Neutrophil Structure

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• Neutrophil Structure Function

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Learning Objectives

• 1. List contents of neutrophils that affect its
  function.
• 2. Describe neutrophil function in terms of the
  three phases of phagocytosis (chemotaxis,
  opsonization, ingestion/intracellular killing)
• 3. Identify briefly describe selected
  disorders with abnormal neutrophil function.
• 4. Describe interpret methods for the
  investigation of neutrophil function.

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Neutrophil Granules

• Four types of granules present
• a. primary -azurophilic, non-specific,normally
  not visible
• b. secondary - pink, specific
• c. tertiary -identified using electron
  microscopy
• d. secretory vesicles -contain enzymes, other
• proteins endocytosed from plasma
• Contents of granules are involved in killing of
  microorganisms

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• secondary granules
• lysozyme
• cytochromes to produce NADPH oxidase
• lactoferrin
• B12 binding protein
• complement activators
• tertiary granules
• gelatinase / membrane glycoproteins
• secretory vesicles
• plasma proteins
• phosphasome
• alkaline phosphatase

Structure - So whats in them granules anyway?

• primary granules
• myeloperoxidase
• lysozyme
• proteases
• defensins

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Function - What do neutrophils do?

• Protect the body against infectious agents
• part of the inflammatory response
• Bacteria stimulate macrophages and basophils to
  release cytokines and histamine which results in
  increased blood flow to infected area
• Spaces open up between endothelial cells
  resulting in increased vascular permeability
• Leukocytes migrate into site of infection
• Collection of fluid and cells causes swelling,
  heat pain of inflammation

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Function - So how do they do it?

• 1. Migration diapedesis
• 2. Opsonization recognition
• 3. Ingestion intracellular killing

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I. Migration Diapedesis - Neutrophils moving to
the site

• chemotaxis - directed movement of phagocytic
  cells to a site of injury via a concentration
  gradient of chemotactic substances
• General Mechanism
• neutrophils have receptors for
• adhesion (CD11/Cd18 complex)
• complement (C3a, C3b, C567)
• immunoglobulin
• kinin-coagulation system products (increases
  permeability of endothelial cells)
• bacterial products
• phagocyte released products (cytokines proteins)

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Chemoattractants
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Adhesion Receptors

• cell adhesion molecules (CAMs)
• neutrophil receptor CD11a-c/CD18
• endothelial receptors CD54 (ICAM1)

CD54
CD11a-c/CD18
What do you think happens when you lack these
receptors?
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II. Opsonization Recognition

• to prepare for dining
• opsonin - any agent in serum which acts on a
  particle to induce phagocytosis
• IgG
• IgM
• C3b

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III. Ingestion Intracellular Killing

• ingestion needs ATP from our favorite pathway!

30 seconds
pH 6.5 - 7.4
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A few minutes later...

• myeloperoxidase released from primary granules

pH 4.0
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Biochemistry of Phagocytosis

• 1. Oxygen Dependent
• O2 uptake
• also called a respiratory burst

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myleoperoxidase

• H2O2

halide
hypohalite
hypohalite good ole bleach
oxidation of bacteriahalogenation of bacterial
cell walldecarboxylation of cell wall amino acids
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2. Oxygen Independent

• low pH
• lysozyme
• lactoferrin
• granular cationic proteins

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Save the Neuts!!

• 2O2-

2H2O2
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Deranged Neuts!

• 1. Abnormal adhesion
• 2. Impaired chemotaxis
• lazy leukocyte syndrome
• Chediak Higashi
• 2. Poor opsonization / phagocytosis
• newborns
• hypogammaglobulinemia
• viruses
• 4. Abnormal organelles
• may hegglin, chediak higashi, alder reilly,
  pelger huet
• 3. Lack of Intracellular killing
• chronic granulomatous disease
• myeloperoxidase deficiency

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Chediak-Higashi Disease

• Autosomal recessive
• patients have recurrent infections,abnormal
  bleeding,mental retardation,enlarged spleen and
  liver
• defective bacterial killing due to abnormal
  chemotaxis and degranulation
• giant primary granules in neutrophils and
  monocytes
• prolonged bleeding times due to defective
  platelet aggregation

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Chronic Granulomatous Disease

• sex linked recessive
• symptoms of disease appear in 1st year of life
• often progresses to death in early childhood
• predisposition to infections by gram s
• Neutrophils and monocytes can ingest, and
  degranulate but cannot kill
• defect in respiratory burst, decreased H2O2,
• 02-, and HOCL production
• bacteria dont die, released when neutrophil dies

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Lazy Leukocyte Syndrome

• Neutrophils show defective chemotaxis
• (defective response to chemotactic factors)
• recurrent infections
• Abnormal Opsonization
• hypogammaglobulinemia
• newborns - low levels of IgG, IgM

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Investigating Neuts

• 1. Clinical information
• 2. white cell count morphology
• 3. immunoglobulin levels
• 4. rebuck skin test
• 5. Nitroblue tetrazolium
• 7. chemiluminescence

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Rebuck Skin Window

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Nitroblue Tetrazolium Test

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Other Function Tests

• Myeloperoxidase stain
• defect of MPO is primary granules
• flow cytometry
• check for adhesion molecules
• ingestion assays
• check for problems with opsonization

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Ingestion