Inflammation Protocols [Methods in Molec Bio 225] - P. Winyard, D. Willoughby (Humana, 2003) WW.pdf - książki - Bio Med 14 - bibiariel

Methods in Molecular Biology TM

VOLUME 225

Inflammation

Protocols

Edited by

Paul G. Winyard

Derek A. Willoughby

HUMANA PRESS

Key Stages in the Acute Inflammatory Response

and Their Relevance as Therapeutic Targets

Introduction to Part 1

Paul G. Winyard

1. Introduction

Inflammation, in its broadest sense, is a host response to tissue injury. The

four ancient, cardinal, signs of inflammation are redness, heat, swelling, and

pain. These clinical signs of inflammation are, of course, the macroscopic cul-

mination of molecular and cellular processes, many of which have become

well defined over the last 120 years, and many of which may be reproduced in

convenient experimental systems in vitro. In collecting the chapters for this

section of the book, our aim is to provide a repository for experimental proto-

cols for the in vitro study of key stages of the inflammatory response. For the

reader who is unfamiliar with the field of inflammation, it is perhaps helpful to

summarize and contextualize some of the key events of the inflammatory res-

ponse, as it is these that may be reproduced in the form of in vitro model sys-

tems by using the protocols that follow.

Within the vast array of interdigitating and iterative molecular and cellular

pathways that constitute inflammation, there are certain linear pathways that have

recently received great attention as, perhaps, the “main highways” of the inflam-

matory response. By way of indicating the significance of some of the experi-

mental protocols in this section, it is convenient to refer to one of these pathways

( see Fig. 1 ), which begins (at least for the purposes of this discussion) with the

stimulation of vascular endothelial cells by key “pro-inflammatory” cytokines

such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The

importance of this pathway described later is validated, at least in part, by the

promise of the therapeutic targets that have been defined at the different stages

along its route, although there are numerous bifurcations away from this path

From: Methods in Molecular Biology, vol. 225: Inflammation Protocols

Edited by: P. G. Winyard and D. A. Willoughby © Humana Press Inc., Totowa, NJ

Winyard

Fig. 1. Grossly simplified schematic diagram representing some of the key events

of the inflammatory response. The letters correspond to some of the processes that can

be investigated using protocols described within this first section of the book (or, if in

brackets, elsewhere in this book). A, intracellular kinase activation by cytokines; B,

activation of NF-κB; C, adhesion molecule expression; D, migration (of specific leu-

kocyte subsets); E, stimulation of inflammatory phagocytes, including changes in

cytosolic Ca 2+ and plasma membrane NADPH oxidase; F, opsonisation involving

activation of the complement cascade; G, release and activation of MMPs within the

extracellular matrix; H, breakdown of the extracellular matrix by proteinases—e.g.,

degradation of cartilage by a MMPs and aggrecanase.

that are not discussed here. In many cases, the individual protocols described

in this section will allow the in vitro screening of candidate therapeutic com-

pounds.

Returning to a description of our illustrative “main highway” of inflamma-

tion, the binding of pro-inflammatory cytokines to their cognate plasma mem-

brane receptors results in the activation of intracellular kinases that catalyze a

cascade of phosphorylation events, including the phosphorylation (catalyzed

by the IKK complex) of IκB, an inhibitory protein subunit bound to the tran-

scription factor NF-κB. The control of gene transcription by specific proteins—

Key Stages in the Acute Inflammatory Response

transcription factors—involves the binding of these proteins to short DNA

sequence elements located adjacent to the promotor or in the enhancer regions

of genes. The phosphorylation of IκB targets it for proteolytic degradation by

the 26S proteasome, leaving the active NF-κB complex to be translocated to

the nucleus, where it binds to a specific DNA motif and is thereby involved,

together with other transcription factors, in controlling the transcription of an

array of inflammation-associated genes. Indeed, a key feature of inflammatory

diseases such as rheumatoid arthritis (RA) is the increased expression of cer-

tain genes that encode “inflammatory” proteins. These include a variety of

adhesion molecules, as well as TNFα, IL-1β, and inducible nitric oxide syn-

thase (iNOS) among many others.

Dr. Mireille Delhase describes methods for measuring cytokine-induced

IKK activity and NF-κB activation in cultured cells. By stably transfecting

cells with a construct containing the NF-κB-binding motif upstream of the

luciferase gene, a convenient system for screening potential inhibitors of NF-

κB may be created, as described by Dr. Deborah Phippard and Dr. Tony Man-

ning. Recently, there has been considerable interest in the idea that transcription

factors may be useful targets for novel therapeutic strategies in the treatment of

human diseases, including inflammatory diseases.

