Microparticles are plasma membrane-derived vesicles shed from stimulated cells, in the broad sense of the term. Their presence is interpreted by proximal or remote cells in fundamental physiological processes including intercellular communication, hemostasis, and immunity. On the other hand, variations of their number or characteristics are frequently observed in pathophysiological situations.
In multicellular organisms, homeostasis results from a subtle balance between cell proliferation and degenerescence. Cells differentiate, expand, fulfill particular functions, then undergo programmed death and are finally cleared by phagocytosis. At each stage of its life, the cell is subjected to a variety of stimulations leading to the release of submicron fragments from the plasma membrane, usually termed microparticles or microvesicles (MPs). MPs hijack membrane constituents and cytoplasmic content and survive the cell (FIGURE 1⇓). Owing to the plasticity of the lateral organization of the plasma membrane into raft domains, known to segregate particular proteins and lipid species, a given stimulus can be expected to elicit a “private” response resulting in an inclusive or exclusive sorting. This explains how MPs of the same cellular origin may have different protein and lipid compositions. Hence, such fragments are interpreted in intercellular communication and participate in the maintenance of homeostasis under physiological conditions, or they can initiate a deleterious process in case of excess or when carrying pathogenic constituents. MPs can therefore be considered a disseminated storage pool of bioactive effectors, the nature and proportion of the latter accounting for duality, more particularly evidenced in vascular disease, inflammation, and immunity.
Mechanisms Governing the Plasma Membrane Remodeling
Each of the two leaflets of the membrane bilayer has a specific lipid composition. Aminophospholipids [phosphatidylserine (PS) and phospha-tidylethanolamine] are specifically segregated in the inner leaflet, whereas phosphatidylcholine and sphingomyelin are enriched in the external one. The transbilayer lipid distribution is under the control of three major players: an inward-directed pump, a flippase, specific for PS and phos-phatidylethanolamine, known as aminophospholipid translocase; an outward-directed pump referred to as “floppase”; and a lipid scramblase, promoting unspecific bidirectional redistribution across the bilayer (8). A significant and sustained increase of cytosolic Ca2+ accompanying cell stimulation may lead to the collapse of the membrane asymmetry by stimulating scramblase and floppase activities and concomitantly inhibiting the flippase. The most prominent change in lipid distribution is surface exposure of PS, followed by MP release allowed by cytoskeleton degradation by Ca2+-dependent proteolysis (FIGURE 2⇓). However, it must be added that MPs can be produced under less-controlled situations and that a proportion of circulating MPs probably originates from necrotic cells upon loss of membrane integrity or from mechanical destruction of cells following injury. PS can be expected to be borne by such MPs because asymmetric phospholipid distribution is no more maintained. Once accessible, at the cell as well as at the MP surface, PS serves at least two important physiological functions: it promotes blood coagulation, and it constitutes a recognition signal for clearance of senescent cells by the reticuloendothelial system. Although actors of externalization may be the same in both situations, regulatory mechanisms are probably different. PS transmembrane redistribution occurs in apoptotic yeast (35). Hence, membrane remodeling and MP shedding seem to be fundamental processes, probably shared by virtually all cell types, that have been interpreted at different stages of evolution of multicellular organisms, for instance in the elaboration of phagocytic and clotting functions. New functions for PS and MPs can be considered in a context in which the receptor (Ptdsr) was recently reported to have an unexpected developmental function as an important differentiation-promoting gene (10).
Microparticles, exosomes, and protein sorting
A basic role for MPs in intercellular communication implies that the cell of origin controls MP content and amount. In this respect, it has been suggested that different agents are able to induce the in vitro release of phenotypically distinguishable MPs (6). The lateral organization of membrane lipids and proteins in raft domains, resulting in inclusive or exclusive sorting, provides a rationale for the shedding of particular lipids and proteins within MPs (24).
