells has been proposed as an immunosurveillance escape mechanism

CD31 expressed on endothelial cells, platelets, macrophages and Kupffer cells, granulocytes,
lymphocytes (T cells, B cells, and NK cells), megakaryocytes, and osteoclasts binding normally leads to repulsion signals. During apoptosis CD31 signalling is disabled so that the phagocyte is not actively rejected

Question 28

What are the consequences of apoptotic cell removal?

Answer 28

Secretion of ‘pro-healing’ anti-inflammatory cytokines (e.g. IL-10, TGF-β)
-reduce inflammation
-promote wound healing

Presentation of antigens
-maintenance of self tolerance
-release of unprocessed pathogens/antigens

Question 29

Describe the Defects in apoptotic cell clearance - links with disease

Answer 29

 Leakage of content from non-cleared apoptotic cells act asninflammatory factors

 Lack of suppression of inflammatory and immune responses
* no release of anti-inflammatory cytokines e.g. TGF-b, IL-1

 Onset of autoimmune disorders linked to inefficient removal of apoptotic cells
* knock-out mice studies (systemic lupus erythematosis)

Question 30

Describe the Defects in phagocytosis- links with disease

Answer 30

 Chronic granulomatous disease Missing NADPH oxidase components

 Chediak-Higashi Syndrome Hyperpigmentation/albinism delayed phagosome-lysosome fusion low chemotaxis

Both lead to recurrent infections

Question 31

List the Macrophage subsets and their role

Answer 31

Question 32

Describe the M1 phenotype

Answer 32

Tissue damage/microbial entry triggers TH1 driven inflammation

Monocytes recruited from circulation/bone-marrow
Monocytes differentiate into macrophages which can help activate DC’s to stimulate Th1/NK cells which then release IFN-γ
IFN-γ promotes Macrophages to become M1-phenotype
M1 inflammatory phenotype produce NO, ROS, IL-1, TNF & MMPs
Inflammatory microbial response

Question 33

Describe the M2 phenotype

Answer 33

Alarmins (IL-25, IL-33 TSLP) stimulate Th2 and
other cell subsets to release IL-4 & IL13

Macrophages develop M2 and later regulatory
phenotypes which begin to accumulate

M2 Macrophages antagonise M1
Macrophages and promote wound healing

Regulatory Macrophages stop wound healing when
complete

Question 34

Describe how Helminth infection can also promote Macrophage
proliferation in situ

Answer 34

Nematode worm infection drives high levels of IL-4
through Th2 and other cell types

High IL-4 promotes M2 macrophage proliferation in
situ
M2 macrophages kill the pathogen

High IL4 and M2 macrophage presence can also produce a M1/M2 combined phenotype that is proliferative