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Macrophages
and Monocytes
Feb 18, 2016
Craig Jenne
IMM5 Macrophages
Phagocytes
•Discovered by Ilya Mechnikov
•Described “eating as defence”
•Experiments involving wooden splinters and starfish larvae
•Shared the first Nobel Prize for Medicine in 1908
•“Cellular Immunity”
IMM5 Macrophages
Mononuclear Phagocyte System
• Mononuclear phagocyte system (MPS)
– Circulating monocytes and tissue macrophages
– 10-15% of cells in every organ in the body
– Take up, process and present antigen to T cells
– The tissue phagocytes are traditionally split into
two groups:
• professional phagocytic macrophages
• antigen-presenting, ‘Dendritic,’ cells (APC)
IMM5 Macrophages
Monocyte and Macrophage
IMM5 Macrophages
Transformation to Macrophage
• Differentiation of the monocyte into the tissue
macrophage involves a number of changes:
– Enlarges 5 - 10 x
– Increases intracellular organelles
– Increases phagocytic ability
– Increased amounts of hydrolytic enzymes
– Begins to secrete a variety of soluble factors
• Macrophages serve different functions in
different tissues.
IMM5 Macrophages
Macrophages function at the
interface between innate and
adaptive immunity
IMM5 Macrophages
What is the role of the MPS in
the host immune response?
• Detect pathogen
• Clear infection
• Activate adaptive immune system
• Shape/direct adaptive immune response
• Resolution
IMM5 Macrophages
What is the role of the MPS in
the host immune response?
• Detect pathogen
• Clear infection
• Activate adaptive immune system
• Shape/direct adaptive immune response
• Resolution
IMM5 Macrophages
‘Resident’ macrophages
• Myeloid progenitor cells (bone marrow)  promonocytes 
blood monocytes  organs  tissue macrophages.
• Found constitutively in the absence of an overt inflammatory
stimulus in many tissues.
• Relatively long lived (~1 month)
• Considerable RNA and protein synthesis activity/potential
• Liver Kupffer cells, mesangial cells (kidney), alveolar,
serosal, brain microglia, spleen sinus, lymph node,
osteoclasts in bone.
• differ in phenotype and display a wide variety of plasma
membrane receptors
• Create a network at tissue interfaces between the host and the
environment (lung, gut, liver) ready to “capture” infectious
organisms.
IMM5 Macrophages
F4/80 Staining of Jejunal Villi
IMM5 Macrophages
“MacGreen”
(M-CSF-1R eGFP)
Lung section
IMM5 Macrophages
Lung
F4/80 Staining Liver
IMM5 Macrophages
Pathogen
Detection
Figure 2-5
IMM5 Macrophages
Macrophage Pathogen Receptors
Figure 2-10
IMM5 Macrophages
Macrophage Pathogen Receptors
• MFR (mannosyl-fucosyl receptor)
• Bind to sugars on microorganisms and effete cells.
• Tim-1, Tim-4 (phosphatidyl serine “receptor”)
• T-cell Immunoglobulin Mucin Proteins (Tim)
• Bind apoptotic cells
• CD14 receptor (LPS/LBP receptor)
• Toll-like receptors (TLR)
• TLR 1, 2, 3, 4, 6, 7, 9.
• Scavenger receptors
IMM5 Macrophages
• SR-A
• Bind LPS, LTA, polyribonucleotides
Macrophage Pathogen Receptors
• Immunoglobulin Fc receptors
• FcγRI (CD64)
– high affinity for IgG
– homologous to FcγRII in the mouse
• FcγRII (CD32)
– Medium affinity for IgG
– homologous to FcγRIIb/I receptor in mouse
• FcγRIII (CD16)
– Low affinity for IgG
• FcεRII (CD23)
– Low affinity for IgE
– Present on activated macrophages
• Complement receptors
IMM5 Macrophages
•
•
•
•
•
•
CR1 - C3b receptor (CD35)
CR3 - iC3b receptor (CD11b, MAC-1)
CR4 - iC3b receptor (CD11c)
C5aR – C5a receptor
C3aR – C3a receptor
CRIg – C3b, iC3b receptor
Pathogen Receptors
Cytoplasmic PRR
Cell Surface PRR
Toll-Like Receptors (TLR)
e.g. TLR2 (Vaccinia virus virion)
Endosomal PRR
Toll-Like Receptors (TLR)
e.g. TLR3 (viral dsRNA, pIC)
Rig-I-like Receptors (RLR)
e.g. MDA5 (viral dsRNA, pIC)
What is the role of the MPS in
the host immune response?
