Presentation: Identification and Characterization of a Differentially Expressed Novel Gene Family in Activated Rheumatoid Arthritis Monocytes (2007)

142 Identification and Characterization of a Differentially Expressed Novel Gene Family in Activated Rheumatoid Arthritis Monocytes

Background: In rheumatoid arthritis (RA), the pannus tissue is characterized by massive infiltration with activated macrophages (Mf). In addition to tissue MO circulating monocytes (MO) in the peripheral blood of patients with active RA are activated and spontaneously release high levels of IL1, IL6, TNFa, PGE2, and neopterin in vitro.
Objectives: Repeated leukapheresis, a therapeutic approach aimed at removing MO from circulation is very efficient in decreasing activation in RA. Because of clear changes in their activation status, MO collected upon 1st-leukapheresis (activated pool) and repeated leukapheresis (non-activated pool) represented an excellent tool to study differential gene expression.
Methods: A λgt11 cDNA library was constructed from the 1st-leukapheresis MO of a highly active RA patient and differentially hybridized with probes from 1st- and 3rd-leukapheresis RA-MO.
Results: In activated MO, IL-1a, IL1b, IL6, TNFa, GRO-alpha, GRO-beta, IL-8, ferritin, alpha-1-antitrypsin, lysozyme, transaldolase, etc. were detected. Furthermore, clones homologous to unknown/functionally undefined genes (BSK-4, 17, 66, 67, 80, 83, 86, and 89) were found in the activated MO cell population. In 3rd-leukapheresis-MO, the clones mainly included differentiation genes (HOX-B3, thymosin-beta-4, PU.1, glucocerebrosidase, MEL18), and over 15 functionally undefined sequences.
Furthermore, 11 different splicing variants encoding transmembrane and non-transmembrane proteins of a novel E3-ligase family plus an in-cis acting “natural antisense transcript” were characterized. Northern blot analysis resulted in different transcript lengthes (1.8-3kB). By 3’/5’ RACE-PCR and sequence comparison algorithms full length’s nucleic acid sequences were characterized. To verify whether this transcript family is representative in RA, RT-PCR quantifications were performed. mRNA transcript levels were measured in: i) normal donors (n=12), ii) patients with RA (n=32), iii) Psoriasis (n=3), and iv) Morbus Bechterew (MB; n=6). When compared with normal MO, a significant differential gene expression was obtained in RA, Psoriasis and MB-MO. More interestingly gene expression was reversed in RA back to normal levels after anti-TNFα treatment (n=10). To define gene functions, siRNA silencing assays were performed, followed by Affymetrix microarray experiments (U133-2 Plus) and pathway defining analyses using KEGG and Ingenuity software tools. Pathway analyses also demonstrated an involvement in the ubiquitination process.
Conclusion: Future, more detailed functional definitions of this gene family might open new avenues for a specific novel therapeutic strategy.
This work is supported by the DFG grant Stu2/1 and the BMBF grants TP29 and TP48

 B. Stuhlmuller, None; L. Matinez-Gamboa, None; D. Machuy, None; T. Haupl, None; T. Rudel, None; G.R. Burmester, None.