Views
3 years ago

Gene expression profiling of human alveolar macrophages of ...

Gene expression profiling of human alveolar macrophages of ...

Normalized

Normalized expression level2.52.01.51.00.50Non-smokersSmokersNS S NS S NS S NS SNS S NS SArray TaqMan Array TaqMan Array TaqManOsteopontinADAM10Chemokine (C-X-C motif)ligand 6Fig. 2 Examples of the confirmation of microarray results withTaqMan real-time RT-PCR. For five nonsmokers and five smokers,expression levels in alveolar macrophages were quantified usingmicroarrays. For all five nonsmokers and four of the five smokers,expression levels of three genes were also measured using TaqManreal-time RT-PCR on the same RNA samples used for themicroarrays. To allow direct comparisons of values obtained usingthe two independent methods, expression levels were normalizedseparately for the microarray and TaqMan analysis by dividingindividual values by the average expression level of all nonsmokers(NS) and smokers (S) for that method. A two-way ANOVA withsmoking status (smokers vs nonsmokers) and method (microarrayvs TaqMan) as independent factors confirmed that expression levelsof these three genes were significantly affected by smoking status(p0.7, all cases). Relative expression levels are shown forosteopontin and ADAM10, genes with greater expression levels insmokers compared to nonsmokers, and chemokine (C-X-C motif)ligand 6 (CXCL6), a gene with greater expression levels innonsmokers compared to smokersinflammation and elevated numbers of AM in the lungs,our approach was to use microarray technology to carry outan unbiased assessment of AM gene expression in normalsmokers compared to nonsmokers. The rationale in usingphenotypically normal smokers with an average smokinghistory of 20 pack-years was two-fold: (1) using phenotypicallynormal smokers obviates the interpretation of theresults from being complicated by secondary processesassociated with responses to COPD per se, and (2) 20 packyearsis on the cusp of epidemiologic data associating theextent of smoking with the increased incidence of COPD[4–7]. The data demonstrates that smoking does not causeextensive changes in the global gene expression pattern ofAM, but affects the expression of a number of specificgenes. Either the gene product or the gene itself had beenpreviously shown to be altered by smoking and/or COPDin the lungs (lung tissue, epithelium, sputum, or blood) foronly 13 of the 75 genes shown to be modulated by smokingin our analysis, and of those 13, only 6 have been shown tobe specifically related to AM (Table 1). The 69 novel genesobserved to be up- or down-regulated in association withsmoking/COPD in AM belong to defined functionalcategories that are important for macrophage function,including immune/inflammatory responses, adhesion andthe extracellular matrix, proteolysis and antiproteolysis,signal transduction, transcription factors, and antioxidantrelatedfunction.Genes previously noted to be associated with smokingand/or chronic obstructive pulmonary diseaseThe use of microarray technology allows the unbiasedassessment of thousands of genes simultaneously usingsmall amounts of biological materials. However, as withany high throughput technology, the number of falsepositives is a concern. One measure of the validity ofresults obtained from microarray analysis is consistencywith previous studies that employed other methodologiesto examine changes in gene expression [17]. In thiscontext, our analysis verified the up-regulation in AM ofsix genes that have been identified in previous studies ofAM as linked to smoking and/or COPD [6, 7, 13, 18–23].Among the immune response and inflammatory genesnoted in the present study to be up-regulated in AM inassociation with smoking were chemokine (CC motif)ligand-2 [CCL2, also known as monocyte chemoattractantprotein 1 (MCP-1)] and colony stimulating factor 1 [CSF1,also known as macrophage colony stimulating factor (M-CSF)]. MCP-1 mRNA levels have been shown to beelevated in lung tissue sections of smokers/ex-smokerswith COPD [19]. Increased MCP-1 levels have also beenobserved in the sputum and in BAL fluid of individualswith COPD [18, 20]. M-CSF has been noted to be upregulatedin AM isolated from healthy smokers [21]. Theobservation in the present study of up-regulation in the AMof smokers of the scavenger receptor type A and cluster ofdifferentiation 36 (CD36) antigen is consistent with previouslypublished observations linking these genes/geneproducts with smoking, including data showing elevatedlevels of CD36 in the BAL cells of smokers compared tononsmokers.Our observation of 3.5-fold up-regulation of the macrophage-producedmatrix metalloprotease 12 (MMP12)(macrophage elastase) in cigarette smokers is in agreementwith published reports linking MMP12 to the pathogenesisof emphysema [6]. MMP12 has been found to be expressedin the AM of normal cigarette smokers and of patients withemphysema [22], and single nucleotide polymorphisms inthe MMP12 gene have been linked to susceptibility tosmoking-induced emphysema [7]. MMP12-knockout miceexposed to cigarette smoke do not develop emphysema [6].The data also shows up-regulation of α2-macroglobulin(α2M) expression in the AM of smokers. α2M is aprotease inhibitor and cytokine transporter that inhibitsmany types of proteases, including collagenases andelastases [24]. The levels of α2M in culture supernatantsfrom the AM of smokers is approximately five-foldgreater than that in the AM of nonsmokers [23], and α2Mis increased in the plasma of smokers compared tononsmokers.Interestingly, we also found up-regulation of threeantioxidant-related genes (glutathione reductase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase)in the AM of smokers, in agreement with theconcept that up-regulation of genes coding for antioxidant

