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Chapter 42 : Chemokine and Chemokine Receptor Analysis

Authors: Benjamin D. Medoff, Andrew D. Luster
Content Type: Reference
Publication Year: 2006

Category: Immunology

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Abstract:

This chapter describes the methodological approaches to studying the role of chemokines and chemokine receptors in the physiology of immune and inflammatory responses. Chemokines share the common function of attracting leukocytes to sites of an inflammatory or immune response. The G protein-coupled cell surface receptors (GPCRs) as signal transducers of chemotaxis appear to be highly conserved in evolution, being present on amoebae and slime molds, and are involved in the signaling of chemotaxis in these simple eukaryotes. Activation of chemokine receptors is usually accompanied by a transient rise in the level of intracellular calcium. The attraction of leukocytes to sites of inflammation and infection is an essential component of the host response to disease. Chemokines and chemokine receptors have been shown to be an integral part of this process and have been implicated in the pathophysiology of many infectious diseases and inflammatory disorders. The recruited monocytes and T lymphocytes are thought to play a key role in the pathogenesis of atherosclerotic plaque, and thus the molecular signals that attract these cells into the lesions are likely important for lesion formation. The majority of cells recruited into the synovium are neutrophils and mononuclear leukocytes that presumably help propagate the inflammation and joint destruction. Chemokine expression in the lung or blood could be used as a reliable noninvasive marker of inflammation in the airways and thus would be of great value in the management of asthma.

Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42
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Chemokine and Chemokine Receptor Analysis
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Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42
/content/10.1128/9781555815905.ch42.ch42fig01
FIGURE 1
Chemokine receptor signal transduction. Chemokine receptors are a subfamily of seven-transmembrane-spanning GPCRs. They are coupled to heterotrimeric G proteins of the Gi subclass, which are distinguished by their pertussis toxin sensitivity. Chemokine receptor activation leads to the stimulation of multiple signal transduction pathways, including the activation of PI 3-kinase (PI-3K) and phospholipase C, leading to the generation of inositol triphosphates, intracellular calcium release, and PKC activation. Chemokine signaling also induces the upregulation of integrin affinity and the activation of Rho, leading to cytoskeletal reorganization. Agonist-stimulated receptors also activate G protein receptor kinases, which leads to receptor phosphorylation, arrestin binding, G protein uncoupling (desensitization), and clathrin-mediated receptor endocy-tosis (internalization). LTB4, leukotriene B4; C5a, complement fragment 5a; PAF, platelet activating factor; cAMP cyclic AMP; PLC β2, phospholipase C β2; DAG, diacylglycerol.
10.1128/9781555815905/f0373-01_thmb.gif
10.1128/9781555815905/f0373-01.gif
Image of FIGURE 1
FIGURE 1

Chemokine receptor signal transduction. Chemokine receptors are a subfamily of seven-transmembrane-spanning GPCRs. They are coupled to heterotrimeric G proteins of the Gi subclass, which are distinguished by their pertussis toxin sensitivity. Chemokine receptor activation leads to the stimulation of multiple signal transduction pathways, including the activation of PI 3-kinase (PI-3K) and phospholipase C, leading to the generation of inositol triphosphates, intracellular calcium release, and PKC activation. Chemokine signaling also induces the upregulation of integrin affinity and the activation of Rho, leading to cytoskeletal reorganization. Agonist-stimulated receptors also activate G protein receptor kinases, which leads to receptor phosphorylation, arrestin binding, G protein uncoupling (desensitization), and clathrin-mediated receptor endocy-tosis (internalization). LTB4, leukotriene B4; C5a, complement fragment 5a; PAF, platelet activating factor; cAMP cyclic AMP; PLC β2, phospholipase C β2; DAG, diacylglycerol.

Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42
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FIGURE 2
Transmigration assay system. Positive, negative, and absent gradients of a chemokine are established in order to assess chemotaxis (movement towards a chemokine) and chemokinesis (random movement of cells in response to a chemokine in the absence of a gradient). Cells are plated into the upper chamber of the transwell system or Boyden chamber, and the proportion of migrating cells is determined by accurate counting of cells which migrate to the lower chamber. The upper and lower chambers are separated by a polycarbonate membrane of standard pore size (between 3 and 8 μm), dependent on the migrating cell type.
10.1128/9781555815905/f0377-01_thmb.gif
10.1128/9781555815905/f0377-01.gif
Image of FIGURE 2
FIGURE 2

Transmigration assay system. Positive, negative, and absent gradients of a chemokine are established in order to assess chemotaxis (movement towards a chemokine) and chemokinesis (random movement of cells in response to a chemokine in the absence of a gradient). Cells are plated into the upper chamber of the transwell system or Boyden chamber, and the proportion of migrating cells is determined by accurate counting of cells which migrate to the lower chamber. The upper and lower chambers are separated by a polycarbonate membrane of standard pore size (between 3 and 8 μm), dependent on the migrating cell type.

Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42
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FIGURE 3
Digitized time lapse photography of cells moving in the presence of a gradient of a chemokine. Positive and negative gradients of a chemokine can be established in methylcellulose as previously described ( ). Cells are plated into methylcellulose, and a gradient is established by inoculating the methylcellulose at a fixed point with the chemokine. Cells are then visualized migrating in response to the gradient by using time lapse video microscopy.
10.1128/9781555815905/f0378-01_thmb.gif
10.1128/9781555815905/f0378-01.gif
Image of FIGURE 3
FIGURE 3

Digitized time lapse photography of cells moving in the presence of a gradient of a chemokine. Positive and negative gradients of a chemokine can be established in methylcellulose as previously described ( ). Cells are plated into methylcellulose, and a gradient is established by inoculating the methylcellulose at a fixed point with the chemokine. Cells are then visualized migrating in response to the gradient by using time lapse video microscopy.

Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42
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Tables

Chemokine and Chemokine Receptor Analysis
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Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42
/content/10.1128/9781555815905.ch42.ch42tab01
TABLE 1
Chemokines and receptors
Generic image for table
TABLE 1

Chemokines and receptors

Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42
/content/10.1128/9781555815905.ch42.ch42tab02
TABLE 2
Chemokines identified in human atherosclerotic lesions
Generic image for table
TABLE 2

Chemokines identified in human atherosclerotic lesions

Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42
/content/10.1128/9781555815905.ch42.ch42tab03
TABLE 3
Chemokines and chemokine receptors with importance in RA based on human and animal studies ( )
Generic image for table
TABLE 3

Chemokines and chemokine receptors with importance in RA based on human and animal studies ( )

Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42
/content/10.1128/9781555815905.ch42.ch42tab04
TABLE 4
Chemokines and chemokine receptors with importance in asthma based on human and animal studies ( )
Generic image for table
TABLE 4

Chemokines and chemokine receptors with importance in asthma based on human and animal studies ( )

Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42
/content/10.1128/9781555815905.ch42.ch42tab05
TABLE 5
Chemokines and chemokine receptors with importance in transplantation based on human and animal studies ( )
Generic image for table
TABLE 5

Chemokines and chemokine receptors with importance in transplantation based on human and animal studies ( )

Citation: Medoff B, Luster A. 2006. Chemokine and Chemokine Receptor Analysis, p 371-384. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch42

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