Macrophages

Question 1

Who discovered Macrophages?

Answer 1

Ilya Mechnikoff was a russian developmental biologist who developed the theory of cellular immunity; studying the phagocytosis of bacteria and combating infection. He observed mobile cells in starfish after injury.

Question 2

Describe a macrophage discovery timeline

Answer 2

1800- Mechnikoff discovery of phagocytes
1970s- mononuclear phagocyte system and classical activation of macrophages (T cells needed to signal to macrophages.)
1980s-macrophage heterogeneity shown to be in different subsets by monoclonal antibodies and specific markers (no one macrophage is similar to another. They are plastic.)
2003- Monocyte subsets shown by markers; 2 important subsets with different functions.
2008- A common precursor of monocytes and dentritic cells in vitro.
2012-Self-renewal of many tissue macrophages; Macrophages can divide and maintain their own cell numbers without monocyte input.
2014-Many tissue macrophages derived from yolk sac precursors and are long lived in tissues and self-renew themselves.

Question 3

In what way are macrophages plastic cells?

Answer 3

They can turn genes on or off and acquire phenotypes depending on the conditions to mediate certain functions.

Question 4

Dendritic cells

Answer 4

Important in antigen presentation but now considered part of the monophagocyte system

Question 5

What defines macrophages? Summary of phagocyte function

Answer 5

They have a large cell size, mononuclear. Phagocytosis occurs when receptor binds to bacterium ligands, triggers a phagocytic cup (a small into the membrane) for engulfment. This is an energy dependent process that forms vacuole called a phagososme, which fused with lysosomes (containing proteases) forming a phagolysosome in which the bacterium is degraded.

Question 6

What do they look like in tissues? And their different functions?

Answer 6

Tissue prepared as a section, fixed and stained with an specific macrophage antibody called F480. Appear as brown

In liver; they are called KUPFFER CELLS which line the sinuses within the liver in order to clear circulating pathogens, old RBC, debris. Important in regulating inflammatory response.

In lung: alveolar macrophages in alveolar spaces (where gaseous exchange takes place) Phagocytose inhaled pathogens, and regulate a protein that influences surface tension regulation and gaseous exchange. Therefore have a role in homeostasis of tissue function. No macrophages here leads to disease called protinosis.

In brain: microglia. Important in neuronal communication function, early development by clearing apoptotic cells and in inflammatory disease (Alzheimer) the macrophages remove toxic components like Alzheimer protein (alpha-beta protein.)

Question 7

List functions of macrophages (generally)

Answer 7

1) Developmental biology; the body makes too many cells early on, these are removed.
2) Metabolism- release of soluble protein adipokines which regulate adipocytes important in metabolism (diabetes)
3) Haematopoiesis-recognition and uptake of RBC nuclei (erythoplast) excluded (nuclei with cytoplasm containing haemoglobin) This allows macrophages to recycle iron. Macrophages produce trophic factors, molecules that promote development of red blood cells.
4) Neurobiology- cross talk with neuronal cells
5) Acute and chronic inflammation
6) self defense of the innate immune system; kill naturally from birth but can be activated to kill.
7) Antigen transfer and presentation. However not as efficient as dendritic cells.

Question 8

Macrophage development

Answer 8

order of precursors: first yolk sac, the foetal liver develops after yolk sac, then bone marrow which sustains adult haematopoiesis.
Monocytes produced in the bone marrow are released with a half life of 2-3 days. Small number are recruited if there is a inflammatory response where they differentiated into macrophages. However, monocytes do not repopulate macrophage populations already present in issues; so monocytes mainly for self defence purposes.

Question 9

List specific macrophage populations in the different organs

Answer 9

1) Liver- Kupffer cells
2) Spleen-red pulp macrophages
3) Peritoneal cavity- peritoneal macrophages
4) Lung-alveolar macrophages
5) Bone-osteoclasts (derived from macrophage precursors which undergo fusion and secrete proteases which digest material)
6) Central Nervous system- mycroglia

Question 10

What are the differences between monocytes and macrophage appearance

Answer 10

Monocytes are smaller with a kidney shaped nucleus

Question 11

What does M-CFU stand for?