enzymes is a likely protective mechanism of the lungsagainst the oxidative stress of cigarette smoke [3]. We havepreviously observed the up-regulation of 16 antioxidantrelatedgenes in the airway epithelium of phenotypicallynormal smokers compared to nonsmokers [25], includingthe three antioxidant-related genes noted to be up-regulatedin the AM of smokers in the present study.Novel genes not previously associated with smokingand/or chronic obstructive pulmonary diseaseIn addition to identifying six smoking-modulated geneswhich had been previously identified in AM in associationwith smoking and/or COPD, microarray analysis identified62 genes not previously linked to smoking or COPD at all,plus seven genes previously associated with smoking and/or COPD but not directly linked with AM. Because adetailed discussion of the potential role of each of these 69genes in smoking-induced COPD is beyond the scope ofthis study, their possible relevance to COPD will be discussedin the context of the relevant functional categories.Immune response and inflammation Nine genes in thiscategory have not been previously associated with smokingand/or COPD in any fashion, and one was previouslyobserved in regard to COPD but not in AM (lipocalin 2, amarker of neutrophil activation) [26–28]. Of the novelgenes identified in our study, osteopontin had the highestdifferential expression in smokers vs nonsmokers (>5-fold), an observation that was confirmed by TaqMan realtimeRT-PCR. Also known as secreted phosphoprotein 1,osteopontin is an arginine-glycine-aspartic acid motifcontainingprotein that is produced by different cell types,including activated macrophages, T cells, and osteoclasts[29–31]. This multifunctional protein has a role in chemotaxis,cell adhesion and proliferation of macrophages,smooth muscle cells, and epithelial cells [29–31]. Activatedalveolar macrophage-produced osteopontin has beenimplicated as a fibrogenic cytokine in a bleomycininducedmouse model of lung fibrosis [32, 33]. In contrastto the marked up-regulation of osteopontin, chemokineCXC ligand 6 (CXCL6; also known as granulocyte chemotacticprotein 2) was significantly down-regulated inthe AM of smokers as compared to nonsmokers. CXCL6is a chemotactic factor for granulocytes [34]; it is possiblethat the down-regulation of CXCL6 is part of themechanism of defense against inflammation in smokers.Adhesion/extracellular matrix Our analysis identified fivegenes in the category of adhesion and extracellular matrix(four up-regulated: α E -integrin, α2-collagen type 6, vinculin,and activated leukocyte cell adhesion molecule; onedown-regulated: intracellular adhesion molecule 3) notpreviously associated with smoking/COPD, suggestingthat the extent of modulation of expression levels inadhesion/extracellular matrix genes by cigarette smokemay be more widespread than previously thought.Proteases and antiproteases The up-regulation of proteolyticenzymes by AM in response to smoking has beenpostulated as pivotal in the pathogenesis of COPD, inparticular in the alveolar destruction that characterizesemphysema [10, 35, 36]. In addition to confirming previousobservations of up-regulation of MMP12, a proteasepreviously associated with smoking/COPD [6, 7], we alsoobserved two proteases to be up-regulated in the AM ofthe smokers that have not been previously noted, includingADAM10, a member of the disintegrin and metalloproteasedomain family of proteases and insulin-degradingenzymes. This observation was confirmed by TaqMan RT-PCR. In another study derived from this observation, wehave shown that overexpression of ADAM10 in the lungsof mice is associated with the development of emphysema[37]. Interestingly, we also found that serpin B5, a serineprotease inhibitor (serpin), member 5 of clade B of theserpin family (also known as maspin), was markedlydown-regulated (8.5-fold) in the AM of smokers. Thisserpin has been postulated to act as a tumor suppressor, byinhibiting cell motility, adhesion, and metastasis.Signal transduction and regulation of transcription Wefound that smoking modulates the expression in AM ofnine genes belonging to the general category of signaltransducers and six genes encoding proteins involved intranscriptional regulation. With the exception of the transcriptionfactor β-retinoid X receptor, previously linked tosmoking in lung cancer precursor lesions [38] and in thebronchial epithelium of heavy smokers [39, 40], thesegenes have not been previously related to smoking and/orCOPD. The observations that smoking alters the expressionlevels of genes coding for signal transduction ortranscription factors is not surprising, because cigarettesmoke, like other environmental stimuli, has been reportedto affect signal transduction pathways and transcriptionfactors in the lungs, including the mitogen activated proteinkinase (MAPK) pathway [14, 41–43]. Among thetranscription factors, we observed down-regulation of thehairy and enhancer of split 1 (HES-1). Enhanced expressionof the HES-1 gene in the early stages of macrophagedevelopment and differentiation leads to inhibition of thegrowth of macrophage progenitors [44]; and thus, thedecreased expression of HES-1 in smokers may be relatedto the increased numbers of AM in the lungs of smokers[8, 9].Overall implicationsThe present study provides further evidence that smokingspecifically modulates alveolar macrophage gene expressionin phenotypically normal smokers, before there is anyevidence of lung disease. COPD is a complex disease, andthere are likely multiple mediators involved in the evolutionof the COPD phenotype in individuals who smoke.Microarray analysis is likely to aid in the identification ofgenes involved in the pathological processes leading to

Gene-expression Profiles as Gene-expression Profiles as
Gene Expression Profiles of Multiple Independent Cadmium- and ...
Gene expression profiling in DNA repair- deficient Xpa ... - Cefic LRI
HIV-1 Infection Does Not Impair Human Alveolar Macrophage ...
Human Alveolar Macrophage Phagocytic Function is ... - Precaution
Comprehensive gene expression profiles reveal pathways related to ...
bacterial products in human alveolar macrophages ... - Progetto LIBRA
in Activated Alveolar Macrophages - Medicina Biomolecular
Characterization and Gene Expression Profiling of Human ...
Gene expression profiling of human oocytes following in ... - Sismer
Gene Expression Profiling of Human Lung to Elucidate the Genetic ...
Gene expression profiling in the human hypothalamus-pituitary ...
3'tag digital gene expression profiling of human brain and universal ...
Gene expression profiling for adult human olfactory neuroepithelial ...
Gene expression profiling in human insulinoma tissue - Endocrine ...
A quality-controlled microarray method for gene expression profiling
Methodological considerations for gene expression profiling of ...
Gene expression profiling and qRT-PCR expression of RRP1B ...
Mycobacterium tuberculosis gene expression profiling ... - UCLA-DOE
Global gene expression profiles reveal significant nuclear ...
Gene expression profiling in the stem of young maritime pine trees ...
Gene Expression Profiling of Xeroderma ... - BioMed Central
Gene expression profiles in liver of zebrafish treated with microcystin ...
TNF- Release from Alveolar Macrophages and Serum Level of sIL ...
Focused microarray analysis of glyco-gene expression in human ...
Interspecific profiling of gene expression informed ... - Hans Hofmann
Gene expression profiles as a prognostic marker in women with ...
Preliminary Analysis of Gene Expression Profiles in HepG2 Cell ...
Gene expression profiling of chronic lymphocytic leukemia can ...