MICR 304 Immunology &

Serology

MICR 304 Immunology &

Serology

Lecture 4Phagocytes

Chapter 2.4, Primary Literature

Lecture 4Phagocytes

Chapter 2.4, Primary Literature

Overview of Today’s Lecture

• Definition of phagocytes• Phagocyte development• A closer look at neutrophils,

monocytes and macrophages• Phagocyte activation and

phagocytosis

Key Players in Immunology

Innate Adaptive

Cells PhagocytesEpithelial Cells

NK Cells

Lymphocytes(B-Ly, T-Ly)

Effector Molecules

ComplementAntimicrobial (Poly)Peptides

Antibodies

Phagocytes• Cells that take up microbes to kill and

digest them• The professionals

– Neutrophil granulocytes (not present in healthy tissue)

– Monocytes, macrophages (present in healthy tissue)

• Cells with phagocytic activities– Dendritic cells (specialized in antigen

presentation)– Basophil granulocytes, mast cells

(specialized mediators of inflammation)

Neutrophils

Granulocyte Development

The Development of Granulocytes

• Important cytokines– IL-3– GM-CSF– G-CSF

GEMM-Progenitor

Myeloblast

MonocytePromyelocyte

Myelocyte(granules appear)

Metamyelocyte

Band Neutrophil

PolymorphonuclearNeutrophil (segmented)

(Eosinophils and Basophils mature similarly)

Granulocytes

• Polymorphonuclear• Granule rich

– Neutrophil– Eosinophil– Basophil

Neutrophil granulocytes = polymorphonuclear cells = PMNs = Polys

Two Major Types of Neutrophil Granules

• Primary (Azurophil)– Appear first during

mitotic development– Stain blue– Elastase, cathepsin

G, myeloperoxidase, defensins,LL37, lysozyme, glucuronidase

– Fuse with phagosome

– pH optimum 4-5

• Secondary (Specific)– Appear after mitotic

development– Outnumber the

primary granules– Lactoferrin,

lysozyme, C3/C5 proteases, receptors for fMLP, complement, Cytochrome b 558

– Secreted

The Fate of Neutrophils

• Short lived cells (days)• Half Life in circulation 6 – 8 h• High turn over rate (1011/per day)• If unstimulated: migrate to

respiratory and digestive mucosal surfaces, apoptotic death

• If activated: will ultimately become necrotic pus

Neutrophil Abundance in Pus

Monocytes and Macrophages

8957B

8957B

Monocyte Development

The Development of Monocytes and Macrophages

• Important cytokines– IL-3– GM-CSF– M-CSF

– IFN-from activated TH cells

GEMM-Progenitor

Monocyte

Macrophage

Neutrophil

Myeloblast

Monoblast

Promonocyte

Monocytes and Macrophages

• Mononuclear cells• Longer living (weeks – months)• Monocytes: in blood, exit into tissue to

differentiate into macrophages• Functions:

– Phagocytosis– Antigen-presentation– Primary activation of T-lymphocytes– Pivotal role in initiating an inflammatory

response

Maturation and Differentiation of

Monocytes• Monocytes only found in bone marrow,

peripheral blood• Immature Cells• Monocytes are limited in receptor

expression, phagocytosis and cytokine production

• Main stimulators of maturation and differentiation to macrophages:– Interferon-gamma (T-Helper cells, NK cells)– GM-CSF (T-cells, macrophages)

Macrophages : Interface to Adaptive Immunity

• Lymphocyte attraction and activation

• Antigen presentation through MHC II

Specialized Macrophages

• Dendritic Cells: subepithelial, in solid organs, lymph nodes and lymphatic tissue

• Langerhans cells: in skin• Kupffer cells: in liver• Alveolar macrophages: in lung• Microglia cells: in brain

Dendritic Cells

• Lymphoid and myeloid progenitor cells– Plasmocytoid DC: interferon producing

in response to viral infections– Conventional DC: antigen presentation

and activation of naïve T cells• Recognize common structures on

pathogens• Macropinocytosis : Receptor

independent• Highly specialized in antigen

presentation• After contact with antigen

migration to lymph nodes• Interact with T-lymphocytes in

lymph nodes

Cytokines Secreted by Macrophages and Dendritic

CellsCytokine Target Cell Effect

IL-1 Lymphocytes Enhances responses

IL-6 Liver (Hepatocytes)

Induces acute phase protein secretion

CXCL8 (IL-8)

Neutrophils Chemoattractant

IL-12 NK cellsNaïve T cells

Activation of NK

TNF Vascular Endothelium

Cell adhesion and PermeabilityBlood clotting

From Microbial Invasion to Successful Pathogen

Removal • Phagocytes are attracted to site of

invasion– Chemotaxis– Transmigration from blood vessel into tissue

• Physical contact between microbe and phagocyte– Opsonization

• Microbial uptake– Phagocytosis

• Killing

Chemotaxis• Directed movement of phagocytes

towards the source of infection• Induced by chemoattractants:

