Title: Increase of circulating neutrophil and platelet microparticles during acute vasculitis and hemodialysis
Abstract: Release of microparticles (MPs) from blood cells may occur upon various activation signals. MPs from neutrophil and platelet have been studied in systemic infectious diseases and cardiovascular diseases, respectively. They are here investigated in common nephropathies including vasculitis and dialysis, two conditions characterized by neutrophil activation. Flow cytometry analysis of neutrophil-derived (CD66b-positive) and platelet-derived (CD41a-positive) MPs was performed on 213 plasma samples from patients with various nephropathies, including 46 patients with vasculitis and 40 hemodialysis patients. MPs released ex vivo, during neutrophil activation in whole blood, were also measured in these patients. Correlations with clinical parameters and creatinine clearance were evaluated. The results show that MPs present in plasma from patients or healthy controls are from various origins: platelet-derived (38±22%), neutrophil-derived (2.8±3.8%) MPs, mixed aggregates of neutrophil/platelet MPs (28±15%) or neither from neutrophil or platelet (null) 31±20%. Acute vasculitis showed the highest level of all types of MPs, while other nephropathies did not result in significant changes of MP levels. A significant increase was observed during hemodialysis sessions. In patients with renal failure, no correlation was seen between MP levels and creatinine clearance. In conclusion, neutrophil and platelet MP levels are non-specific markers of neutrophil activation during vasculitis acute phase and dialysis-induced inflammation. Circulating aggregates of neutrophil/platelet MPs co-express adhesion molecules of both cell types and may be thus endowed with inflammation and coagulation- thus modulating properties. Release of microparticles (MPs) from blood cells may occur upon various activation signals. MPs from neutrophil and platelet have been studied in systemic infectious diseases and cardiovascular diseases, respectively. They are here investigated in common nephropathies including vasculitis and dialysis, two conditions characterized by neutrophil activation. Flow cytometry analysis of neutrophil-derived (CD66b-positive) and platelet-derived (CD41a-positive) MPs was performed on 213 plasma samples from patients with various nephropathies, including 46 patients with vasculitis and 40 hemodialysis patients. MPs released ex vivo, during neutrophil activation in whole blood, were also measured in these patients. Correlations with clinical parameters and creatinine clearance were evaluated. The results show that MPs present in plasma from patients or healthy controls are from various origins: platelet-derived (38±22%), neutrophil-derived (2.8±3.8%) MPs, mixed aggregates of neutrophil/platelet MPs (28±15%) or neither from neutrophil or platelet (null) 31±20%. Acute vasculitis showed the highest level of all types of MPs, while other nephropathies did not result in significant changes of MP levels. A significant increase was observed during hemodialysis sessions. In patients with renal failure, no correlation was seen between MP levels and creatinine clearance. In conclusion, neutrophil and platelet MP levels are non-specific markers of neutrophil activation during vasculitis acute phase and dialysis-induced inflammation. Circulating aggregates of neutrophil/platelet MPs co-express adhesion molecules of both cell types and may be thus endowed with inflammation and coagulation- thus modulating properties. Polymorphonuclear neutrophils play a central role in inflammation acute phase, via their degranulation response and respiratory burst.1.Witko-Sarsat V. Rieu P. Descamps-Latscha B. et al.Neutrophils: molecules, functions and pathophysiological aspects.Lab Invest. 2000; 80: 617-653Crossref PubMed Scopus (835) Google Scholar During early activation, neutrophils are capable, as platelets, to release microparticles (MPs).2.Hess C. Sadallah S. Hefti A. et al.Ectosomes released by human neutrophils are specialized functional units.J Immunol. 1999; 163: 4564-4573PubMed Google Scholar MPs, or ectosomes, are plasma membrane vesicles which express markers of the parent cells outer membrane and are released by many cell types upon activation or during apoptosis.3.Freyssinet J.M. Cellular microparticles: what are they bad or good for?.J Thromb Haemost. 