Apoptosis or programmed cell death is vital for the resolution of inflammation, and phagocytosis of apoptotic cells triggers the discharge of actively anti-inflammatory responses from the phagocytes. Eosinophils are among the most potent inflammatory cells within the body and it is linked to numerous diseases, in most cases connected with parasitic infections and allergic diseases. Apoptosis in eosinophils thus remains probably the most significant systems to prevent inflammation. The purpose of the current study was to look at the mechanisms behind, and the outcomes of this process in eosinophils. Apoptotic eosinophils have a unique surface receptor expression which shows capabilities to communicate with T-, B- and antigen presenting cells. They have a novel expression of CD49f, implying an importance for binding to laminin or unfamiliar functions of the VLA-6 receptor, possibly in the idea of phagocytosis of the apoptotic cell. In apoptotic eosinophils the granules are translocated to the periphery of the cell, most likely via a disruption of the cytoskeleton…
Contents: Eosinophil Apoptosis
INTRODUCTION
The History of Apoptosis
Programmed cell death
Morphological characteristics of apoptosis
DNA fragmentation
Phosphatidylserine exposure
Recognition by phagocytic cells and disposal of cellular corpses
Genetics and regulating factors
Bcl-2 Family proteins
p53 gene expression
Caspases
Death receptors
The signalling process of death receptor activation
The Eosinophil Granulocyte
Morphology and functions
Release of granule proteins
Oxidative Metabolism and Lipid Mediators
Adhesion
Eosinophil apoptosis
Eosinophil pro- and anti-apoptotic cytokines
The importance of phosphorylation
Eosinophil roles in health and disease
Parasite infections
Asthma and allergy
Eosinophils and cancer
Tissue repair and fibrosis
AIM
METHODS
Isolation of eosinophils (Studies I-IV)
Culture of the purified eosinophils (Studies I-IV)
Culture with sodium azide (study I)
Culture in the presence of IL-2
Culture in laminin-coated plate
Isolation of Annexin V positive cells using the Apoptotic Cell Isolation Kit (Studies I-IV)
Staining with FDA and PI (Studies I-IV)
Staining with anti-CD95 (Study I)
Staining with Annexin V
Staining of surface receptors
Flow cytometry (Studies I-IV)
Cell sorting by flow cytometry (Study I)
Examination by light microscopy (Study I)
Examination by electron microscopy (Studies I and III)
Analysis of DNA fragmentation by agarose gel electrophoresis (Study I)
Density gradient centrifugation (Study I)
Release of granule proteins
ECP and EPX assay
Adhesion to endothelial cell adhesion receptors
Adhesion to laminin and albumin
Induced sputum
Statistics
REFERENCE CONTROLS AND PATIENTS
Reference Controls (Studies I-IV)
Patients (Study II)
Pollen Season Study design
Bronchial challenge with inhaled birch pollen extract
Induction of sputum
RESULTS
Presence of apoptotic cells with degraded DNA measured by flow cytometry (Study I)
Forward and side scatter pattern
Staining with PI and FDA
Examination by electron microscopy
Analysis of DNA fragmentation by agarose gel electrophoresis
Use of other techniques to further confirm apoptosis
Functions of Apoptotic Eosinophils (Studies II-IV)
Adhesion to E-selectin, VCAM-1 and ICAM-1 (Study IV)
Adhesion to laminin (Study IV)
Survival in laminin-coated plates (Study IV)
Release of ECP (Study III)
Release of EPX (Study III)
Examination by electron microscopy (Study III)
Release of ECP during culture (Study III)
Cell surface expression of β2-integrins (Studies III and IV)
Cell surface expression of β1-integrins (Studies III and IV)
Cell surface expression of CD44 and CD66b
Cell surface expression of CD9
Eosinophil expression of Fc receptors…
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