A specialized category of MPs with known functions in protein sorting and in the immune response are the exosomes. More homogenous in size and composition, exosome characteristics in different cell types indicate the following minimal requirements for discrimination with MPs: a diameter of 30–90 nm; a density in sucrose gradients of 1.13–1.19 g/ml; an endocytic origin (exosomes do not derive from the plasma membrane but from internal membranes); and enrichment in tetraspaning molecules (48). PS is also present at the surface of exosomes that are derived from platelets and dendritic cells but at (much) lower levels. The aim of this review is not to describe exosomes, which has been done by other authors (48). Briefly, they have a clearing function because of enrichment in some proteins known to decrease or disappear from the cell surface during maturation. Perhaps more importantly, they are also known to harbor major histocompatibility complex molecules, making them responsible for the stimulation of T cells in an antigen-specific manner, promoting an antitumor immune response in vivo. Interestingly, exosomes were recently shown to bear the pathogenic form of the prion protein PrPSc (16), as previously described for the normal cellular form, PrPc, in MPs (21).
Membrane Microparticles in Intercellular Communication
Antigen transfer and intercellular cross-talk
Several studies point to MP-induced cell stimulation. Leukocyte MPs activate endothelial cells or transfer leukocyte antigens to epithelial cells. This passive acquisition of leukocytic phenotypes is associated with changes in phosphorylation of cellular proteins and cell-cell adhesion properties (37, 47). Platelet-derived MPs modulate monocyte-endothelial cell interactions and stimulate proliferation, survival, adhesion, and chemotaxis of hematopoietic cells, and they enhance engraftment of hematopoietic progenitor cells (6). MPs bearing Fas ligand can also be shed from the cell surface in a bioactive configuration providing a mechanism for long-range signal-directed apoptosis (3).
MPs may also be used to address morphogens into a developing tissue. These particular MPs, derived from the basolateral membrane of cells, have been termed “argosomes” (23). We have recently observed that MPs generated from apoptotic T cells harbor morphogens of the Hedgehog family and are able to induce differentiation of pluripotent hematopoietic cells toward megakaryocytic lineage (unpublished observations).
These studies demonstrate that MPs can be efficient vectors of biological information from one cell type to another within proximal or remote tissues.
Microparticles and vascular function
Under pathological situations associated with vascular damage such as myocardial infarction or preeclampsia, MPs isolated from the plasma of patients were found to cause damage in isolated arteries (11, 50).
We recently observed that MPs from apoptotic T cells induce endothelial dysfunction in both conductance and resistance arteries by alteration of nitric oxide (NO) and prostacyclin pathways. In addition, circulating MPs from diabetic patients induced endothelial dysfunction and decreased NO synthase expression (36). These data provide a rationale to explain the paracrine role of MPs as vectors of bioactive effectors promoting vascular dysfunction through transcellular exchanges during inflammatory disease. Endothelial NO dysfunction was not observed in all of the studies (11), but they all share the conclusion that MPs impair endothelial function (12, 43). Discrepancies might originate from quantitative and qualitative differences in MPs, stemming from different tissues challenged by different stimuli.
Together, these studies contribute to a better understanding of the deleterious effects of enhanced circulating levels of MPs observed in cardiovascular or immune disorders. Very recently, it has been proposed that MPs from endothelial cells impair endothelial function by altering the redox balance (12).
Microparticles in angiogenesis
MPs shed from tumor cells stimulate metastasis by promoting in vivo neovascularization. Sphingo-myelin seems to be the active component responsible for this proangiogenic potential by stimulating endothelial cell migration, invasion, and tube formation. Some matrix metallo-proteinases present in endothelium-derived MPs and platelet MPs were shown to be potent angiogenic stimulators in vitro (for a recent review, see Ref. 19).
Tissue factor (TF), the major initiator of blood coagulation, serves as a regulator of angiogenesis, tumor growth, and metastasis (34). TF mediates upregulation of the proangiogenic vascular endothelial growth factor, and MPs harboring TF on their surface could in that way act on endothelial cells to promote vessel formation. Such MPs were shown to circulate in vivo (14), but their proangiogenic activity remains to be established, especially in a context in which the signaling activity of TF seems to be linked to its cytoplasmic domain, which is probably sequestered within MPs (7).
Membrane Microparticles in Blood Coagulation
Microparticles are essential for hemostasis
PS present at the surface of MPs provides a catalytic surface promoting the assembly of the characteristic enzyme complexes of the coagulation cascade. MPs shed from activated platelets constitute the main circulating population. They harbor major membrane glycoproteins, including functional adhesive receptors, and consequently disseminate a procoagulant potential that can be targeted according to the nature of counterligands (18). They can bind to soluble or immobilized fibrinogen and aggregate with platelets (26). The procoagulant potential of MPs is not, however, restricted to platelet MPs. MPs from monocytes, lymphocytes, or endothelial cells also present PS at their surface. Other effectors were also detected at the surface of circulating endothelial MPs, for instance von Willebrand factor and E-selectin (29).