Answer 11

Macrophage colony forming units; it is a macrophage precursor.

Question 12

OLD! Describe a general pathway of development including precursors.

Answer 12

HSC= GM-CFU=M-CFU (in both fetal liver and bone marrow)
In fetal liver: M-CFU= Monoblast= Pro-monocyte= Monocye= Macrophage
In bone marrow: M-CFU= Monoblast= Pro-monocyte= Ly6C + (inflammatory monocyte) = into tissues= Ly6C (negative) resident mmonocyte= Macrophage, Dendritic cell or osteoclast

Question 13

What does Ly6C +/- indicate?

Answer 13

+ indicates an inflammatory monocyte. It has function to kill pathogens/self defence

• indicates monitoring/control resident monocytes..

Question 14

What technique is used to determine Transcription factor influence on development?

Answer 14

Knock out mice- mice lacking in Myb TF will not make haemopoietic cells but will make yolk sac macrophages.

Question 15

What are the Transcription factors involved?

Answer 15

• Myb: needed fo haemopoietic stem cells
• PU.1: needed for full myeloid cell development; without it septicema causes death.
• NR4A1: needed for LyC6- resident monocytes.
• Mafb: needed to have tissue macrophages but not monocytes.
• Spi-C: needed for splenic red pulp macrophages.

Question 16

What is the importance of transcription factors?

Answer 16

Different subsets of macrophages in different tissues may have certain genes switched on by these specific T.F which cause their function.

Question 17

List the growth factors involved in macrophage development

Answer 17

• M-CSF/CSF1: needed for blood monocytes so less osteoclasts so less bone reasorption. There is selective loss of tissue macrophage population.
• IL34 (interleukin): needed for microglia and Langerhan cells

Question 18

Describe functions of macrophages:

Answer 18

1) waste disposal
2) Lung
3) Initiation and resolution of inflammation
4) Adipose tissue
5) Bone

Question 19

Waste disposal

Answer 19

Aged blood cells have surface properties that are recognised by red pulp/Kupffer macrophages. If not impaired iron recycling.

erythroid nuclei is taken up by bone marrow and fetal liver. If not IFN-b produced and hematopoiesis is disrupted in bone marrow.

Clearance of apoptotic cells; known as the silent clearance as macrophages don’t switch on an immune response. This must be controlled to prevent immune response for this process and then autoimmunity like stemic lupus (SLE)

Question 20

Lung Function

Answer 20

To clear surfactant protein. If not leads to disease called alveloar proteinosis

Question 21

Inflammation function

Answer 21

1) pathogen recognition
2) inflammation response involving TNF, IL-6 ect.
3) resolution involving lipid mediators, IL-10
if not there is chronic failure, tissue damage and fibrosis

Question 22

Metabolism function

Answer 22

Macrophages in white adipose tissue regulate lipolysis. If not causes insuin resistance.

In brown adipose tissue regulates thermogenesis. If not causes loss of adaptive thermogenesis

Question 23

Bone function

Answer 23

Osteoclasts, relates to phagocytic cells, undergo a fusion. They secrete proteases which degrade the bone matrix. This process depends on cytokines M-CSF and RANKL(ligand).
If no growth factors, it causes osteopetrosis, where bone becomes too big, as it is not degraded.

Question 24

What diseases can be caused in various parts of body?

Answer 24

In brain- normal: fluid balance, neuronal patterning. Pathology = neurodegeneration.

In bone- normal bone remodelling and haematopoiesis. Pathology = leukemia, osteoporosis, osteopetrosis.

Heart- normal-artherosclerois

Liver- Kupffer cells normally remove toxins, lipid metabolism. Pathology = fibrosis.

Pancreas- pathology = cancer and metastasis. Cancer; macrophages aid the cancer to grow.

