12-hydroxy-5-8-10-14-eicosatetraenoic-acid and Arthritis--Rheumatoid

Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arthritis, Experimental; Arthritis, Rheumatoid; Blood Platelets; Cell Line; Cell-Derived Microparticles; Cells, Cultured; Endocytosis; Group II Phospholipases A2; Humans; Immunoblotting; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Confocal; Microscopy, Electron; Mitochondria; Neutrophils; Reverse Transcriptase Polymerase Chain Reaction; RNA; Synovial Fluid

In the present study, we have demonstrated platelet-type 12-lipoxygenase (12-LOX) expression in human rheumatoid arthritis (RA) type B synoviocytes by reverse-transcription polymerase chain reaction (RT-PCR). The presence of 12-LOX mRNA in these cells was revealed by classical RT-PCR analysis using platelet-type 12-LOX cDNA primers and the PCR fragment (246 bp) was purified, amplified and sequenced. By sequence analysis, this fragment was determined to be 100% identical to that from platelet-type 12-LOX cDNA. Immunofluorescence data demonstrate that interleukin-1beta (IL-1beta) increases cellular 12-LOX protein. Other results associate specific inflammatory cytokines with the activity of 12-LOX in human RA type B synoviocytes. IL-1beta increased 12S-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) production (4-fold) and we also observed an increase in 12-HETE production (2.5-fold) after incubation of human RA type B synoviocytes with TNF alpha. In contrast to the action of IL-1beta on 12-HETE synthesis, IL-4 and IL-6 did not enhance 12-HETE production. This is the first demonstration of platelet-type 12-LOX cDNA derived from the mRNA of cultured human RA type B synoviocytes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Arthritis, Rheumatoid; Blood Platelets; Calcimycin; Cells, Cultured; Cloning, Molecular; DNA Primers; Fluorescent Antibody Technique; Gene Expression; Humans; Interleukin-1; Interleukin-4; Interleukin-6; Microscopy, Fluorescence; Polymerase Chain Reaction; RNA, Messenger; Sequence Analysis, DNA; Synovial Membrane; Tumor Necrosis Factor-alpha

We have examined the ability of interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-1 receptor antagonist protein (IL-1ra) to regulate spontaneous interleukin-8 (IL-8) production in cultured SF mononuclear cells (SFMC) from RA. Furthermore, we examined whether IL-4, IL-10, or IL-1ra could influence the production of the arachidonic acid products leukotriene B4 (LTB4), 12-hydroxy-eicosatetraenoic acid (12-HETE) and 15-hydroxy-eicosatetraenoic acid (15-HETE). IL-4 induced a maximal suppression of 75% in the IL-8 secretion in SFMC from 10.0 ng/ml down to 2.5 ng/ml after 24 h and from 17.2 ng/ml to 4.2 ng/ml after 72 h of culture. IL-10 induced a 55% inhibition of the IL-8 secretion at 24 h and a 40% inhibition at 72 h. IL-1ra did not change the spontaneous IL-8 secretion from rheumatoid SFMC. We also examined, whether addition of IL-4, IL-10 or IL-1ra was able to modulate formation of the arachidonic acid products LTB4, 12-HETE and 15-HETE in cultured SF cells, stimulated with the calcium ionophore A23187. 15-HETE was not detected in untreated cultures, nor in IL-10 or IL-1ra treated cultures. IL-4, however, stimulated the formation of the anti-inflammatory mediator; 15-HETE (23 ng/10(6) cells). These results suggest that IL-4 or IL-10, could have beneficial anti-inflammatory effects in RA.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arthritis, Rheumatoid; Humans; Hydroxyeicosatetraenoic Acids; Interleukin 1 Receptor Antagonist Protein; Interleukin-10; Interleukin-4; Interleukin-8; Leukotriene B4; Sialoglycoproteins; Synovial Fluid

The inhibition of 5-lipoxygenase could be involved in the mechanism of action of methotrexate (MTX). We studied 8 patients with active rheumatoid arthritis (RA) immediately before and the day after the first dose of MTX (10 mg intramuscularly). Leukotriene B4 (LTB4) formation by polymorphonuclear leukocytes was significantly decreased (32%, p less than 0.01). This essentially involved released LTB4. A slight decrease was also obtained in omega-oxidation products. Similar results were obtained for plasma LTB4 (30%, p less than 0.02). A non-significant decrease in 5-HETE was noted. Conversely, 12-HETE was not modified. Our results suggest MTX has an effect on the 5-lipoxygenase pathway, particularly at the LTA4 epoxide hydrolase step, since 5-HETE and 6-trans-LTB4 isomers are not involved.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Arthritis, Rheumatoid; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Female; Humans; Hydroxyeicosatetraenoic Acids; Injections, Intramuscular; Leukotriene B4; Male; Methotrexate; Middle Aged; Neutrophils; Time Factors