Arachidonate 15-lipoxygenase of reticulocyte-type in human rheumatoid arthritis type B synoviocytes and modulation of its activity by proinflammatory cytokines.
OBJECTIVE
Lipoxygenases (LOX) are lipid-peroxidating enzymes that are implicated in the pathogenesis of a variety of inflammatory disorders such as arthritis, psoriasis, and asthma. 15-LOX catalyzes the oxygenation of free arachidonic acid to 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), which is reduced to 15-hydroxyeicosatetraenoic acid (15-HETE). The biological role of 15-HETE is less clear. We sought to determine if cultured human rheumatoid synovial cells were able to express 15-LOX mRNA, leading to the synthesis of 15-HETE, and to examine the effect of different cytokines on 15-LOX activity.


METHODS
Adherent synovial cells were obtained by enzymatic digestion of rheumatoid synovium, isolated from patients with rheumatoid arthritis (RA) undergoing hip synovectomy. Between passages 4 and 8, reticulocyte-type 15-LOX expression in these cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) in situ and confirmed by classical RT-PCR analysis followed by enzymatic digestion. The PCR fragment was purified, amplified, and sequenced. Cultured synovial cells were incubated with or without different cytokines and exogenous [1-(14)C] arachidonic acid metabolism of synoviocytes was analyzed by reverse phase high performance liquid chromatography (RP-HPLC).


RESULTS
RT-PCR results showed that human RA type B synoviocytes expressed a reticulocyte-type 15-LOX. By sequence analysis, the PCR fragment (474 bp) was determined to be 100% identical to that of reticulocyte-type 15-LOX cDNA. Other results associated specific inflammatory cytokines with the activity of 15-LOX in these cells. RP-HPLC analysis showed that interleukin 4 (IL-4) increased 15-HETE production (2.4-fold); we also observed an increase in 15-HETE production (1.2-fold) after incubation of the cells with IL-1beta.


CONCLUSIONS
Human RA type B synoviocytes are able to express 15-LOX mRNA leading to the synthesis of 15-HETE, which is modulated by various cytokines that play a major role in the pathophysiology of RA, especially IL-4 and IL-1.
Liagre
B
B
University of Medicine, and the Department of Rheumatology, CHRU Dupuytren, Limoges, France.
Vergne
P
P
Rigaud
M
M
Beneytout
J L
JL
D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001095:Arachidonic Acids / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D016207:Cytokines / Q000378:metabolism*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007375:Interleukin-1 / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012156:Reticulocytes / Q000201:enzymology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000201:enzymology* / Q000473:pathology
D001095:Arachidonic Acids; D016207:Cytokines; D006893:Hydroxyeicosatetraenoic Acids; D007375:Interleukin-1; D012333:RNA, Messenger; D015847:Interleukin-4; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D001093:Arachidonate 15-Lipoxygenase