Adipogenesis- Pathology = obesity and diabetes

Spleen- immunity. Pathology = arhritis, EAE, IBD

Question 25

Describe macrophage and cancer relationship

Answer 25

Macrophages are present in many different area in a tumor and many are attracted to the hypoxia regions (lack of oxygen.) Here macrophages produces growth factors which stimulate production of blood vessels which are needed by tumor.

Killing off macrophages therefore may stop tumour from developing normally.

Question 26

Functions of monocytes.

Answer 26

1) Most tissues dont need monocytes, however some tissues like the GUT, where there are a very large number of microorganisms, monocytes needed as macrophage precursor in adults.

2) In the blood, there are two subsets; i) inflammatory LyC6 [high] which are rapidly recruited to inflammatory sites.
ii) Patrolling LyC6 [low] which maintain endothial cell integrity in blood vessels. This acts as a vaccum to remove debris from endothelial cells. But it can be activated in the presence of a dangerous pathogen, causing the monocyte to recruit neutrophils to kill cell and allows endothelial cell neighbour to produce a new endothial cells.

Monocyte responds to danger!

Question 27

What are the functions of macrophages in innate immunity and acquired immunity

Answer 27

INNATE- pattern recognition receptor, complement receptor

ACQUIRED- Fc and complement receptors to bind immune complexes

INAATE AND ACQUIRED
-production of cytokines and other soluble mediators for promoting inflammatory responses.

-antimicrobial activities: reactive oxygen species and lysosomal enzymes

Question 28

General Macrophage functions

Answer 28

• Phagocytosis/endocytosis of non self microbes
• Antimicrobial activity
• Production of cytokines and other soluble mediators
• antigen presentation and T cell stimulation as so have a role in adaptive immune system.

Question 29

List Macrophage receptors involved in host and pathogen recognition

Answer 29

1) TLR- Toll like receptor-binds to ligands on bacterial cell wall
2) LPS or TLR 4 or CD14 binds to polysaccharides on bacteria.
3) Manose- binds to carbohydrates
4) Glucan- binds to receptors on yeast
5) Scavenger- scavenge range of molecules for recognition
6) CR3- complement

Receptors that lead to phagocytosis can also be stimulate signal which lead to gene expression and production of cytokines

Question 30

List the main types of pattern recognition receptors; patterns found on different pathogens

Answer 30

• Toll-like receptors
• NOD-related proteins
• Scavenger receptors
• Lectins

Question 31

Pattern recognition: Scavenger receptor functions

Answer 31

Receptors have different ligands found on bacteria, fungi, and host proteins.

CD14- binds to apoptotic cell, polysaccharides on cell walls and important for recognition of LPS and clearance

CD36- binds to host glycoprotein, eg, Ox-LDL and phosohatidylserine exposed on apoptotic cells where it is important for clearance.

SRA- binds to polyanionic ligands (important for processing lipoprotein cholestrol and apoptotic cells.)

MARCO (a scavanger receptor) that specifically recognizes bacteria and important in host defense

Question 32

Describe the macrophage-mannose receptor, (phagocytic) ?

Answer 32

Large extracellular domain that can bind to carbohydrates.

It is a C-type lectin with 4 active and 4 inactive recognition domains; the cystene rich domain will remove hormones of pregnancy such as lutropin.

It binds to a broad range of different pathogens, like yeast (Candida alibcan), parasites, virus like HIV.

Question 33

Describe Dectin 1 receptor

Answer 33

Beta-glucan found on many fungal cell walls; C- type lectin specific for beta-1,2/1,6-glucans. It defends host from fungal pathogens.

Binding triggers pathways leading to production of pre-inflammatory cytokines like TNF-a via activation of receptors ITAM signalling motif. It also triggers NADH oxidase activation and phagocytosis of Beta-glucan.

Question 34

Describe an antibody receptor

Answer 34

Fc receptors on macrophage bind to Fc portion of antibody (attached to bacterium.)
Binding triggers signal hat leads to phagocytosis: complete internalisation where macrophage membrane surrounds bacterium, phagosome and lysosome fusion making phagolysosome and degradation.