• Detect pathogen
• Clear infection
• Activate adaptive immune system
• Shape/direct adaptive immune response
• Resolution
IMM5 Macrophages
Clearance Of Infection
• Direct clearance
• Phagocytosis of pathogen, killing
• Enhance inflammatory response
•
•
•
•
Contain/isolate pathogen
Secrete cytokines
Raise body temperature
Recruit additional cells (neutrophils)
IMM5 Macrophages
Phagocytosis
• Macrophages can ingest and digest…
– Exogenous material; microorganisms, insoluble particles
– Endogenous material; injured or dead host cells, debris
• Steps in phagocytosis
– Chemotaxis - move towards chemoattractants
– Adherence of antigens to macrophage cell membrane
– Membrane protrudes forming pseudopodia
• Extends around the attached material
– Fusion of the pseudopodia encloses the material within a
membrane bound structure called a phagosome
– Phagosome fuses with a lysosome  phagolysosome
• The lysosome containes lysozyme and other hydrolytic
enzymes that digest the ingested material
– Elimination of waste by exocytosis
IMM5 Macrophages
IMM5 Macrophages
Phagocytosis
Figure 2-32
IMM5 Macrophages
Phagocytosis
Figure 2-31
IMM5 Macrophages
Clearance Of Circulating Pathogens
Clearance Of Circulating Pathogens
Bacteria Catching in the Liver
CRIg – complement receptor from the Ig-superfamily
Binds targets tagged with C3b or iC3b (generated from the activation of C3)
Killing and Digestion of
Phagocytosed Material
• A number of antimicrobial and cytotoxic substances are
produced by activated macrophages
Killing and Digestion of
Phagocytosed Material
• Types of killing: O2 dependent and O2 independent
• Oxygen dependent killing - Respiratory Burst
• Reactive Oxygen Intermediates (ROI)
– membrane bound NADPH Oxidase catalyzes the
reduction of O2 to superoxide anion – produces
– hydroxyl radicals and hydrogen peroxide
– myeloperoxidase produces hypochlorate from hydrogen
peroxide and Cl- (bleach)
• Reactive Nitrogen Intermediates (RNI)
– inducable Nitric Oxide Synthase (iNOS)
– oxidizes L-arginine to L-citrulline and NO
IMM5 Macrophages
Oxygen Dependent
Figure 2-7 Killing of
Phagocytosed Material
Killing and Digestion of
Phagocytosed Material
• Oxygen independent killing
• Acidification of phagosomes by H+ ion pumps
• Macrophages make lysozyme and various other hydrolytic
enzymes that do not require oxygen
• Defensins
– Cysteine-rich cationic peptides containing 29-35 aa
residues
– Tend to cause bacterial permeability and osmotic lysis
IMM5 Macrophages
Enhanced Inflammatory Response
• Macrophage activity can be further enhanced by
• Cytokines from activated NK cells, TH cells
– IFNγ is a classical macrophage activator
• Mediators of inflammation, cytokines from other macrophages
or inflammatory cells
• Component from bacterial cell walls
• Activated (classically) macrophages are more effective in
eliminating potential pathogens
• Greater phagocytic activity
• Increased killing capacity
• Increased secretion of inflammatory mediators
• Increased ability to activate T cells
• Express higher levels of MHC class II molecules - increased
antigen presentation.
IMM5 Macrophages
Enhanced Inflammatory Response
Figure 2-15
IMM5 Macrophages
Enhanced Inflammatory Response
Figure 2-47 part 1 of 2
IMM5 Macrophages
Enhanced Inflammatory Response
Figure 2-39
IMM5 Macrophages
Enhanced Inflammatory Response
Figure 2-46
IMM5 Macrophages
What is the role of the MPS in
the host immune response?
• Detect pathogen
• Clear infection
• Activate adaptive immune system
• Shape/direct adaptive immune response
• Resolution
IMM5 Macrophages
Activation of Adaptive Immunity
• Macrophages are professional antigen presenting cells (APC).
• APC capture, process, transport, present and retain antigen
• Phagocytosed antigen is digested within the endocytic
processing pathway into peptides that associate with class II
MHC molecules and the complexes move to the macrophage
membrane surface.
• Activated macrophages express high levels of B cell (CD40L)
and T cell (B7.1, B7.2) co-stimulatory molecules.
• These class II MHC-antigen complexes and the co-stimulatory
B7 family of membrane molecules render the macrophage
more effective in activating TH cells.
– Essential in both humoral (T cell help for B cells) and cellmediated immunity (Th1 and activation of cytotoxic T cells).
Activation
of
Adaptive
Immunity
Figure 2-41 part 1 of 2
• Dedritic cells capture Ag in the periphery.
• Activated dendritic cells transport these Ag to local lymph
nodes via draining lymphatics.
• Ag is transferred to subcapsular sinus macrophages.
• These subcapsular macrophages serve as an Ag depot
and allow for the transfer of to germinal centers.
• Germinal centers facilitate B cell activation, affinity
maturation and antibody isotype class-switch
recombination.
