Abstract
The P2X7 receptor (P2X7R) is a key pro-inflammatory plasma membrane receptor responsible for NLRP3 inflammasome activation and IL-1β release. Various inflammatory plasma membrane receptors (e.g., IL-1 type I receptor, TNF type I and II receptors, IL-2 receptor) are shed under different pathophysiological conditions. In the present study, we show that the full length P2X7R is released into circulation in patients as well as in healthy subjects. Blood levels of shed P2X7R (sP2X7R) correlate to those of the inflammatory marker C reactive protein (CRP). Blood sP2X7R ranged from 16.74 to 82.17 ng/L, mean ± SE 40.97 ± 3.82 (n = 26) in healthy subjects, from 33.1 to 484.0 ng/L, mean ± SE 114.78 ± 12.22 (n = 45) in patients with CRP <3 mg/L, and from 63.65 to 1092.3 ng/L, mean ± SE 204.2 ± 30.94 (n = 42) in patients with CRP >3 mg/L. sP2X7R in plasma was largely associated to microvesicles/microparticles. Peripheral blood monocytes from healthy subjects released sP2X7R upon stimulation with the semi-selective P2X7R agonist benzoyl ATP. These data show that the P2X7R can be released into circulation, and that its blood levels increase in various disease conditions.
Highlights
Plasma membrane receptors for cytokines or growth factors can be shed due to protease-mediated cleavage, or released in association with plasma membrane-derived microvesicles/microparticles (MVs/MPs) [1, 2], deeply affecting signaling by the cognate cytokine [3]
Serum and cell lysate samples were tested for the presence of sP2X7R by ELISA. sP2X7R concentration in plasma from healthy subjects (n = 26) ranged from 16.7 to 82.17 ng/L
Specificity of the ELISA assay was validated with cell lysates from wt-HEK293 (P2X7R-null), HEK293-P2X7 receptor (P2X7R), and ACN cells
Summary
Plasma membrane receptors for cytokines or growth factors can be shed due to protease-mediated cleavage, or released in association with plasma membrane-derived microvesicles/microparticles (MVs/MPs) [1, 2], deeply affecting signaling by the cognate cytokine [3]. Gorecki and co-workers showed that P2X7R activation triggers release of matrix metallo-proteinase-2 (MMP-2), which in turn cleaves and releases a truncated form of P2X7R itself [17] It is not known whether the P2X7R is released into circulation. It has been previously reported that full length P2X7 subunits are associated to MVs/MPs released by immune cells [18], or that a C-terminal cleaved form can be shed following MMP-2 activation [17]. Increased circulating levels of the P2X7R might be associated to inflammation, and possibly be a useful diagnostic or prognostic marker. Our data show that the full-length P2X7R was released into circulation in association to plasma membrane-derived MVs/MPs. sP2X7R blood levels were comprised within the 16.74 to 82.17 ng/L, and 33.10 to. Cell lysates were centrifuged for 5 min at 5000xg at 4◦ C and supernatants collected and immediately assayed
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