Limits...
The exposure of autoantigens by microparticles underlies the formation of potent inflammatory components: the microparticle-associated immune complexes.

Cloutier N, Tan S, Boudreau LH, Cramb C, Subbaiah R, Lahey L, Albert A, Shnayder R, Gobezie R, Nigrovic PA, Farndale RW, Robinson WH, Brisson A, Lee DM, Boilard E - EMBO Mol Med (2012)

Bottom Line: Rather, mpICs display autoantigens vimentin and fibrinogen, and recognition of these targets by anti-citrullinated peptide antibodies contributes to the production of mpICs.Functionally, platelet mpICs are highly pro-inflammatory, eliciting leukotriene production by neutrophils.Taken together, our data suggest a unique role for platelet MPs as autoantigen-expressing elements capable of perpetuating formation of inflammatory ICs.

View Article: PubMed Central - PubMed

Affiliation: Faculté de Médecine de l'Université Laval, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Québec, Québec, Canada.

Show MeSH

Related in: MedlinePlus

Platelet mpICs promote leukotriene production by neutrophilsQuantifications of the leukotriene biosynthesis by neutrophils incubated in presence of RA SF mpICs [n = 3 distinct patients ***p < 0.0001 patients 1 and 2; **p = 0.0011 patient 3 (unpaired t-test, relative to PBS control)]. Data are mean ± SEM. Leukotrienes measured represent the sum of LTB4, 6-trans-LTB4, 12-epi-6-trans-LTB4, 20-OH-LTB4 and 20-COOH-LTB4.Platelet mpICs form promptly upon co-incubation of platelet MPs with anti-fibrinogen in vitro. Representative FSC-PMT and SSC dot plot of mpICs. The presence of mpICs formed in vitro was demonstrated using a combination of FITC-conjugated Annexin-V and Alexa 647-conjugated secondary antibody. The four-quadrant gates were positioned according to the isotypic controls. The MPs (Annexin-V+-IgG−) presented in blue show dimensions ranging mostly from 100 to 500 nm (lower inset) while the mpICs (Annexin-V+-IgG+) presented in purple display dimensions ranging from 800 to 3000 nm (upper inset). The relative dimensions are presented according to size-defined microsphere calibrations.Quantifications of the leukotriene biosynthesis by neutrophils incubated in presence of in vitro mpICs, anti-fibrinogen and platelet MPs. The stimulatory potency of mpICs compared to the others conditions was statistically significant [n = 3 ***p ≤ 0.0001 (unpaired t-test)]. Data are mean ± SEM. Leukotrienes measured represent the sum of LTB4, 6-trans-LTB4, 12-epi-6-trans-LTB4, 20-OH-LTB4 and 20-COOH-LTB4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3569640&req=5

fig07: Platelet mpICs promote leukotriene production by neutrophilsQuantifications of the leukotriene biosynthesis by neutrophils incubated in presence of RA SF mpICs [n = 3 distinct patients ***p < 0.0001 patients 1 and 2; **p = 0.0011 patient 3 (unpaired t-test, relative to PBS control)]. Data are mean ± SEM. Leukotrienes measured represent the sum of LTB4, 6-trans-LTB4, 12-epi-6-trans-LTB4, 20-OH-LTB4 and 20-COOH-LTB4.Platelet mpICs form promptly upon co-incubation of platelet MPs with anti-fibrinogen in vitro. Representative FSC-PMT and SSC dot plot of mpICs. The presence of mpICs formed in vitro was demonstrated using a combination of FITC-conjugated Annexin-V and Alexa 647-conjugated secondary antibody. The four-quadrant gates were positioned according to the isotypic controls. The MPs (Annexin-V+-IgG−) presented in blue show dimensions ranging mostly from 100 to 500 nm (lower inset) while the mpICs (Annexin-V+-IgG+) presented in purple display dimensions ranging from 800 to 3000 nm (upper inset). The relative dimensions are presented according to size-defined microsphere calibrations.Quantifications of the leukotriene biosynthesis by neutrophils incubated in presence of in vitro mpICs, anti-fibrinogen and platelet MPs. The stimulatory potency of mpICs compared to the others conditions was statistically significant [n = 3 ***p ≤ 0.0001 (unpaired t-test)]. Data are mean ± SEM. Leukotrienes measured represent the sum of LTB4, 6-trans-LTB4, 12-epi-6-trans-LTB4, 20-OH-LTB4 and 20-COOH-LTB4.

Mentions: The inflammatory lipid mediators leukotrienes are present in RA synovial fluid (Klickstein et al, 1980) and its production by neutrophils, a cellular lineage dominant in the synovial fluid from RA patients (Edwards & Hallett, 1997), contribute to the pathophysiology of inflammatory arthritis in in vivo mechanistic models (Chen et al, 2006). Similarly to MPs, the mpICs can be centrifuged and used as cellular stimulus. We thus centrifuged SF from RA patients and used the resulting pellet to determine its potency on human neutrophils. We demonstrate that this fraction, which contains both MPs and mpICs, can stimulate neutrophils to produce leukotrienes (Fig 7A). We next used in vitro generated mpICs to examine whether they could elicit the release of leukotrienes from neutrophils. We find that mpICs, which spontaneously form after the co-incubation of platelet MPs with anti-fibrinogen (Fig 7B), induce robust leukotriene production by neutrophils (Fig 7C). For comparison, platelet MPs and anti-fibrinogen are only modestly active when added to neutrophils separately (Fig 7C). To assess the relative pro-inflammatory activity of mpICs, we fractionated mpICs, MPs and anti-fibrinogen ICs via size exclusion filtration (800 nm pores). Interestingly, we show that the mpIC depletion abrogates much of the leukotriene stimulation in this assay (Fig 7C), establishing that the platelet mpICs are active and highly pro-inflammatory.


The exposure of autoantigens by microparticles underlies the formation of potent inflammatory components: the microparticle-associated immune complexes.

Cloutier N, Tan S, Boudreau LH, Cramb C, Subbaiah R, Lahey L, Albert A, Shnayder R, Gobezie R, Nigrovic PA, Farndale RW, Robinson WH, Brisson A, Lee DM, Boilard E - EMBO Mol Med (2012)

Platelet mpICs promote leukotriene production by neutrophilsQuantifications of the leukotriene biosynthesis by neutrophils incubated in presence of RA SF mpICs [n = 3 distinct patients ***p < 0.0001 patients 1 and 2; **p = 0.0011 patient 3 (unpaired t-test, relative to PBS control)]. Data are mean ± SEM. Leukotrienes measured represent the sum of LTB4, 6-trans-LTB4, 12-epi-6-trans-LTB4, 20-OH-LTB4 and 20-COOH-LTB4.Platelet mpICs form promptly upon co-incubation of platelet MPs with anti-fibrinogen in vitro. Representative FSC-PMT and SSC dot plot of mpICs. The presence of mpICs formed in vitro was demonstrated using a combination of FITC-conjugated Annexin-V and Alexa 647-conjugated secondary antibody. The four-quadrant gates were positioned according to the isotypic controls. The MPs (Annexin-V+-IgG−) presented in blue show dimensions ranging mostly from 100 to 500 nm (lower inset) while the mpICs (Annexin-V+-IgG+) presented in purple display dimensions ranging from 800 to 3000 nm (upper inset). The relative dimensions are presented according to size-defined microsphere calibrations.Quantifications of the leukotriene biosynthesis by neutrophils incubated in presence of in vitro mpICs, anti-fibrinogen and platelet MPs. The stimulatory potency of mpICs compared to the others conditions was statistically significant [n = 3 ***p ≤ 0.0001 (unpaired t-test)]. Data are mean ± SEM. Leukotrienes measured represent the sum of LTB4, 6-trans-LTB4, 12-epi-6-trans-LTB4, 20-OH-LTB4 and 20-COOH-LTB4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569640&req=5

fig07: Platelet mpICs promote leukotriene production by neutrophilsQuantifications of the leukotriene biosynthesis by neutrophils incubated in presence of RA SF mpICs [n = 3 distinct patients ***p < 0.0001 patients 1 and 2; **p = 0.0011 patient 3 (unpaired t-test, relative to PBS control)]. Data are mean ± SEM. Leukotrienes measured represent the sum of LTB4, 6-trans-LTB4, 12-epi-6-trans-LTB4, 20-OH-LTB4 and 20-COOH-LTB4.Platelet mpICs form promptly upon co-incubation of platelet MPs with anti-fibrinogen in vitro. Representative FSC-PMT and SSC dot plot of mpICs. The presence of mpICs formed in vitro was demonstrated using a combination of FITC-conjugated Annexin-V and Alexa 647-conjugated secondary antibody. The four-quadrant gates were positioned according to the isotypic controls. The MPs (Annexin-V+-IgG−) presented in blue show dimensions ranging mostly from 100 to 500 nm (lower inset) while the mpICs (Annexin-V+-IgG+) presented in purple display dimensions ranging from 800 to 3000 nm (upper inset). The relative dimensions are presented according to size-defined microsphere calibrations.Quantifications of the leukotriene biosynthesis by neutrophils incubated in presence of in vitro mpICs, anti-fibrinogen and platelet MPs. The stimulatory potency of mpICs compared to the others conditions was statistically significant [n = 3 ***p ≤ 0.0001 (unpaired t-test)]. Data are mean ± SEM. Leukotrienes measured represent the sum of LTB4, 6-trans-LTB4, 12-epi-6-trans-LTB4, 20-OH-LTB4 and 20-COOH-LTB4.
Mentions: The inflammatory lipid mediators leukotrienes are present in RA synovial fluid (Klickstein et al, 1980) and its production by neutrophils, a cellular lineage dominant in the synovial fluid from RA patients (Edwards & Hallett, 1997), contribute to the pathophysiology of inflammatory arthritis in in vivo mechanistic models (Chen et al, 2006). Similarly to MPs, the mpICs can be centrifuged and used as cellular stimulus. We thus centrifuged SF from RA patients and used the resulting pellet to determine its potency on human neutrophils. We demonstrate that this fraction, which contains both MPs and mpICs, can stimulate neutrophils to produce leukotrienes (Fig 7A). We next used in vitro generated mpICs to examine whether they could elicit the release of leukotrienes from neutrophils. We find that mpICs, which spontaneously form after the co-incubation of platelet MPs with anti-fibrinogen (Fig 7B), induce robust leukotriene production by neutrophils (Fig 7C). For comparison, platelet MPs and anti-fibrinogen are only modestly active when added to neutrophils separately (Fig 7C). To assess the relative pro-inflammatory activity of mpICs, we fractionated mpICs, MPs and anti-fibrinogen ICs via size exclusion filtration (800 nm pores). Interestingly, we show that the mpIC depletion abrogates much of the leukotriene stimulation in this assay (Fig 7C), establishing that the platelet mpICs are active and highly pro-inflammatory.

Bottom Line: Rather, mpICs display autoantigens vimentin and fibrinogen, and recognition of these targets by anti-citrullinated peptide antibodies contributes to the production of mpICs.Functionally, platelet mpICs are highly pro-inflammatory, eliciting leukotriene production by neutrophils.Taken together, our data suggest a unique role for platelet MPs as autoantigen-expressing elements capable of perpetuating formation of inflammatory ICs.

View Article: PubMed Central - PubMed

Affiliation: Faculté de Médecine de l'Université Laval, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Québec, Québec, Canada.

Show MeSH
Related in: MedlinePlus