As aforementioned, induction of the expression of several adhesion mol-

ecules, such as intercellular adhesion molecule-1 (ICAM-1), and E-selectin

vascular cell adhesion molecule-1 (VCAM-1), represents a significant inflam-

matory event and a potential therapeutic target. The increased transcription of

these proteins ultimately results in their upregulation on the plasma membrane

surface of endothelial cells, and the levels of adhesion molecules may be mea-

sured using the techniques described by Dr. Susan Cuvelier and Dr. Kamala

Patel. The increased expression of adhesion molecules, in turn, facilitates the

“rolling” and adhesion to the vascular endothelium of inflammatory phago-

cytic cells at vessel sites adjacent to the site of inflammation. The phagocytic

cells, such as neutrophils, then migrate through the vessel wall, via the process

of diapedesis, and arrive at the focus of “acute” inflammation. The reader is

referred to the second section of this book for a description of an in vivo tech-

nique for the study of this process, as provided by Professor Mauro Perretti and

Dr. Stephen Getting. Later, in the “chronic” phase of inflammation, other cell

types (e.g., macrophages and lymphocytes) will be recruited to the site of inflam-

mation by an analogous process. As part of the classic, acute inflammatory

response, neutrophils are activated by immune complexes, the complement

system and/or pathogens to produce both free radicals (such as superoxide)

and proteinases (such as elastase and cathepsin G) which act in concert to kill

invading fungi or bacteria, but which may also cause tissue damage. This stimu-

lation of neutrophils can be reproduced in vitro using isolated cells and the

extent of the response in the presence of putative antiinflammatory compounds

Winyard

may be tested using the methods described by Dr. Maurice Hallett et al. This

group also describes specific methods for the measurement of neutrophil intra-

cellular Ca 2+ fluxes and O 2 •– production by a plasma membrane NADPH oxi-

dase. The killing of bacteria by neutrophils involves their opsonization by plasma

proteins, including various components of the complement cascade. Furthermore,

certain components of the complement cascade are chemotactic for neutrophils.

A protocol by which the extent of complement activation may be determined is

described by Professor Tom Eirik Mollnes.

Phagocytic cells, and sometimes resident cells at the site of inflammation,

are also stimulated by cytokines such as TNF and IL-1 to release matrix

metalloproteinases (MMPs). In respect of the inflamed joint, the resident cells

of the synovial membrane—the so-called type B synoviocytes—are activated

by cytokines to release MMPs. The MMPs involved in inflammatory tissue

destruction include collagenases, gelatinases, and stromelysins. The activities

of these proteinases may be determined, again in an in vitro system, according

to the zymographic technique detailed by Dr. Linda Troeberg and Professor

Hideaki Nagase. Among the expressed metalloproteinases is aggrecanase,

which plays an important role in the degradation of cartilage within the rheu-

matoid joint. Professor Bruce Caterson et al. describe a protocol for the mea-

surement of aggrecanase activity, whereas Dr. Bill Shingleton describes an in

vitro model of articular cartilage degradation.

In finishing this introduction to in vitro protocols for the study of inflamma-

tion it should be stressed again that, for the sake of simplicity, the description

above refers to one of many pathways of acute inflammation. Many of the exper-

imental systems described in this section of the book are relevant to more than

one stage or type of inflammation, e.g., acute versus chronic, immune vs

nonimmune, and so on. Although, for convenience, the inflammation protocols

in this book have been divided between in vitro and in vivo methods, it is vital

to have both. It is, of course, impossible to reproduce inflammation in vitro. In

vivo, the environment at the site of inflammation changes millisecond by milli-

second—this can never be reproduced in a test tube. However, the complemen-

tary use of both in vitro and in vivo techniques is a powerful strategy in the

study of inflammation and antiinflammatory drug development: For example,

the cytokines can be identified in vivo, whereas the signal transduction path-

ways leading to the production of such cytokines can be characterized in vitro.

The combination of the outputs from these two approaches may then allow the

demonstration of the importance of a particular pathway by the in vivo testing

of selective inhibitors.

Inflammation Protocols [Methods in Molec Bio 225] - P. Winyard, D. Willoughby (Humana, 2003) WW.pdf

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika: