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Identifying genes in lung fibroblasts

15th December 2014

Posted By Paul Boughton


Fig. 1. Table shows donor characteristics of primary cells used.
Fig. 2. Genes up- or downregulated in asthmatic sample vs. normal cells.
Fig. 3. Genes up- or downregulated in COPD sample vs. normal cells.

Ludger Altrogge, Lubna Hussain and Rochelle Myers report on asthma- and COPD-related differential gene expression in primary human lung fibroblasts.

With the prevalence of asthma and chronic obstructive pulmonary disease (COPD) growing worldwide, the availability of primary human cells from these respiratory diseases is critical to increase research at a cellular level. In this study, we sought to identify genes differentially regulated in asthma and COPD lung fibroblasts. Primary lung fibroblasts are isolated from normal, asthma and COPD human donors in accordance with all informed consent rules and regulations. 

The cells were cultured for seven passages in optimised media and gene expression analysis was performed. This article summarises a study comparing normal versus COPD lung fibroblasts and normal versus asthma lung fibroblasts to explore differences in gene expression among the donor samples.

In this study, we grew primary lung fibroblasts from normal, asthmatic and COPD tissues in standard submerged culture (see Fig. 1) to assess gene expression changes associated with diseased states.
Each lot of cryopreserved P2 normal and diseased lung fibroblasts was thawed and plated at a density of 2,500 cells/cm2 in FGM-2 Growth Medium. Growth media were changed after 24 hours and the cells were subcultured through seven passages. At P7, cells were pelleted and cell lysates were obtained. For each cell lysate, RNA extraction and cDNA synthesis was performed according to the specifications of individual suppliers. 

cDNA samples were distributed into Human Immune System Phenotyping 96 StellARray qPCR Array Plate. Arrays were run on the BioRad CFX 96 using a standard qPCR program. To generate data with biologically relevant variance, three replicate samples were independently assayed using qPCR for each cell type.

Results and discussion
In each diseased cell type, statistically relevant differences in gene expression were detected in the diseased cells compared to the normal cells. Genes with P-values of <0.05 or better are reported.

The asthmatic fibroblast cells had two upregulated genes as compared to the normal – CD14 (upregulated 4.97 fold) and IL7R (upregulated 2.8 fold). Both of these genes play a role in the innate immune system that is activated in response to stimuli in asthmatic patients(1). CD14 is a surface antigen that is expressed on several immune cells and IL7R is critical in the development and activation of lymphocytes(2). The two downregulated genes in the asthmatic donor were CD22 and CD40 (3.2 fold and 4.6 fold respectively). CD40 encodes for a protein involved in a variety of immune and inflammatory responses2.

The COPD sample had several upregulated genes compared to the normal sample. Two of these genes, TNFSF4 (upregulated 15.1 fold) and TNFSF18 (upregulated 10.4 fold), encode for proteins that are cytokines and part of the tumor necrosis factor (TNF) ligand family. TNFSF4 is involved in T cell antigen-presenting cell interactions and is reported to mediate adhesion of activated T cells to vascular endothelial cells(2).TNFSF18 has been observed to modulate T lymphocyte survival in peripheral tissue and is expressed in endothelial cells(2). CD40 (upregulated 4.2 fold) encodes for a protein belonging to the TNF-receptor family. 

The receptor is critical in mediating a broad variety of immune and inflammatory responses(2). CD36 (upregulated 15.1 fold) encodes a protein that serves as a receptor for thrombospondin in platelets(2). One study found that long-term cigarette smoke exposure is associated with fibrosis and inflammation via increased levels of thrombospondin(3). KLRD1, expressed by natural killer cells that mediate cytotoxic activity and secrete cytokines upon immune stimulation, was upregulated 7.1 fold(2). One significantly downregulated gene was CD24 (25.3 fold), which encodes a sialoglycoprotein that is expressed on mature granulocytes and also in many B cells2.

Summary
We have described a method in which normal, asthmatic, and COPD lung fibroblasts were expanded and gene expression was analysed. The resulting data revealed differences in gene expression between the normal and diseased cell types. Access to these diseased cell types provides a convenient, biologically relevant model to assess the genetic pathways involved in the disease and may help to indicate other potential drug targets. Lonza currently offers a selection of various cell types from normal, asthmatic, and COPD donors. The cells are tested and guaranteed to perform with the suggested Clonetics Media Kits and Reagents.

References:

1  Klaassen et al. Allergy, Asthma & Clinical Immunology, 9:10, 2013.
2  Pubmed GENE IDs
3  Ishikawa T. et al. Correlation Between Thrombospondin-1 And Pulmonary Function After Long-Term Cigarette Smoke Exposure. Am J Respir Crit Care Med. 18:5, 2012.

For more information visit www.scientistlive.com/eurolab

Ludger Altrogge is with Lonza Cologne, Germany, Rochelle Myers and Lubna Hussain are with Lonza Walkersville, USA. www.lonza.com






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