In the present study, we investigated the plasma level of circulating MPs in patients with the AIDs pSS, SLE and RA and found a higher level of total and platelet-derived circulating MPs as compared with HCs. A specific feature of pSS was an elevated level of leukocyte-derived MPs, which was not observed in other AIDs. Interestingly, in severe pSS with extraglandular manifestations, the level of platelet MP was less increased than those in pSS patients with glandular involvement only. In addition, we found an inverse correlation between level of MPs and disease activity in RA and SLE. Moreover, we found the level of MPs inversely correlated with two other quantitative biomarkers, serum β2 microglobulin level in pSS and anti-dsDNA IgG antibodies in SLE.
The patients in our study were slightly older than those in the HC group. No correlation has been observed between the total MPs levels and the age of patients in each disease group (P
> 0.1). Likewise, no data in the literature suggest any impact of age on MP levels except in subjects younger than 18 years old [19
]. The relatively small number of men in each group may probably not have an impact on MP levels: the comparison of the MP levels between men and women with AIDs has shown no difference according to the sex for each subtype of MPs (P
> 0.16). To avoid the confounding effects of other factors susceptible to increase the level of MPs, such as cardiovascular risk factors or infection [20
], we verified that associated cardiovascular co-morbidities might not have influenced the increased number of MPs. In addition, we have not included patients with recent thrombosis, acute or chronic infection who represent confounding factors disturbing the interpretation of results in AIDs. Finally, some patients have very high levels of MPs, suggesting that MP levels may be heterogenous in a defined disease group. However, after excluding patients with total and platelet MP levels above 20 nM PS Eq and leukocyte MP levels above 10 nM PS Eq in all groups, the statistical analysis remained unchanged (data not shown).
Circulating MPs originate from cell plasma membranes and are generated after cell stimulation (apoptosis or activation). In AIDs, MPs could be released at a systemic level by cytokine stimulation according to the same mechanism demonstrated in vitro
]. The increase in the level of platelet MPs suggests that platelets were activated in the three diseases we studied. To confirm this feature in AIDs, the plasma concentrations of sCD40L and sCD62P, which are released by platelets upon stimulation and considered the two typical biomarkers of platelet activation [23
], were increased in all AID groups as compared with HCs. This increase has been reported for RA and SLE [3
], whereas in pSS, sCD40L has been reported only once [4
], and sCD62P measurement in pSS has never been assessed. These data emphasize the known role of platelets in RA and SLE. Of note, activated platelets in SLE could activate plasmacytoid dendritic cells for interferon-alpha production [26
]. These latter cells are also detected in labial salivary glands of patients with pSS [27
]; hence, platelets could also contribute to plasmacytoid dendritic-cell activation in pSS.
As MPs can be detected by several non-standardized methods [28
], we assessed MPs with two different methods simultaneously, solid-phase capture assay and flow cytometry, the results being positively correlated. Of interest, capture assay detects leukocytes and platelet MPs as being annexin V positive, whereas quantification of these subtypes of MPs by flow cytometry does not use annexin V ligation and thus involves annexin V-positive MPs as well as the small fraction of annexin V-negative MPs [2
]. However, no clinical association with results obtained on flow cytometry was tested because few patients were tested with this method. Furthermore, tissue factor-positive MPs were not assessed in this study because of the low frequency of thombotic manifestations in pSS.
As MPs are generated after cell activation and/or apoptosis, it is not possible to discriminate between these two mechanisms to explain the increase in MPs in AIDs. If apoptosis play a role, it is probably not linked to immunosuppressive agents because the patients treated with these drugs did not have higher levels of MPs.
Increased plasma MP levels have been reported in metabolic, cardiovascular, infectious, neoplastic and autoimmune diseases [29
]. In autoimmune diseases, MPs have been found elevated in RA [3
], SLE [14
], Crohn's disease [32
], systemic sclerosis [22
], vasculitis [33
] and myositis [36
]. Here we report the first assessment of circulating MPs in pSS. An interesting finding was the significantly decreased level of platelet MPs in pSS patients with more severe disease corresponding to extra-glandular involvement compared with those with glandular involvement only. A similar feature was also shown in patients with more severe SLE and RA disease as assessed by the SLEDAI and DAS28, respectively. However, in the three AIDs, the level of MPs in patients with more severe disease remained greater than in HCs. In fact, similar results have been recently reported in systemic sclerosis [22
] and Crohn's disease [32
] on assessment of MPs by flow cytometry and solid-phase capture assay, respectively. These results and the present results suggest that the level of circulating MPs might be inversely related to severity of disease as a general biological mechanism. In RA, discordant results have been reported: MP level was found increased or not different from that in HCs [30
]. Finally, for other acute inflammatory diseases such as severe sepsis or multiple organ dysfunction syndrome, the number of platelet and endothelial MPs was found to be lower than that for controls [39
] and a low level of MPs in severe sepsis was associated with a poorer prognosis [40
Several hypotheses could explain these discordant findings. First, the decreased plasma level of MPs could be a result of consumption or confinement of MPs by adhesion in the tissue target of the AID such as the synovium in RA [41
]. Second, MPs can aggregate circulating leukocytes and platelets, thus leading to the formation of leukocyte-platelet complexes. Thus, MP measurements do not take these MPs sequestered in cell aggregates into account, which leads to an underestimation of their amount [3
]. These aggregates were found in higher levels in SLE and RA patients than in controls, but no association with disease activity has been reported to date [3
]. Finally, the decreased level of MPs in active disease could be explained by the destruction of circulating MPs in the peripheral blood by phospholipases, especially sPLA2, which targets its aminophospholipid substrates in shedded membrane particles to generate lysophosphatidic acid [16
Interestingly, we found a significant inverse correlation between levels of total MPs or platelet-derived MPs and sPLA2 activity in patients. We hypothesised that plasma MPs could be destroyed by increased sPLA2 through the degradation of their aminophospholipids in active disease. Thus, previous in vitro
experiments showed that cell-derived microvesicles provide a preferential substrate for sPLA2 by the transformation of phospholipids present in MPs into lysophosphatic acid [16
]. New experiments assessing a direct consumption of MPs by sPLA2 would be very interesting to perform.
sPLA2 activity was increased in all patients, especially pSS patients with extra-glandular involvement who showed a significantly decreased level of platelet MPs. Furthermore, although high level of sPLA2 has been reported in RA [44
], we report for the first time in pSS and SLE the increased functional activity of sPLA2, despite the absence of increased levels of other classical biological markers of systemic inflammation (C-reactive protein and fibrinogen; Table ). Thus, the exact role of sPLA2 in AIDs, in addition to its pro-inflammatory role, remains to be elucidated, especially in the context of cardiovascular complications observed in these diseases. Of note, we did not use a quantitative but rather a functional assay of sPLA2, which may better explain MP destruction in case of active disease.
To date, plasma level of MP has been considered a biomarker reflecting cell activation and could participate in the accelerated atherosclerosis observed in AIDs, but involvement of MPs in the cross-talk between resident cells in target organs of autoimmunity and inflammatory infiltrating cells has been sparsely reported. In RA, leukocyte MPs can activate synovial fibroblasts [21
], but no data are available for pSS and SLE. Only exosomes, another kind of circulating vesicles containing specific auto-antigens and generated by salivary gland epithelial cells, have been identified [48
]. As we found elevated MP level in pSS, the functional role of MPs remains to be elucidated, as does the role of platelet activation, despite the absence of increased thrombosis in this disease.