cDNA Representational Difference Analysis of Human Neutrophils Stimulated by GM-CSF

doi:10.1006/bbrc.2000.3678

Biochemical and Biophysical Research Communications

Volume 277, Issue 2, 22 October 2000, Pages 401-409

https://doi.org/10.1006/bbrc.2000.3678 Get rights and content

Abstract

Neutrophils are the first cell type to migrate out of the vascular space and into the inflammatory site during an acute inflammation. However, in chronic inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), a lack of clearance of neutrophils, imbalance between inflammatory mediators produced by neutrophils and their natural inhibitors make these cells a potential cause of tissue destruction in lung disease. Neutrophilic inflammation is generally characterised by high levels of local expression of activating cytokines (e.g., GM-CSF). Only a few studies have been published so far that have investigated the expression of genes preferentially expressed in activated neutrophils. The isolation of such genes, however, can lead to a better understanding of inflammatory disease and the identification of potential novel therapeutic targets or markers of the disease. We performed representational difference analysis of cDNA, a sensitive PCR-based subtractive enrichment procedure, and isolated 12 genes, 1 EST clone, and 3 sequences not represented in the public databases. Differential expression for 9 of these clones was confirmed by Northern hybridisation. Of the above nine transcripts three were chosen and shown to be up-regulated in neutrophils cocultured with stimulated primary human bronchial epithelial cells using a semiquantitative RT-PCR approach. Among the known genes identified were HM-74, CIS1, Cathepsin C, α-enolase, CD44, and the gene Translocation Three Four (TTF), most of them previously not known to be involved in GM-CSF induced neutrophil activation. Along with its tissue and cellular distribution we also derived the complete cDNA sequence and genomic structure of CIS1 using an in silico approach. In addition, we also report the initial characterisation of a novel gene, P1-89 that is primarily expressed in granulocytes and is up-regulated in activated cells. Our results identify several important genes associated with neutrophil activation and can lead to a better understanding of the molecular mechanisms of neutrophilic inflammations.

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^☆: Abbreviations used: granulocyte macrophage colony stimulating factor, GM-CSF; chronic obstructive pulmonary disease, COPD; complementary deoxyribonucleic acid, cDNA; difference product 1, DP1; difference product 2, DP2; expressed sequence tag, EST; glyceraldehyde-3 phosphate dehydrogenase, GAPDH; messenger ribonucleic acid, mRNA; nanogram, ng; polymerase chain reaction, PCR; polymorphonuclear, PMN; representational difference analysis, RDA; human bronchial epithelium cell, HBEC.

¹: To whom correspondence should be addressed. Fax: 44-1403-323253. E-mail: [email protected].

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Regular ArticlecDNA Representational Difference Analysis of Human Neutrophils Stimulated by GM-CSF☆

Abstract

Lancet

Trends Pharmacol. Sci.

Immunol. Today

Cytokine Growth Factor Rev.

Proc. Nat. Acad. Sci. USA

Blood

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Clin. Chim. Acta.

Biochim. Biophys. Acta

Granulocyte apoptosis and its role in the resolution and control of lung inflammation

Am. J. Respir. Crit. Care Med.

Regulation of transendothelial neutrophil migration by endogenous interlukin-8

Science

Circulating granulocyte macrophage colony-stimulating factor in plasma of patients with the systemic inflammatory response syndrome delays neutrophil apoptosis through inhibition of spontaneous reactive oxygen species generation

Shock

Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: A general mechanism to accumulate effector cells in inflammation

Proc. Nat. Acad. Sci. USA

Regular Article
cDNA Representational Difference Analysis of Human Neutrophils Stimulated by GM-CSF☆