Identification of known and novel genes in activated monocytes from patients with rheumatoid arthritis.
OBJECTIVE
To define gene activation patterns of monocytes (MO) in patients with rheumatoid arthritis (RA).


METHODS
A complementary DNA (cDNA) library was constructed from first-leukapheresis MO obtained from an RA patient with active disease; 32P-labeled cDNA from first-leukapheresis MO (activated pool) and third-leukapheresis MO (nonactivated pool) were used as probes for differential hybridization. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess gene activation in MO from an additional 26 RA patients and 6 normal controls.


RESULTS
Subtraction of genes from first- and third-leukapheresis MO resulted in 482 differentially expressed clones. In first-leukapheresis MO, these clones included the following: 1) interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, tumor necrosis factor alpha, growth-related oncogene alpha (GROalpha)/melanoma growth-stimulatory activity, macrophage inflammatory protein 2/GRObeta, ferritin, alpha1-antitrypsin, lysozyme, transaldolase, Epstein-Barr virus-encoded RNA 1 (EBER-1)/EBER-2-associated-protein, thrombospondin 1, an angiotensin receptor II (ATRII) C-terminal homolog, and RNA polymerase II elongation factor (elongin); 2) two clones homologous to functionally undefined genes (BSK-67 and BSK-83); and 3) three unknown cDNA sequences (BSK-66, 80, 89). In third-leukapheresis MO, the clones included differentiation genes (HOX-B3, thymosin-beta4, PU.1, glucocerebrosidase, MEL-18, and glucose-6-phosphate dehydrogenase) and 3 unknown/functionally undefined sequences. Differential expression of most genes from the activated pool was confirmed in leukapheresis samples from 2 additional RA patients. In MO from RA patients, not only were IL-1beta and the ATRII homolog significantly overexpressed (maximum 36-fold), but also 4 of the unknown/functionally undefined genes (maximum 102-fold). Notably, messenger RNA levels of BSK-89 correlated positively with the erythrocyte sedimentation rate (ESR), whereas those of BSK-83 correlated negatively with the ESR and C-reactive protein level.


CONCLUSIONS
The combined strategy of gene subtraction and semiquantitative RT-PCR may allow the definition of MO activation patterns during different disease phases (including therapy-induced remission) and the identification of novel MO genes with pathogenetic relevance in RA.
Stuhlmüller
B
B
Department of Rheumatology and Clinical Immunology, Charité University Hospital, Humboldt University of Berlin, Germany.
Ungethüm
U
U
Scholze
S
S
Martinez
L
L
Backhaus
M
M
Kraetsch
H G
HG
Kinne
R W
RW
Burmester
G R
GR
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001172:Arthritis, Rheumatoid / Q000097:blood* / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D005786:Gene Expression Regulation; D015723:Gene Library; D006801:Humans; D007937:Leukapheresis; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation
D012333:RNA, Messenger