Phagocytic Cells & Their Function

Purdue University

J.Paul Robinson

Purdue University Cancer Center&

Purdue University Cytometry Laboratories

Optical Design

PMT 1

PMT 2

PMT 5

PMT 4

DichroicFilters

BandpassFilters

Laser

Flow cell

PMT 3

Scatter

Sensor

Sample

Flow cytometry measurements

L

M

G

SCATTER 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

marrow

Recruitment

Circulation Tissue Circulation

macrophage

liver

Stimulation

We can monitor phagocytic cells

• Flow Cytometry– Cell numbers– Cell function

Scatter Pattern of Human leukocytes

Lymphocytes

Monocytes

NeutrophilsA flow cytometryscattergram

For

war

d sc

att e

r (s

ize)

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 glyco-

phosphatidylinositol 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)

CD16 “bright”

CD16 “dim”

Normal PMN

Older PMN

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.

CD16 “dim”

CD16 “bright”

The loss of CD16 “bright” peak is a signal for macrophages to phagocytose the neutrophils- they signal that they are apoptotic

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• FCRIIA 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

represents internal particlesFITC-Labeled Bacteria

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

Histograms showing neutrophils labeled with primary antibodies to neutrophil adhesion markers: CD11b (Mo-1-FITC) at a dilution of 1:8, CD18 (DAKO-CD18) 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.

CO

UN

TS

Log FITC

0 1

00

75

50

25

CD11b CD18

.1 1000 100 10 1

CD11a

.1 1000 100 10 1.1 1000 100 10 1

Log FITC Log FITC

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!!!

O2 H2O2 OH• H2O

e e e e

OxygenXanthine

L-Arginine SuperoxideHydrogenPeroxide Water

HydroxylRadical

ONOO

NO•

MPO +

Hali

de

HOCl

NADPHOxidase

SuperoxideDismutase

O2

OCl-

SingletOxygen

Xanthine

L-Arginine NOS

HNO3

- H+

NO3-

NO2•

OH•

XO

Fe2+Fe3+

H+

CATALASE

GSH

GSSG

GlutathionePeroxidase

GlutathioneReductase

NADP

NADPH

-

-

- - - -

Human Neutrophil

Phagosome

O2

O2-

H2O2

NADPH + H+

NADP+

HMP

NADPH

Oxidase

GSSG

GSH

GRGP

SODO2

-

H+

Catalase

H2O + O2

SOD

Stimulant

PKC(PMA)

Lipid Peroxidation

Phospolipase A2 activity

H2O2

H2O

H2O2

+O2

-

OH.

Leukotrienes

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

FADNDPH

p67-phoxp47-phox

p21 rac1

cytosol

membrane

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 Hydroethidine

• Hydrogen Peroxide Dichlorofluorescein

• Glutathione levels Monobromobimane

• Nitric Oxide Dichlorofluorescein ?

DCFH-DA DCFH DCFDCF

COOHH

Cl

O

O-C-CH3

O

CH3-C-O

Cl

O

COOHH

Cl

OHHO

Cl

O

COOHH

Cl

OHO

Cl

O

Fluorescent

Hydrolysis

Oxidation

2’,7’-dichlorofluorescin

2’,7’-dichlorofluorescin diacetate

2’,7’-dichlorofluoresceinCellular Esterases

H2O2

DCFH-DA

DCFH-DADCFH-DA

DCFHDCFH

DCF

H OH O 2 22 2Lymphocytes

Monocytes

Neutrophils

log FITC Fluorescence.1

1000

100 10

1

0

20

40

60

cou

nts

PMA-stimulated PMNControl

80

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