First Lecture: 3rd year students

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Lecture 1
First Lecture (3rd year students) Phagocytic
Cells and their function This lecture will
discuss the principles of phagocytosis and
neutrophil function. We will discuss the basic
principles of how phagocytic cells function to
bind, ingest and destroy invading bacteria. As
part of this lecture we will discuss current
research techniques such as measurements of the
respiratory burst as well as clinical
applications. In addition we will discuss the
latest understanding of neutrophil receptors and
how these receptors impact the role of the
neutrophil in health and disease.
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Phagocytic Cells Their Function
Purdue University
J.Paul Robinson
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Purdue University Cancer CenterPurdue
University Cytometry Laboratories
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Optical Design
PMT 5
PMT 4
Sample
PMT 3
Dichroic
Filters
Flow cell

PMT 2
PMT 1
Scatter
Laser
Sensor
Bandpass
Filters
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Flow cytometry measurements
SCATTER FLUORESCENCE IMAGE
G
M
L
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Presentation Overview

• Role of Neutrophils
• Mechanisms of Action and Function
• Models for studying Neutrophil Function

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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

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Factors that Increase PMN s

• Stress
• injury
• infection
• ? cytokines

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Mechanisms of Action

• Phagocytosis
• Recognition
• Attachment and binding
• Ingestion
• Destruction
• Clearance of phagocytes

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General Pathway
Storage
Circulation
Tissue
Circulation
Stimulation
Recruitment
marrow
macrophage
liver
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We can monitor phagocytic cells

• Flow Cytometry
• Cell numbers
• Cell function

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Scatter Pattern of Human leukocytes
A flow cytometry scattergram
Forward scatter (size)
Side scatter (granularity)
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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 organisms surface from hydrophilic
  (relative to the PMN) to more hydrophobic ?
  ingestion

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IgG Receptors

• Fab-region reacts with organism
• Fc domain on PMN - Fc? R
• 3 Classes
• Fc? RI (CD64)
• Fc? RII (CD32)
• Fc? RIII (CD16)

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Fc? RI (CD64)

• Binds IgG1 and IgG3 with high affinity
• Not expressed on resting neutrophils
• ? expression by IFN-g, G-CSF, infection
• 3 immunoglobulin like binding domains

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Fc? RII (CD32)

• Low affinity binding
• IgG1IgG3gtgtIgG2IgG4
• 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

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Fc? RIII (CD16)

• Binds IgG1 and IgG3 with intermediate affinity
• 2 genes code for Fc? RIII
• Fc? RIIIB - molecule with a glyco-phosphatidylinos
  itol 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)

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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 bright
CD16 dim
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.
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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
• FCgRIIA polymorphism is important for capacity to
  ingest
• Hemophilus influenzae type b
• S.aureus Wood 46
• encapsulated group B Streptococci

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Summary of Fc

• Fc? RII is the main Fc? R
• primarily mediates ingestion
• triggers the oxidative burst

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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
• represents internal particles

FITC-Labeled Bacteria
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Trypan Blue is added to remove the external
fluorescence
FITC-Labeled Bacteria
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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

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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)

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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

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CD11b
CD18
CD11a
100
75
COUNTS
50
25
0
Log FITC
Log FITC
Log FITC
Histograms showing neutrophils labeled with
primary antibodies to neutrophil adhesion
markers CD11b (Mo-1-FITC) at a dilution of 18,
CD18 (DAKO-CD18) at a dilution of 150, and CD11a
(DAKO-CD11a) at a dilution of 1100. The gray
lines show the expression after 30 min at 37C,
while the black lines show the expression on
neutrophils stimulated with 10 ng/ml PMA for 30
min at 37C.
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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)

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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!!!
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NO3-
-
- H
ONOO
HNO3
H
NO2
L-Arginine
NOS
HOCl
NO
OH
Xanthine
GSH
NADP
MPO Halide
Glutathione Reductase
Glutathione Peroxidase
?
?
?
XO
?
GSSG
NADPH
-
-
-
-
-
e
e
e
e
H2O
O2
O2
H2O2
OH
NADPH Oxidase
Superoxide Dismutase
?
?
Fe2
?
Fe3
?
OCl-
CATALASE
Oxygen Xanthine L-Arginine
Hydrogen Peroxide
Hydroxyl Radical
Singlet Oxygen
Superoxide
Water
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Human Neutrophil
Phospolipase A2 activity
Leukotrienes
Lipid Peroxidation
OH.
H2O2
O2-
H2O O2

Phagosome
H
H2O2
Stimulant
Catalase
O2
SOD
(PMA)
O2-
O2-
SOD
PKC
H2O2
GSSG
NADPH
GP
Oxidase
GR
H2O
GSH
NADPH H
NADP
HMP
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Membrane Complexes

• NADPH - Oxidase - originally described in 1973 by
  Babior - based on b558 cytochrome
• heterodimer
• 2 subunits-gp22-phox (a unit)
• 1 ? subunit - gp91-phox

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NADPH Oxidase of Neutrophils
membrane
?
?
?
FAD
NDPH
p67-phox
p47-phox
cytosol
p21 rac1
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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

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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??

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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

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Oxidative Reactions

• Superoxide Hydroethidine
• Hydrogen Peroxide Dichlorofluorescein
• Glutathione levels Monobromobimane
• Nitric Oxide Dichlorofluorescein ?

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DCFH-DA DCFH DCF
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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

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Acknowledgements

• Padma Narayanan
• Nian-Yu Li
• Wayne Carter
• Kathy Ragheb
• Gretchen Lawler
• Steve Kelley
• Monica Shively
• Stephanie Sincock
• Karin Kooreman

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Thank you for your attention

• These slides will be available on our website at
• www.cyto.purdue.edu/meetings