Key Points: When activated and in platelet storage bags, platelets release respiratory-competent mitochondria, a recognized damage-associated molecular pattern. Mitochondria, descendant of Rickettsia prowazekii, serve as substrate for bactericidal sPLA2-IIA to promote inflammation.

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.

Real-time polymerase chain reaction (PCR) constitutes a significant improvement over traditional end-point PCR, as it allows the quantification of starting amounts of nucleic acid templates, in real-time. However, quantification requires validation through numerous internal controls and standard curves. We describe in this paper a simple protocol which uses real-time PCR to compare mRNA levels of a gene of interest between different experimental conditions. Comparative real-time PCR can be a relatively low-cost method and does not require sequence-specific fluorescent reporters. Moreover, several genes from a set of experiments can be assessed in a single run. Thus, in addition to providing a comparative profile for the expression of a gene of interest, this method can also provide information regarding the relative abundance of different mRNA species.

The impact of lipoxin A₄ (LXA₄) and aspirin-triggered lipoxins (ATLs) was investigated in tumor necrosis factor (TNF)-α–initiated neutrophil (polymorphonuclear leukocyte) responses in vitro and in vivo using metabolically stable LX analogues. At concentrations as low as 1–10 nM, the LXA₄ and ATL analogues each inhibited TNF-α–stimulated superoxide anion generation and IL-1β release by human polymorphonuclear leukocytes. These LXA₄-ATL actions were time and concentration dependent and proved selective for TNF-α, as these responses were not altered with either GM-CSF– or zymosan-stimulated cells. TNF-α–induced IL-1β gene expression was also regulated by both anti-LXA₄ receptor antibodies and LXA₄-ATL analogues. In murine air pouches, 15R/S-methyl-LXA₄ dramatically inhibited TNF-α–stimulated leukocyte trafficking, as well as the appearance of both macrophage inflammatory peptide 2 and IL-1β, while concomitantly stimulating IL-4 in pouch exudates. Together, these results indicate that both LXA₄ and ATL regulate TNF-α–directed neutrophil actions in vitro and in vivo and stimulate IL-4 in exudates, playing a pivotal role in immune responses.

Platelets are a source of extracellular DNA and mitochondrial antigens in systemic lupus erythematosus.