2003; 1: 1655-1662Crossref PubMed Scopus (365) Google Scholar They are distinct from exosomes, smaller membrane vesicles which result from the exocytosis of endocytic multivesicular bodies.4.Thery C. Zitvogel L. Amigorena S. Exosomes: composition, biogenesis and function.Nat Rev Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3145) Google Scholar Neutrophil MPs carry a large set of cell adhesion molecules and proteases such as proteinase 3 or elastase.5.Gasser O. Hess C. Miot S. et al.Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils.Exp Cell Res. 2003; 285: 243-257Crossref PubMed Scopus (196) Google Scholar The pro-inflammatory or anti-inflammatory functions of these neutrophil MPs are still under discussion.6.Mesri M. Altieri D.C. Leukocyte microparticles stimulate endothelial cell cytokine release and tissue factor induction in a JNK1 signaling pathway.J Biol Chem. 1999; 274: 23111-23118Crossref PubMed Scopus (292) Google Scholar, 7.Gasser O. Schifferli J.A. Activated polymorphonuclear neutrophils disseminate anti-inflammatory microparticles by ectocytosis.Blood. 2004; 104: 2543-2548Crossref PubMed Scopus (274) Google Scholar The release of neutrophil MPs is well described during systemic infectious diseases or in severely injured patients.8.Joop K. Berckmans R.J. Nieuwland R. et al.Microparticles from patients with multiple organ dysfunction syndrome and sepsis support coagulation through multiple mechanisms.Thromb Haemost. 2001; 85: 810-820PubMed Google Scholar, 9.Fujimi S. Ogura H. Tanaka H. et al.Increased production of leukocyte microparticles with enhanced expression of adhesion molecules from activated polymorphonuclear leukocytes in severely injured patients.J Trauma. 2003; 54 (discussion 119-120): 114-119Crossref PubMed Scopus (38) Google Scholar On the other hand, platelet MPs are involved in cardiovascular diseases with coagulation abnormalities as acute coronary syndromes10.VanWijk M.J. VanBavel E. Sturk A. Nieuwland R. Microparticles in cardiovascular diseases.Cardiovasc Res. 2003; 59: 277-287Crossref PubMed Scopus (510) Google Scholar, 11.Diamant M. Tushuizen M.E. Sturk A. Nieuwland R. Cellular microparticles: new players in the field of vascular disease?.Eur J Clin Invest. 2004; 34: 392-401Crossref PubMed Scopus (313) Google Scholar or diabetes.12.Sabatier F. Darmon P. Hugel B. et al.Type 1 and type 2 diabetic patients display different patterns of cellular microparticles.Diabetes. 2002; 51: 2840-2845Crossref PubMed Scopus (327) Google Scholar Similarly, endothelial MPs are relevant markers of endothelium dysfunction.13.Horstman L.L. Jy W. Jimenez J.J. Ahn Y.S. Endothelial microparticles as markers of endothelial dysfunction.Front Biosci. 2004; 9: 1118-1135Crossref PubMed Scopus (260) Google Scholar During chronic renal failure (CRF) progression, pro-inflammatory cytokines are increased and inflammation could explain in part the decline of renal function because of accelerated atherosclerosis. Platelet MPs have been reported to be increased during CRF14.Mercier E. Branger B. Vecina F. et al.Tissue factor coagulation pathway and blood cells activation state in renal insufficiency.Hematol J. 2001; 2: 18-25Crossref PubMed Scopus (35) Google Scholar and could participate in producing hypercoagulation. So far, neutrophil MPs have not been studied in patients with nephropathies or CRF. The aim of the present study was to evaluate neutrophil and platelet MPs in nephropathies, including vasculitis and end-stage renal failure, since neutrophils are particularly involved in both the development of active vasculitis and the effect of the dialysis membranes on inflammation. We show that platelet and neutrophil MPs are significantly increased in patients with active vasculitis and during hemodialysis. Figure 1 shows flow cytometry dot blots analysis of plasma MPs recovered from whole blood of a healthy donor, before and after in vitro incubation with tumor necrosis factor alpha (TNFα) and the formyl-peptide fMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine). Figure 1a shows the size (forward scatter channel, FSC) and densitometry (side scatter channel, SSC) of MPs (R2), as compared to the 3 μm calibrated latex beads (R1). Events present in R2, which included most spun-down MPs except for rare high size events that could be contaminating blood cells, were gated to count immunolabeled MPs (Figure 1b). CD66b-labeled MPs represent neutrophil-derived MPs (Neutro-MPs) and CD41a-labeled MPs are platelet-derived MPs (Pl-MPs). Double-labeled CD66b- and CD41a-positive MPs reveal the presence of neutrophil/platelet MP aggregates (Mixed MPs). One should point out that in vitro formation of MP aggregates had been prevented by adding ethylene diamine tetra-acetic acid to the collected plasma, to block novel Ca2+-dependent adhesion via P-selectin. CD66b- and CD41a-negative events, included in the lower left quadrant, are called null MPs. FSC/SSC dot blots of each MP population (Pl-, Mixed, Neutro, and Null), in Figure 1c, show that they are all similar in size, but MPs with 100–1000 FSC, presumably representing MP aggregates, were mostly observed in mixed MPs and absent in the platelet MPs gate. Quantification of each MP type was performed by using the latex beads (R1) as an internal standard, as described in Materials and Methods. Figure 2 analyses the release of MPs resulting from ex vivo neutrophil activation in whole blood from healthy donors. Figure 2a shows that a 20-min incubation of whole blood at 37°C with 1 μM fMLP induces a significant release of Neutro- and Mixed MPs, as defined above, while the number of platelet MPs does not change. Figure 2b shows the effect of whole blood activation by TNF-α and fMLP on total CD66b-positive MPs, that is, Neutro- and Mixed MPs. The addition of 1 ng/ml TNF-α or 1 μM fMLP was sufficient to induce a significant increase of these MPs (91±44 and 118±49 × 103 MPs/ml respectively, as compared to 52±19 × 103 MPs/ml in control unactivated blood, P≤0.01). The highest level (276±198 × 103 MPs/ml) was observed when fMLP 1 μM was associated with 10 ng/ml TNF-α, revealing the priming effect of TNF-α on neutrophil responses.15.Elbim C. Chollet-Martin S. Bailly S. et al.Priming of polymorphonuclear neutrophils by tumor necrosis factor alpha in whole blood: identification of two polymorphonuclear neutrophil subpopulations in response to formyl-peptides.Blood. 1993; 82: 633-640Crossref PubMed Google Scholar, 16.Hallett M.B. Lloyds D. Neutrophil priming: the cellular signals that say 'amber' but not 'green'.Immunol Today. 1995; 16: 264-268Abstract Full Text PDF PubMed Scopus (227) Google Scholar The number of released MPs in this condition was greatly variable among donors but significantly higher than with fMLP alone. As reported by Gasser et al.,5.Gasser O. Hess C. Miot S. et al.Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils.Exp Cell Res. 2003; 285: 243-257Crossref PubMed Scopus (196) Google Scholar we confirmed by flow cytometry that neutrophil MPs express membrane PR3 on their membrane (data not shown). The number of null MPs also increased after neutrophil activation by fMLP in whole blood (151±132 × 103 up to 474±248 × 103, n=17, P<0.0001, data not shown). The phenotype of these null MPs was investigated using antibodies to non-platelet and non-neutrophil markers (data not shown): more than 80% of null MPs expressed phosphatidylserine on their surface, as shown by the binding of annexin V and confirming previous reports.17.Nieuwland R. Berckmans R.J. Rotteveel-Eijkman R.C. et al.Cell-derived microparticles generated in patients during cardiopulmonary bypass are highly procoagulant.Circulation. 1997; 96: 3534-3541Crossref PubMed Scopus (319) Google Scholar They are thus membrane particles rather than protein aggregates such as fibrin or von Willebrand factor polymers. They were negative in lymphocyte marker CD3, monocyte marker CD14, endothelial marker CD146, and negative in CD63, a marker of exosomes recently described in plasma.18.Caby M.P. Lankar D. Vincendeau-Scherrer C. et al.Exosomal-like vesicles are present in human blood plasma.Int Immunol. 2005; 17: 879-887Crossref PubMed Scopus (830) Google Scholar In all, 40–80% of null MPs expressed an erythrocyte marker, glycophorin C. The reddish color of the plasma, after ex vivo neutrophil activation in whole blood, indeed shows that neutrophil activation induces hemolysis, presumably via the release of reactive oxygen species. Null MPs could thus include erythrocyte ghosts or erythrocyte-derived ectosomes, similar to those released following complement attack.19.Stein J.M. Luzio J.P. Ectocytosis caused by sublytic autologous complement attack on human neutrophils. The sorting of endogenous plasma-membrane proteins and lipids into shed vesicles.Biochem J. 1991; 274: 381-386Crossref PubMed Scopus (139) Google Scholar MP levels according to the pathological classification are given in Table 1 and schematized in Figure 3. The distribution of circulating MPs was similar in all pathologies as in healthy controls, that is, 2.8±3.8% Neutro- (1.1–4.5%), 28±15% mixed Pl/neutro-MPs (22–38%), 38±22% platelet MPs (27–43%), 31±20% null MPs (18–39%). The most striking result was obtained in acute vasculitis, where the levels of all MP types were increased six times as compared to normal controls. An important point was a large individual variation within subgroups (Table 1). When using a multiple comparison test, highly significant differences were observed between acute vasculitis and healthy controls, for all types of MPs. A modest but significant increase of mixed MPs was also observed in chronic vasculitis and in tubulo-interstitial nephritis. Neutro-MPs were found to be significantly increased in both acute and chronic vasculitis, in tubulo-interstitial nephritis, and in glomerular nephropathies with immune deposits (IgA nephropathy/membranous nephropathy). The low and highly variable level of neutro-MPs probably explains this lack of specificity. When total CD66b-positive Neutro-MPs (neutro-MPs+mixed MPs) were considered, the difference with healthy controls was again highly significant in acute vasculitis (P<0.001), but less significant in chronic vasculitis and tubulo-interstitial nephritis (P=0.02 and 0.04, respectively; data not shown). When total CD41a-positive Pl-MPs (pl-MPs+mixed MPs) were considered, a significant difference was only observed in acute vasculitis (P<0.001; data not shown). An increase of Null MPs number was also observed in acute vasculitis (645±819 × 103) and, to a lesser extent, in tubulo-interstitial nephritis (289±481 × 103) and IgA/MN (330±666 × 103), when compared to controls (179±152 × 103) (data not shown).Table 1MP levels in nephropathiesnAge (years)Creatinine clearanceNeutro-MPs (× 103/ml)Mixed-MPs (× 103/ml)Pl-MPs (× 103/ml)Controls20NRNR2±1.549±18147±238Nephrosclerosis2060±1641±286±5.789±62269±514Diabetic nephropathy1057±1534±246±7.376±51115±175Tubulo-interstitial nephritis1945±2140±2918±22 (P<0.001)99±78 (P=0.001)208±250MCN/FSG736±1488±464±3.792±58190±226IgAN/MN1851±1452±2514±15 (P=0.001)88±43156±176Lupus nephropathy1639±2165±358±1372±41210±144Chronic vasculitis2951±1561±247±4.7 (P=0.01)96±60 (P=0.001)445±581Acute Vasculitis1754±1459±2123±19 (P<0.001)238±83 (P<0.001)1025±1982 (P=0.005)Urological diseases1749±2042±237±1176±47226±343Others2162±1536±1611±27101±80291±415FSG, focal segmental glomerulosclerosis; IgAN, IgA nephropathy; MCN, minimal changes nephropathy; MN, membranous nephropathy; Pl-MPs, platelet-derived MPs. Clearance was reported in ml/min. NR, not recorded; MP, microparticles. Bold characters underline numbers significantly different from normal controls (P<0.05). Open table in a new tab FSG, focal segmental glomerulosclerosis; IgAN, IgA nephropathy; MCN, minimal changes nephropathy; MN, membranous nephropathy; Pl-MPs, platelet-derived MPs. Clearance was reported in ml/min. NR, not recorded; MP, microparticles. Bold characters underline numbers significantly different from normal controls (P<0.05). Concerning clinico-biological parameters, no correlation was observed of MP levels with age (r=0.06; P=0.4, for neutrophil-derived, CD66b-positive MPs; r=0.05; P=0.45 for platelet-derived, CD41a-positive MPs) or with serum C reactive protein (r=0.15; P=0.06 for neutrophils; r=0.07; P=0.4 for platelets). A slight positive correlation was observed, however, between Neutro-MPs and circulating neutrophil counts (r=0.2, P=0.02) or between Pl-MPs and circulating platelet counts (r=0.3; P<10-4). Decreased creatinine clearance was not correlated with an increase of neutrophil-derived (r=0.1; P=0.2) or platelet-derived (r=0.06; P=0.5) MPs level. We even observed an opposite trend when four patients subgroups were defined according to the creatinine clearance (Figure 4) When chronic and acute vasculitis were compared separately with healthy controls, a significant increase of Neutro- and Mixed MPs was observed in acute and, to a lesser extent, in chronic vasculitis, while platelet MPs were only increased in acute vasculitis (Figure 5a). MP levels were further increased during acute phases of the disease. This appears clearly in Figure 5b, where the level of CD66-positive MPs (neutro-MPs and mixed MPs) was strikingly higher in the plasma of patients with acute vasculitis, when compared to chronic vasculitis. The large standard deviation among patients is a characteristic of MPs detection, even in healthy patients.20.Jy W. Horstman L.L. Jimenez J.J. et al.Measuring circulating cell-derived microparticles.J Thromb Haemost. 2004; 2: 1842-1843Crossref PubMed Scopus (395) Google Scholar Nevertheless, we observed no overlap between patients with acute vasculitis and healthy controls (Figure 5b). Two patients with Wegener granulomatosis were analyzed before and after treatment: In the first patient, an 80% decrease of CD66b-positive MPs and a 6% decrease of CD41a-positive MPs were observed 3 days after steroid pulses. In the second patient, a 48% decrease of CD66b-positive MPs and a 27% decrease of CD41a-positive MPs were observed 1 month later (data not shown). MP levels before and after the hemodialysis session are given in Table 2 and Figure 6a. Neutro-PMs, but not Pl-MPs were significantly enhanced in patients before hemodialysis, when compared to healthy controls (Figure 6b). A further significant increase of both neutrophil MPs and platelet MPs was observed after hemodialysis. The number of null MPs did not change (164±199 before and 123±139 after dialysis, n=33, P=0.28) (data not shown).Table 2MP levels in hemodialysis patientsCD66baNeutro-MPs+Mixed MPs. (× 103)CD41abPl-MPs+Mixed MPs. (× 103)Neutro-MPs (× 103)Mixed (× 103)Pl-MPs (× 103)All patients (BD)115±114264±44413±1498±104156±345P (compared to controls)0.0030.58<0.0010.020.36All patients (AD)197±255687±124741±170156±188532±1139P (compared to BD)0.0010.0080.880.0060.018Before dialysisAfter dialysisCD66baNeutro-MPs+Mixed MPs.(× 103)CD41abPl-MPs+Mixed MPs.(× 103)CD66baNeutro-MPs+Mixed MPs.(× 103)CD41abPl-MPs+Mixed MPs.(× 103)Dialysis >6 months102±74225±187216±261825±1292Dialysis<6 months121±141282±593178±255550±1220With vasculopathies104±58161±142255±302834±1368Without vasculopathies115±135309±536167±228611±1202Synthetic membranes108±147260±593171±254665±1228Celluloid membranes114±64247±194223±261710±1300AD, after dialysis; BD, before dialysis; MP, microparticles; Pl-MPs, platelet-derived MPs.Bold characters underline numbers significantly different from normal controls (P<0.05).a Neutro-MPs+Mixed MPs.b Pl-MPs+Mixed MPs. Open table in a new tab AD, after dialysis; BD, before dialysis; MP, microparticles; Pl-MPs, platelet-derived MPs. Bold characters underline numbers significantly different from normal controls (P<0.05). Concerning subgroups of hemodialysis patients, no significant difference in MP levels was observed between patients with less or more than 6 months of hemodialysis, patients without or with established vasculopathies, or patients hemodialysis with synthetic or celluloid membranes. Ex vivo neutrophil activation in whole blood from patients with acute vasculitis (Figure 7a) or blood recovered after hemodialysis (Figure 7b) did not result in a significant increase of neutrophil MPs, as compared to the initial number of circulating MPs. This contrasted with what was observed with normal controls, with patients with chronic vasculitis, or with patients before hemodialysis. This suggested that the ability of neutrophils to further release MPs upon activation ex vivo was exhausted in patients with acute vasculitis or after the contact between circulating cells and dialysis membranes. This study shows that neutrophil and platelet MPs are important in vivo markers of neutrophil activation, in patients with vasculitis and during hemodialysis-induced inflammation. An increase of circulating endothelial cells and endothelial MPs has been previously reported in patients with vasculitis.21.Brogan P.A. Shah V. Brachet C. et al.Endothelial and platelet microparticles in vasculitis of the young.Arthritis Rheum. 2004; 50: 927-936Crossref PubMed Scopus (158) Google Scholar, 22.Woywodt A. Streiber F. de Groot K. et al.Circulating endothelial cells as markers for ANCA-associated small-vessel vasculitis.Lancet. 2003; 361: 206-210Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar Surprisingly, neutrophil MPs were not investigated, in spite of the central role of neutrophils in anti-neutrophil cytoplasmic antibody-associated vasculitis. In our study, patients with acute vasculitis clearly had the highest levels of circulating neutrophil MPs. These levels were significantly lower in chronic vasculitis and may thus be considered as markers of disease activity. With regard to treatment response, we tested two patients with Wegener granulomatosis before and after steroid treatment and observed a striking decrease of Neutro-MPs and, to a lesser extent, of Pl-MPs. A larger prospective study would be of interest to analyze MP levels in relation to disease activity and treatment. One should keep in mind, however, that neutrophil MPs strongly increase during severe infectious diseases8.Joop K. Berckmans R.J. Nieuwland R. et al.Microparticles from patients with multiple organ dysfunction syndrome and sepsis support coagulation through multiple mechanisms.Thromb Haemost. 2001; 85: 810-820PubMed Google Scholar and should be cautiously used as a marker of acute vasculitis relapses in patients with immunosuppressive treatment. Patients with tubulo-interstitial nephritis, in our study, also had high levels of circulating MPs. Both renal and extra-renal inflammation may be involved in these patients, since acute tubulo-interstitial inflammation was present and primary immune disorders with systemic manifestations were identified in 9/19. Neutrophil MP levels had not been previously studied during CRF progression. Platelet MP increases have been reported, but with a range overlap with the healthy control group and without precise absolute number of MPs.14.Mercier E. Branger B. Vecina F. et al.Tissue factor coagulation pathway and blood cells activation state in renal insufficiency.Hematol J. 2001; 2: 18-25Crossref PubMed Scopus (35) Google Scholar In our study, we found no significant increase of neutrophil MP levels during progression of renal failure. By contrast, we observed increased levels of Neutro-MPs in hemodialysis patients. This discordance is important to consider in the light of the inhibitory effects of uremic toxins, such as p-cresol, on neutrophil activation.23.Vanholder R. De Smet R. Waterloos M.A. et al.Mechanisms of uremic inhibition of phagocyte reactive species production: characterization of the role of p-cresol.Kidney Int. 1995; 47: 510-517Abstract Full Text PDF PubMed Scopus (129) Google Scholar Moreover, in CRF, neutrophil activation does not result in increased cytosolic Ca2+, a mechanism involved in MPs release.24.Caimi G. Canino B. Vaccaro F. et al.Polymorphonuclear cytosolic Ca(2+) concentration before and after activation in chronic renal failure.Nephron. 2000; 85: 371-372Crossref PubMed Google Scholar Thus, it is possible that uremic toxins inhibit MPs release by preventing neutrophil activation. During the hemodialysis session, the levels of all three types of MPs (Pl-MPs, neutro-MPs, and mixed MPs) were significantly enhanced. Detrimental effects of hemodialysis membranes (and/or dialysate itself) on inflammation status are well documented.25.Stenvinkel P. Lindholm B. Heimburger M. Heimburger O. Elevated serum levels of soluble adhesion molecules predict death in pre-dialysis patients: association with malnutrition, inflammation, and cardiovascular disease.Nephrol Dial Transplant. 2000; 15: 1624-1630Crossref PubMed Scopus (165) Google Scholar, 26.Rao M. Guo D. 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This priming effect has also been described for the oxidative burst.15.Elbim C. Chollet-Martin S. Bailly S. et al.Priming of polymorphonuclear neutrophils by tumor necrosis factor alpha in whole blood: identification of two polymorphonuclear neutrophil subpopulations in response to formyl-peptides.Blood. 1993; 82: 633-640Crossref PubMed Google Scholar, 16.Hallett M.B. Lloyds D. Neutrophil priming: the cellular signals that say 'amber' but not 'green'.Immunol Today. 1995; 16: 264-268Abstract Full Text PDF PubMed Scopus (227) Google Scholar We thus anticipated that circulating TNF-α, present in patients with vasculitis,28.Noronha I.L. Kruger C. Andrassy K. et al.In situ production of TNF-alpha, IL-1 beta and IL-2R in ANCA-positive glomerulonephritis.Kidney Int. 1993; 43: 682-692Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 29.Tesar V. Masek Z. 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Characterization of a new form of leukocyte regulation.J Immunol. 1994; 153: 3267-3275PubMed Google Scholar In our study, we found that the increase of neutrophil MPs was constantly accompanied by an increase of platelet MPs. Although platelets do not express receptors for inflammatory stimuli, such as of TNF receptors or the fMLP-receptor,31.Drabikova K. Jancinova V. Nosal R. Danihelova E. Human blood platelets, PMN leukocytes and their interactions in vitro. Responses to selective and non-selective stimuli.Gen Physiol Biophys. 2000; 19: 393-404PubMed Google Scholar, 32.Cambien B. Bergmeier W. Saffaripour S. et al.Antithrombotic activity of TNF-alpha.J Clin Invest. 2003; 112: 1589-1596Crossref PubMed Scopus (69) Google Scholar they can be activated indirectly by neutrophil-derived proteases33.Ferrer-Lopez P. Renesto P. 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