Introduction
Acute Lung Injury (ALI) and its most severe clinical presentation, Acute Respiratory Distress Syndrome (ARDS), occur after a variety of insults, including sepsis, trauma, or aspiration of gastric contents. Despite recent therapeutic advances in the field of mechanical ventilation, 30% to 50% of ARDS patients die [
1]. A recent systematic review suggested that mortality from ARDS has not decreased substantially since the publication of the American-European consensus conference in 1994 [
2,
3].
Converging evidence from clinical and experimental studies shows that leukocytes play a pivotal role in injury during the acute phase of ALI/ARDS. Early in the course of ARDS, lung biopsies and bronchoalveolar-lavage fluid (BAL) show a marked accumulation of neutrophils. Neutrophils are the corner stone of host defenses by releasing proinflammatory cytokines and chemokines that could explain, at least in part, why anti-inflammatory therapies have largely been unsuccessful in ARDS [
4].
The local and systemic proinflammatory responses accompanying ARDS are orchestrated by the interactions between circulating cells such as leukocytes, platelets, and endothelial cells. It is now well admitted that during inflammatory responses, cells release submicron vesicles that bud off from the cell membrane. These cell-derived microparticles (MPs) have proven to be sensitive markers for assessing the activation/apoptotic status of cells in many inflammatory disorders such as SIRS, meningococcal sepsis, severe trauma, and neuropaludism [
5‐
7]. Microparticles are submicron plasma membrane vesicles that express cell surface proteins of the original cells and negatively charged phospholipids such as phosphatidylserine. Moreover, MPs behave as vectors of bioactive molecules, which accounts for their pro-coagulant and pro-adhesive potential. Taken together, the interest in MPs has substantially increased, not only for their clinical relevance as disease markers but also for their role as effectors in the tight tuning of adaptive responses such as inflammation, immunity, or hemostasis [
8].
The critical role of leukocyte-mediated responses led us to hypothesize that the strong local and systemic inflammatory responses associated with ARDS may be associated with altered levels of leukocyte MPs (LeuMP). The objective of the present study was therefore to measure LeuMP and other MP subpopulations both in blood and BAL early in the course of ARDS. Because recent evidence suggests that poor outcome in critically ill patient is associated with "immune paralysis" (endogenous immunosuppresion) [
9], we also postulated that high levels of LeuMP may be associated with better outcome during ARDS.
Discussion
To our knowledge, this is the first study which characterized the cellular origin of MPs in the course of ARDS, both in circulating blood and BAL. The major findings are that 1/MPs originating from platelets, endothelial cells and leukocytes were detectable in the BAL of patients with ARDS 2/among them, only LeuMP and NeuMP were elevated in BAL when compared with the spontaneous breathing group 3/Higher levels of circulating LeuMP detectable early at time of diagnosis of ARDS were associated with better prognosis.
As a result of the local and systemic pro-inflammatory responses associated with ARDS, leukocytes become activated within the general or in the pulmonary circulation. In the present study, the analysis of MP subpopulation reflects the origin and the activation status of the cells present in the BAL. Accordingly the low number of granulocytes in BAL from spontaneous breathing (SB) controls is consistent with the fact that NeuMP represent a minor sub-population. The major influx of neutrophils toward the lungs early in the course of ARDS is suggested by the high levels of NeuMP recovered by BAL.
The general belief is that MPs are conveyers of deleterious information associated with exaggerated inflammatory response [
8]. Indeed, elevated levels of MPs from platelets, granulocytes, and endothelium are found in patients with septic shock, meningococcemia, traumatic brain injury, and severe trauma [
5‐
7,
17,
18]. In contrast, Soriano
et al. have suggested that MPs presented protective effects in patients with septic shock [
19].
Bastarache
et al. [
20] reported the presence of procoagulant microparticles in the lung from ARDS patients. By focusing on the epithelial alveolar origin of microparticles, they reported higher concentrations of these MPs in ARDS patient's edema fluid compared to patients with hydrostatic pulmonary edema. However, this latter study reported a trend for higher concentrations of total MPs in the edema fluid from non-survivors. Although this latter result did not reach statistical significance, differences with our study might be due to the cellular origin of the MPs (alveolar epithelial vs. leukocyte or platelet lineages) or to the different compartments where MPs were isolated (edema fluid vs. blood).
The main finding of the present study is that lower levels of leuMP are detectable in blood (day 1) and BAL (day 3) in non survivors.
