Lecture 1 First Lecture: (3 rd 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.

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 Dichroic Filters Bandpass Filters Laser Flow cell PMT 3 Scatter Sensor Sample

Flow cytometry measurements L M G SCATTER FLUORESCENCEIMAGE

Presentation Overview Role of Neutrophils Mechanisms of Action and Function Models for studying Neutrophil Function

Role of Neutrophils 60 x 10 6 /minute released into circulation max 10 hours (1/2 life = 7 hours) RBC 90 x 10 6 /minute - live 70 days so outnumber PMN by 10 3

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 CirculationTissueCirculation macrophage liver Stimulation

We can monitor phagocytic cells Flow Cytometry –Cell numbers –Cell function

Scatter Pattern of Human leukocytes Lymphocytes Monocytes Neutrophils A flow cytometry scattergram Forward scatter (size) 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 IgG 1 and IgG 3 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 IgG 1 =IgG 3 >>IgG 2 =IgG 4 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 IgG 1 and IgG 3 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 IgG 2 complexes IgG 2 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 represents internal particles FITC-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. COUNTS Log FITC CD11b CD CD11a 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!!!

O2O2 H2O2H2O2 OH H2OH2O         e e e e Oxygen Xanthine L -Arginine Superoxide Hydrogen Peroxide Water Hydroxyl Radical ONOO NO MPO + Halide HOCl NADPH Oxidase Superoxide Dismutase O2O2 OCl - Singlet Oxygen Xanthine L -Arginine NOS HNO 3 - H + NO 3 - NO 2 OH XO Fe 2+ Fe 3+ H+H+ CATALASE GSH GSSG Glutathione Peroxidase Glutathione Reductase NADP NADPH

Human Neutrophil Phagosome O2O2 O2-O2- H2O2H2O2 NADPH + H + NADP + HMP NADPH Oxidase GSSG GSH GR GP SOD O2-O2- H+H+ Catalase H 2 O + O 2 SOD Stimulant PKC (PMA) Lipid Peroxidation Phospolipase A2 activity H2O2H2O2 H2OH2O H2O2H2O2 + O2-O2- OH. Leukotrienes

Membrane Complexes NADPH - Oxidase - originally described in 1973 by Babior - based on b 558 cytochrome heterodimer – 2 subunits-gp22-phox (  unit) –1  subunit - gp91-phox

NADPH Oxidase of Neutrophils   FAD NDPH p67-phox p47-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 O 2 - Other cells??

Other Oxidative systems All other cells contain SOD B-lymphocytes have been shown to produce SOD inhibitable O 2 - Human fibroblasts Kidney mesangial cells Endothelial cells (several) Canine NK cells

Oxidative Reactions SuperoxideHydroethidine Hydrogen PeroxideDichlorofluorescein Glutathione levelsMonobromobimane Nitric OxideDichlorofluorescein ?

DCF DCFH-DA DCFH DCF COOH H Cl O O-C-CH3 O CH3-C-O Cl O COOH H Cl OH HO Cl O COOH H Cl O HO Cl O Fluorescent Hydrolysis Oxidation 2’,7’-dichlorofluorescin 2’,7’-dichlorofluorescin diacetate 2’,7’-dichlorofluorescein Cellular Esterases H2O2H2O2 DCFH-DADCFH-DA DCFH DCF H O Lymphocytes Monocytes Neutrophils log FITC Fluorescence counts PMA-stimulated PMN Control 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: