Phagocytic Cells & Their
Function
Purdue University
J.Paul Robinson
Purdue University Cancer Center
&
Purdue University Cytometry Laboratories
Optical Design
PMT 5
PMT 4
Sample
PMT 3
Flow cell
Dichroic
Filters
Scatter
Sensor
PMT 2
PMT 1
Laser
Bandpass
Filters
Flow cytometry measurements
SCATTER
G
M
L
FLUORESCENCE
IMAGE
Presentation Overview
• Role of Neutrophils
• Mechanisms of Action and Function
• Models for studying Neutrophil Function
Role of Neutrophils
• 60 x 106/minute released into circulation
• max 10 hours (1/2 life = 7 hours)
• RBC 90 x 106/minute - live 70 days so
outnumber PMN by 103
Factors that Increase PMN #s
•
•
•
•
Stress
injury
infection
 cytokines
Mechanisms of Action
• Phagocytosis
•
•
•
•
•
Recognition
Attachment and binding
Ingestion
Destruction
Clearance of phagocytes
General Pathway
Storage
Stimulation
Circulation Tissue Circulation
Recruitment
marrow
macrophage
liver
We can monitor phagocytic cells
• Flow Cytometry
– Cell numbers
– Cell function
Scatter Pattern of Human leukocytes
A flow cytometry
scattergram
Neutrophils
Monocytes
Lymphocytes
Side scatter (granularity)
Opsonins
• Primary Opsonins
– IgG
– Complement factor C3
• Most microorganisms will NOT be phagocytosed
without opsonins (see later)
• Pseudopods extend to cover particle (but only the part
that is opsonized)
• Changes the organism’s surface from hydrophilic
(relative to the PMN) to more hydrophobic  ingestion
IgG Receptors
• Fab-region reacts with organism
• Fc domain on PMN - Fc R
– 3 Classes
•
•
•
Fc RI (CD64)
Fc RII (CD32)
Fc RIII (CD16)
Fc RI (CD64)
•
•
•
•
Binds IgG1 and IgG3 with high affinity
Not expressed on resting neutrophils
 expression by IFN-, G-CSF, infection
3 immunoglobulin like binding domains
Fc RII (CD32)
•
•
•
•
Low affinity binding
IgG1=IgG3>>IgG2=IgG4
Membrane spanning domain and cytoplasmic tail
3 genes code for Fc RII - Fc RIIA is the major
transcript in neutrophils
• Neutrophil Fc RII exhibits genetically determined
structural polymorphism- may have functional
consequences
Fc RIII (CD16)
• Binds IgG1 and IgG3 with intermediate
affinity
• 2 genes code for Fc RIII
– Fc RIIIB - molecule with a glycophosphatidylinositol anchor (Neutrophils only)
• it is shed during activation
• 2 allotypic forms NA1 and NA2
– Fc RIIIA - transmembrane and cytoplasmic
domains (Only on NK cells and Macrophages)
The loss of CD16 “bright” peak is a signal for macrophages to
phagocytose the neutrophils- they signal that they are apoptotic
Normal PMN
CD16 “bright”
CD16 “dim”
Older PMN
CD16 “dim”
CD16 “bright”
The above figure demonstrates CD16 expression on neutrophils, comparing
fresh, normal neutrophils to neutrophils 24 hours old. Clearly the bright
population is severely reduced after 24 hours in culture.
Fc R mediated Phagocytosis
• RII - Most important for phagocytosis of IgG coated
particles and microorganisms
• RII sole class capable of binding human IgG2 complexes
• IgG2 subclass containing antibodies to bacterial capsular
polysaccharides
• FCRIIA polymorphism is important for capacity to
ingest
– Hemophilus influenzae type b
– S.aureus Wood 46
– encapsulated group B Streptococci
Summary of Fc
• Fc RII is the main Fc R
– primarily mediates ingestion
– triggers the oxidative burst
Phagocytosis
• Uptake of Fluorescent labeled particles
• Determination of intracellular or extracellular state of
particles
How the assay works:
•
•
•
•
Bacteria are labeled with a fluorescent probe (eg fluorescein)
The bacteria are mixed with phagocytes so phagocytosis takes place
A fluorescent absorber is added to remove fluorescence from membrane bound
particles (these are not phagocytosed but stick to the surface)
The remaining fluorescence
FITC-Labeled Bacteria
represents internal particles
Trypan Blue is added to remove the
external fluorescence
FITC-Labeled Bacteria
Complement Related Proteins
• Activation of complement cascade causes
proteolytic cleavage of complement factors
creating potential ligands for complement
receptors on neutrophil surfaces
• C3a & C5a --- chemotactic factors
• C3b & C3bi --- main complement derived
opsonins
Neutrophil Complement Receptors
• CR1 (CD35)
– glycoprotein consisting of a single membrane spanning
domain and a short cytoplasmic c-terminal domain
– large extracellular domain of 30 repeated units arranged in
tandem
– 2 pools of receptors - 15% in clusters on surface 85% in
intracellular compartments
– binds dimeric C3bi (not uncleaved C3b)
– Very weak binding (?? physiological importance)
Complement R CR3
• Glycoprotein member of the Integrin family
(CD11b/CD18)
–
–
–
–
noncovalently linked dimer 185kDa  chain (CD11b)
95kDa  chain (CD18)
B chain same as in LFA-1 (CD11a)
-p150 (CD11c)
• Whole molecule termed the CD11/CD18 complex
• Resides in 2 pools in neutrophils as does CR1
50 75 100
25
0
COUNTS
CD11a
CD18
CD11b
.1
1
10
Log FITC
100
1000
.1
1
10
Log FITC
100
1000
.1
1
10
100
1000
Log FITC
Histograms showing neutrophils labeled with primary antibodies to neutrophil
adhesion markers: CD11b (Mo-1-FITC) at a dilution of 1:8, CD18 (DAKOCD18) at a dilution of 1:50, and CD11a (DAKO-CD11a) at a dilution of 1:100.
The gray lines show the expression after 30 min at 37°C, while the black lines
show the expression on neutrophils stimulated with 10 ng/ml PMA for 30 min
at 37°C.
CR3
• Recognizes 4 ligands
– C3bi (opsonin deposited on surfaced of microorganisms)
– ECM -fibrogen, fibrin, laminin - promotes adhesion to ECM
– ICAM-1 (CD54) on endothelial cells (CD18 required for PMN
movement through EC to tissue)
– Some surface structures on microorganisms - ie CR3 can bind in
ABSENCE of opsonin for
• S.aureus, group B Strep, E.coli (via mannose specific ligand), Bordetella
pertussis, Histoplasma capsulatum, Leishmania, Zymosan (yeast cell
wall)
Destruction & Killing
• 2 primary mechanisms
– Oxidative mechanisms
– Non-oxidative
• Enzymes and cytoplasmic granules
• pH change
WARNING: The next slide could be dangerous to your health!!!
NO3-
ONOO
L-Arginine
NOS
HNO3
NO2•
HOCl
OH•
Xanthine
GSH
Glutathione
Peroxidase