– Bacterial products• Formylated peptides like fMLP

– Complement fragment• C5a

– Host derived lipid metabolites• LTB4 (arachidonic acid metabolite, produced upon

stress)

– Chemokines• CXCL-8 (formerly IL-8, acts on neutrophils)• CCL-2 (MCP-1, acts on monocytes)

Classification of Chemokines

• Depending on amino acid structure• Number and spacing of cysteine

residues at N-terminus (C: cysteine; X: any amino acid)

• Chemokine families include– CXC (e.g. CXCL8, CXCL7)– CC (CCL2, CCL11)

Chemokines Acting on Phagocytes

Chemokine

Producer Cells attracted

Major Effects

CXCL8(IL8)

MonocytesMacrophagesFibroblastsKeratinocytesEndothelial cells

NeutrophilsNaïve T-cells

MobilizationNeutrophil activation and degranulation

CXCL7(NAP-2)

Platelets Neutrophils Activates neutrophilsClot resorptionAngiogenesis

CCL2(MCP-1)

MonocytesMacrophagesFibroblastsKeratinocytes

MonocytesNK and T-cellsBasophilsDendritic cells

Activates macrophagesHistamin release by basophilsPromotes TH2 immunity

Change of Cell Shape in Response to Chemokines

PMNs before and 5 sec after stimulation with chemokine

(Olsen et al, 2002)

Phagocyte Movement: Cytoskeleton

Rearrangement

Extravasation

1.Rolling adhesion2.Firm adhesion3.Transmigration

Endothelial cells

Leukocytes

Endothelial cellsLeukocytes

• Leukocyte:– Integrins

• Mac-1• LFA-1

• Endothelial Cell– Intercellular

adhesion molecules• ICAMs

http://www.youtube.com/watch?v=I9zSe0qmXGw&NR=1

Extravasation Requires Activation of Endothelium and

Leukocyte• Endothelial Cell

1. Selectin2. ICAM3. CD31 (PECAM)

• Leukocyte

1. Sialyl-LewisX

2. Integrin3. CD31

(PECAM)

Opsonophagocytosis

Opsonization

• Covering microbial surfaces with molecules recognizable by phagocytes:– Complement factors (C3b)– Immunoglobulins (IgG)– C-reactive protein– Mannose-binding protein and other

collectins– Surfactant

Selected Opsonin Receptors

Complement Receptor for C3b

Fc-Receptor for Antibodies

Opsonization and Engulfment

• Engagement of receptors trigger cytoskeletal movement

• Process continues until pseudopods make contact and seal

• A phagosome has been created

• http://www.youtube.com/watch?v=I_xh-bkiv_c

Phagolysosome Formation and Killing

• Engagement of receptors

• Triggering of killing mechanisms– Oxidative burst – Release of

lysosomal contents into phagosome • Antimicrobial

peptides

Killing and Digestion by Phagocytes

• Reactive oxygen and reactive nitrogen intermediates– O2

-, H2O2

– NO

• Antimicrobial peptides• Low pH• Hydrolases, proteases,

phospholipases

Oxygendependent

Oxygenindependent

Oxidative Burst

• Generation of oxygen radicals under consumption of molecular oxygen

• Initiated by NADPH oxidase– multi component membrane

enzyme complex including cytochrome b 558

• Delivered into phagolysosome and extracellular space

C. albicans

FormazanCrystals

PMN

C. albicans

Opsonophagocytosis of C. albicans and Generation of

ROI

A Second System to Produce Radicals Exist

• Nitric oxide synthase is the key enzyme

• Generates from L-arginine radicals like nitric oxide (reactive nitrogen metabolites or RNI)

• Readily detectable in murine macrophages

• Role in human PMN killing unclear

Principal targets of ROI and RNI

• DNA• Hemes• Thioesters• Alkenes• Sulfhydryls

Phagocytic Killing Mechanisms

Radical attack

Pore formation

Enzymaticattack

Summary of Major Steps in Opsonophagocytosis

1. Opsonization2. Attachment3. Receptor clustering

and engulfment4. Phagosome

formation5. Phagolysosome

formation6. Killing and digestion

1.

2./3.

4. 5.

6.

Today’s Take Home Message

• Chemotaxis is directed movement in response to a stimulus

• Chemokines bind to a seven membrane span receptor and have multiple effects

• Leukocyte extravasation is a three-step process: 1. rolling adhesion, 2. firm adhesion, and 3. transmigration mediated by 1. selectin:sialyl Lewis, 2. ICAM:integrin, and 3. PECAM:PECAM interaction between endothelial cell and leukocyte

Additional Resources

• http://education.vetmed.vt.edu/curriculum/vm8054/Labs/Lab6/IMAGES/MONOCYTE%20IN%20SMEAR.JPG