ARDS is clinically characterized by a strong alteration of ventilator mechanics with decreased lung compliance. During ARDS, plateau pressure must be monitored and maintained as low as possible to reduce ventilator-induced lung injury or right heart failure. This can be achieved by reducing tidal volume on the ventilator and by setting an appropriate level of positive end expiratory pressure (PEEP). In previous studies, clinical parameters such as plateau pressure and quasi-static pulmonary compliance were found being strongly associated with mortality occurring during ARDS [
21,
22]. To assess if levels of microparticles may reflect some part of ventilatory induced lung injury, it would be interesting to search some correlation between levels of MP and the ventilatory mechanics parameters in different ventilatory conditions.
ARDS from pulmonary origin is characterized by a local pro-inflammatory process that first occurs in damaged lung and then contributes to multi-organ dysfunction. A balance between pro-inflammatory and anti-inflammatory effects is observed in the course of acute lung injury. Sustained high levels of pro-inflammatory biomarkers are associated with poor outcome of ARDS [
23,
24]. In the present study, the observation that low levels of MPs are associated with mortality is in agreement with data already reported for severe sepsis [
19].
The initial theory for death-associated sepsis was that multiple organ failure resulted from an excessive or uncontrolled inflammatory response. A more recent concept to explain the different outcomes during sepsis is that normal responses to injury can be immunosuppressive, inducing "an immune paralysis" and leading to health care associated infections, multiple organ failure and, finally, death [
9,
25]. Consistent with this theory one could speculate that the lows levels of MPs in patients with worse outcome, reflect "suppression of vesiculation", given the fact that vesiculation is a response of cells to injury. Although the mechanism supporting this potential protective effect remains to be elucidated, they can be reliable to the anti-inflammatory effect of polymorphonuclear neutrophil derived MPs (NeuMP) reported by the work of Gasser and Schifferli [
26]. They showed that NeuMP block the response of macrophages to LPS and increased the secretion of transforming growth factor beta1, a potent inhibitor of macrophage activation. Thus, in the earliest stage of inflammation, neutrophils cells release MP that convey potent anti-inflammatory effects, by driving the resolution of inflammation. More recently, it was reported that MPs shed from adherent neutrophils bear Annexin 1, an endogenous anti-inflammatory protein able to inhibit neutrophil adhesion to the endothelium [
27]. It could have been interesting to assess some biomarkers that evaluate endothelial permeability (VWF or Ang2), the inflammation (IL-6 or IL-8) or apoptosis like FasLigand or soluble FAS.
Furthermore, our data suggesting that circulating LeuMP are protective upon ARDS onset may also be reliable to the MPs beneficial properties exerted on vascular tone during sepsis. Mostefai
et al. [
28] showed that the number of total circulating and platelet microparticles in patients with septic shock was increased and that these microparticles were protective against vascular hyporeactivity.
The mechanism supporting this protective effect in ARDS is a challenging question. One can hypothesize that blocking inflammatory cytokines such as TNF-α could suppress the release of MPs that may be beneficial in ARDS patients, which may help explain why anti-inflammatory therapies such as anti-interleukin-1R or anti-TNF have largely been unsuccessful in this context [
4]. Thus, if we consider subpopulation of MP such leuMP as protector, our results support the negative impact of decreasing inflammatory response early in the course of ARDS and enlights the potential of therapeutic option aimed to promote the release of these MPs subpopulation.
Mechanical ventilation has been reported for modulate the platelet microparticles release in an experimental context [
29]. Our data do not support this hypothesis to explain the differences observed between the VC group and the ARDS group concerning LeuMP.
Limitations
The first limitation of our study is to extrapolate our results to a mixed population of ARDS. Indeed, the patients had direct lung injury in 90% with pneumonia in 73% of cases. One other limitation of our study is that circulating MPs in ARDS patients were not compared to those of the control groups. However, in contrast to MPs in BAL, low levels of circulating MPs in healthy subjects have been extensively reported in the literature [
6,
30] and such comparison is beyond the question raised by this study focused on the outcome of patients. This study can only provide relationship between microparticles and clinical outcome in patients with established ARDS. It was not designed to assess the association between MPs and the development of ARDS.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CG included all the patients, performed the BAL procedures and drafted the manuscript. RL performed cytometry analysis. JMF and AR participated in the study and study analysis. CG, RL, LCJ, LP and FDG participated in the interpretation of the results and gave the advices for improving the manuscript. LP, LCJ and FDG initiated the study, participated in the design of the protocol and helped to draft the manuscript. All authors read and approved the final manuscript.