Glutathione
Reductase
GSSG
e-
O2

NADP
NADPH
Oxidase
-
e-
O2
e-
H2O2
OH•
NADPH
e-
H 2O
Superoxide
Dismutase
Fe2+
Fe3+
OClCATALASE
Singlet
Oxygen
Oxygen
Xanthine
L-Arginine
Superoxide
Hydrogen
Peroxide
Hydroxyl
Radical
Water
Human Neutrophil
Phospolipase A2 activity
Leukotrienes
Lipid Peroxidation
OH.
H2O2
+
Phagosome
Stimulant
(PMA)
H2O2
SOD
O2
O2-
Oxidase
GR
NADP+
HMP
SOD
GP
GSH
NADPH + H+
-
H2O2
GSSG
NADPH
H2O + O2
+
H
O2
PKC
O2-
H2O
Catalase
Membrane Complexes
• NADPH - Oxidase - originally described in
1973 by Babior - based on b558 cytochrome
• heterodimer
– 2 subunits-gp22-phox ( unit)
– 1  subunit - gp91-phox
NADPH Oxidase
of Neutrophils


membrane

FAD
NDPH
p47-phox
p67-phox
p21 rac1
cytosol
The Oxidase
•
•
•
•
•
Membrane and cytosolic components
gp47-phox
gp67-phox
rac-1 - (GTP binding protein)
NOTE: the mechanism of
activation/pathway is quite different from
the NADH pathway in mitochondria thus
the term PHOX - Phagocyte Oxidase
Major Differences in Phagocytic
Cells
• NADPH Oxidase is unique to phagocytic cells
• Requires assembly from multiple sites
• Midpoint redox potential is very low (-245mV)
so it can reduce molecular oxygen directly to
O2• Other cells??
Other Oxidative systems
• All other cells contain SOD
• B-lymphocytes have been shown to produce
SOD inhibitable O2• Human fibroblasts
• Kidney mesangial cells
• Endothelial cells (several)
• Canine NK cells
Oxidative Reactions
•
•
•
•
Superoxide
Hydrogen Peroxide
Glutathione levels
Nitric Oxide
Hydroethidine
Dichlorofluorescein
Monobromobimane
Dichlorofluorescein ?
DCFH-DA
DCFH
DCF
2’,7’-dichlorofluorescin diacetate
O
O
CH3-C-O
O
O-C-CH3
Cl
2’,7’-dichlorofluorescin
Cl
H
COOH
O
HO
Cellular Esterases Cl
OH
Fluorescent
Cl
H
COOH
Hydrolysis
2’,7’-dichlorofluorescein
O
HO
O
H2O2
Cl
Cl
H
Oxidation
DCFH-DA
COOH
Neutrophils
DCFH-DA
80
Monocytes
DCFH H O
2 2
Lymphocytes
DCF
counts
60
PMA-stimulated PMN
Control
40
20
0
.
1
1log
100
FITC 10
Fluorescence
1000
Summary
• Neutrophils are rather more complex than we might think
• Neutrophils have homogenous response, but heterogenous
function
• They are very reactive and can cause more damage than
they protect from
• Once activated it is difficult to stop them
• Neutrophils are vital components in the immune system,
but we have so many of them that even 50% reduction in
function may not be too harmful
• PMN function can easily be measured by flow cytometry,
microscopy and image analysis tools
Acknowledgements
•
•
•
•
•
•
•
•
•
Padma Narayanan
Nian-Yu Li
Wayne Carter
Kathy Ragheb
Gretchen Lawler
Steve Kelley
Monica Shively
Stephanie Sincock
Karin Kooreman
Thank you
for your attention
These slides will be available on
our website at:
www.cyto.purdue.edu/meetings