IMM5 Macrophages
Activation
of
Adaptive
Immunity
Figure 2-41 part 1 of 2
IMM5 Macrophages
What is the role of the MPS in
the host immune response?
• Detect pathogen
• Clear infection
• Activate adaptive immune system
• Shape/direct adaptive immune response
• Resolution
IMM5 Macrophages
Shaping Adaptive Immunity
• A number of important proteins central to the development of
immune responses are secreted by activated macrophages.
– Cytokines
•
•
•
•
TNFα, IL-1β and IL-6 promote the inflammatory response
IL-10 and TGF-β promote the anti-inflammatory response
IL12, TNFα promote Th1, cell mediated immunity
IL10, IL4 promote Th2, humoral immunity
– Chemokines
• CCL3, CXCL10 promote Th1, cell mediated immunity
• CXCL12, CXCL13 promote Th2, humoral immunity
IMM5 Macrophages
Shaping Adaptive Immunity
Figure 2-41 part 1 of 2
IMM5 Macrophages
Shaping Adaptive Immunity
Figure 2-41 part 2 of 2
IMM5 Macrophages
Shaping Adaptive Immunity
Fibrogenesis, tissue repair and proliferation
Enhanced bactericidal and phagocytic functions
(M1a and M1b?)
and IL-13
Type II adaptive immune responses,
Recruitment of Treg cells
IMM5 Macrophages
Deactivation, resolution of inflammation
Martinez et. al. Frontiers in
Bioscience (2008) p.453-461
What is the role of the MPS in
the host immune response?
• Detect pathogen
• Clear infection
• Activate adaptive immune system
• Shape/direct adaptive immune response
• Resolution
IMM5 Macrophages
Resolution
• After an immune / inflammatory response,
resolution is a critical step in returning the affected
tissue back to its normal functioning status.
• Stop cellular recruitment, clearance of debris,
removal of surplus immune cells, appropriate
restoration of the tissue microenvironment.
• Failure of resolution leads to chronic inflammation
and/or inappropriate healing (fibrosis)
IMM5 Macrophages
Resolution
Front Immunol. 2011; 2: 57.
Resolution
Resolution
Control PLN
Experimental PLN
Resolution
Light (Mantel) Zone
Maturation and
Class Switching
Light Zone
Selection
Displacement of
low affinity clones
off of FDC by
higher affinity
clones displaced
low affinity clones
die via apoptosis
Dark Zone
Proliferation
Hypermutation
Memory Cell
Plasma Cell
Follicular Dendritic
Cell Displaying Ag
Apoptotic Cells
High Affinity
Centrocytes
Low Affinity
Centrocytes
Centroblast
Tingible-body
macrophage
Resolution
http://www.hindawi.com/journals/isrn/2014/363854/fig2/
Resolution
Front Immunol. 2011; 2: 65.
Resolution
http://www.hindawi.com/journals/isrn/2014/363854/fig2/
Resolution
Front Immunol.
2011; 2: 65.
Cellular Recruitment to Sterile Injury
0h
1h
2h
3h
4h
1h
2h
3h
4h
Dead Cells
Neutrophils
0h
Dead Cells
CCR2-RFP
Monocyte Populations
CCR2
CD16
IL-1, IL-6, IL-12
TNF-α, ROS, MMP
“Inflammatory”
Cx3CR1
IL-10R
IL-10
“Anti-Inflammatory”
Tissue Patrolling
CCR2+ Recruitment to Sterile Injury
CCR2+/RFP mice
8h
24 h
48 h
CCR2+ Recruitment to Sterile Injury
Endothelium
CCR2-RFP
Monocyte-Dependent Debris
Clearance in Sterile Injury
WT
CCR2ko
Sytox Green (dead cells)
Cx3CR1 Monocyte Recruitment
at Later Timepoints
Cx3CR1+/GFP mice
8h
8h
24 h
48 h
72 h
Lack of Cx3CR1+ Monocyte
Migration
Endothelium
Cx3CR1-GFP
CCR2 and Cx3CR1 Monocytes
Accumulate at Injury Site
Cx3CR1+/GFP
Anti-inflammatory
Or reparative
Monocytes
CCR2+/RFP
Classic or
Inflammatory
Monocytes
CCR2+/RFP/Cx3CR1+/
Overlay
Green: CX3CR1-GFP+
Red: CCR2-RFP+
“Double Positive” Monocytes
CCR2-RFP
Cx3CR1-GFP
“Double Positive” Monocytes
CCR2-RFP
Cx3CR1-GFP
“Changing” Monocytes In Vitro
0h
2h
CCR2+/RFP/Cx3CR1+/GFP mice
Monocyte Conversion is
Dependent on IL4 and IL10
CCR2-RFP
Cx3CR1-GFP
Collagen
Dead Cells
Monocyte Conversion is Required
for Debris Clearance and Healing