Link to adaptive immune system.

Question 35

What changes to a cell in apoptosis?

Answer 35

Surface changes: molecule phosphatidylserine (PS) normally on inside of cell membrane exposed on outside surface

Question 36

Why are changes caused to apoptotic cells?

Answer 36

infection, Drugs, irradiation, inflammation

Question 37

List receptors important for recognitions

Answer 37

Many receptors that work together to mediat e recognition and phagocytosis.

1) CD14
2) CD36
3) SR-A
4) CD91
5) CR3
6) PS receptor like TIM-4
7) Vitronectin recepor
8) MER tyrosine kinase

Question 38

Why is phagocytosis anti-inflammatory

Answer 38

Anti-inflammatory means more Transformation Growth Factor (TGF) beta produced when phagocytosis occurs which switches off inflammation. TNF-a, IL-8, IL1beta is suppressed.

To prevent macrophage activation and potential activation of auoimmune response

Question 39

List three deficiencies autoimmunity

Answer 39

1) lack of C1q complement
2) lack of seum amyloidp (SAP) component
3) lack of MER (receptor tyrosine kinase) needed for apoptotic cell uptake

In absence in any one results in autoimmunity as there is no ability to suppress a inflammatory response.

Question 40

How are Macrophages activated?

Answer 40

They are activated by cytokines from T cells/lymphocytes, or macrophages themselves

Question 41

What are the two types of macrophage activation?

Answer 41

1) Classic activation (M1) which depends on cytokines from TH1 cells. Classic cytokine is interferon -gamma, a key activating molecule
2) Alternative activaion (M2) mediated by TH2 T cells which make IL-4 and IL-13 that both act on macrophages so they become M2 macrophage.

M1 and M2 differ in properties.

Other cytokines like IL-10 can deactivated macrophages. Not irreversible process.

Question 42

CLASSIC macrophage acivation

Answer 42

-macrophage recognises pathogen and so produces TNF-a and IL12. IL12 triggers production of TH1 cells and activates NK cells. These are sources of interferon(IFN)-gamma.

IFN-gamma acts on macrophage to prime it for activation. In presence of TNFa macrophage is fully activated.

Activated macrophage makes more TNFa, NO (nitric oxide), and H2O2 (hydrogen peroxide) important in host defense.

TNFa can feedback on macrophage to make it more activated so this is why macrophages can be deactivated.

Question 43

ALTERNATIVE macrophage activation

Answer 43

TH2 cells make IL4 and IL13. These act on macrophages to produce a very different phenotype; it NO longer makes toxic proinflammatory cytokines like TNFa, instead they make molecules for tissue repair, endocytosis, pinocytosis, apoptotic cell clearance.

Where epithelial cell is damaged; depending on what stimulus is will result in M1, or M2; if a small parasite causes damage, M2 cells become activated to make molecules for fibroblast proliferation and collagen for wound healing.

Question 44

Give some examples of what would cause M1 activation

Answer 44

Often associated with autoimmune diseases.
Myobacteria
Viruses,
Diabetes
Arthritis
Steatosis
Glomerulo-nephritis

Question 45

Give some examples of what would cause M2 activation

Answer 45

Often associated with parasitic diseases, cancer, defects in wound healing ect.
Helminths
Granuloma
Breast cancer
Fibrosis
Wound healing
Atopic dermatitis
Airway inflammation (asthma)

Question 46

List 4 fates of macrophages

Answer 46

1) innate activation by TLR ligands like LPS causing increased production of pro-inflammatory cytokines
2) Classical activation by IFN-gamma and LPS causing increased production of pro-inflammatory cytokines, MHC II CD86, more antigen presentation, and microbial activity
3) Alternative activation by IL4, IL13 causing increased endocytic activity, more mannose receptor, dectin 1, arginase, more cell growth, tissue repair and parasite killing.
4) Deactivation by IL10, TGF-beta, seriod. causing increase production of IL10, TGF-beta and PGE, reduced expression of MHC II.