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Abstract:
Background
In rheumatoid arthritis (RA) macrophages (Mf) play a pivotal role. They become highly activated in synovitis and at the cartilage–pannus junction. Furthermore, therapeutic neutralization of molecules produced by activated Mf lead to clinical improvement in RA, and circulating monocytes (MO) of the peripheral blood in patients with RA spontaneously express proinflammatory genes (IL-1β, IL-6, TNF).
Methods
A custom RA-MO cDNA microarray was generated using differentially expressed genes obtained from gene subtraction and from comparative whole genome wide U133A analysis in normal donors, active and anti-TNF-α created RA patients. Genes were selected using MAS 5.0, multtest and PAM. The custom microarray consists of 313 genes including guide dots, and positive (housekeeping genes and spike controls) and negative controls for image and statistical analysis. Each probe was spotted in 16 replicates.
Results
The RA-MO chipset-II was validated using the following: non-stimulated and LPS, PMA, Vit.D3+LPS, PMA+LPS stimulated U937 cells; nonstimulated and LPS stimulated healthy donor MO; MO from normal donors (n = 3) and RA patients before and during anti-TNF-α treatment (n = 5 each); and synovial tissue from normal individuals (n = 2) and RA patients (n = 2). Not only LPS/PMA regulated genes but also RA specific and anti-TNF-α regulated genes were validated. In addition, we could clarify whether these genes are differentially transcribed only in MO or whether they can also be found in RA tissue Mf. Our data indicate a high degree of reproducibility that is sufficient for diagnostic applications and therapy monitoring.
Conclusion
The RA-MO chipset-II microarray is competitive and flexible for enlargement of the number of genes. The current gene selection will contribute to validating the role of monocytes in disease activity, to therapeutic interventions, and may improve the knowledge on the regulation of pathways in activated monocytes in chronic inflammation.