Scott syndrome is the direct evidence of the key role of MPs and PS in hemostasis. This inherited disorder, characterized by hemorrhagic complications, is linked to the lack of surface exposure of plasma membrane PS and to the inability of platelets and other blood cells to generate MPs (49). Another bleeding disorder has also been described with decreased capacity of platelets to generate MPs, but at variance with Scott syndrome, with a correct PS exposure (13). These abnormalities highlight the importance of MPs in hemostasis and perhaps more particularly that of platelet MPs.
Microparticles and TF are closely associated in the vascular compartment
TF is the prime cellular initiator of coagulation. TF expression is not restricted to the subendothelium but can also originate from stimulated monocytes or endothelial cells (34). However, an essential point must be taken into account. At the stimulated monocyte cell surface only 10–20% of the total extractable TF activity is expressed. Thus there exists circulating, potentially active blood-borne TF in normal subjects, but most of its activity is latent or encrypted (22). TF activity is highly dependent on its lipid environment (5). Decryption could be mediated through the secretion of TF-rich MPs by monocytes. Such MPs can bind activated platelets or platelet-derived MPs and facilitate fusion events between monocytes (or monocyte MPs) and platelets (or platelet MPs) leading to MP rich in decrypted, active TF (42). Moreover, in experimental thrombi, TF was predominantly colocalized at the surface of membrane vesicles, frequently clustered adjacent to platelet surfaces (22).
Among the non-cell-bound TF present in blood, a variant form that results from alternative splicing of the primary RNA transcript was identified: alternatively spliced TF (asTF). asTF is soluble, circulates in plasma, and is biologically active. During thrombus formation, platelet deposition may separate catalytic enzyme complexes from circulating blood. Binding of asTF as well as TF-bearing monocyte-derived MPs to platelets provides a rationale for thrombus formation and growing (9). Binding can be mediated by platelet P-selectin and monocytic P-selectin glycoprotein ligand-1 (PSGL-1). This indicates one pathway for the initiation of blood coagulation involving the accumulation of TF-containing MPs in the platelet thrombus (15). The importance of the interaction between P-selectin and PSGL-1 was confirmed in vivo in a mouse model of hemophilia A in which soluble P-selectin was able to induce the generation of TF-positive MPs and correct hemostasis that way (27). Functional competence of blood-borne TF could be expressed when MPs and platelets adhere to neutrophils (40). Finally, in vitro interaction of endothelial MPs with monocytic cells was also shown to induce TF-dependent procoagulant activity (45).
Microparticles, thrombosis, and inflammation
It is obvious that pathological dysregulations leading to variations in the nature or proportion of circulating MPs (qualitative and quantitative aspects), and perhaps more frequently an augmentation of the number of circulating procoagulant MPs, will result in an increased thrombotic propensity. Elevated levels of circulating MPs have indeed been reported in various disorders characterized by thrombotic complications and more particularly in cardiovascular diseases (39, 51).
The implication of MPs in inflammation is also well documented. For instance, the presence of interleukin-1β (IL-1β) was observed in shed MPs (33), and MPs are a source of aminophospholipid substrates of secretory phospholipase A2 for the generation of lysophosphatidic acid, a potent proinflammatory mediator and platelet agonist (17). Additionally, MPs were shown to favor endothelial activation and monocyte-endothelium interactions, the two initial steps of the atherosclerotic plaque formation (28). Blood-derived MPs can stimulate release of cytokines from endothelial cells and upregulation of TF expression at their surface (38). Platelet-derived MPs also enhanced expression of cell adhesion molecules in monocytic and endothelial cells and induced production of IL-8, IL-1β, and IL-6 by endothelial cells as well as IL-8, IL-1β, and tumor necrosis factor-α by THP-1 cells (ATCC no. TIB-202) (41).
Together, these studies show that MPs are key actors of blood coagulation, but their pathophysiological role is clearly not restricted to their procoagulant potential.
Membrane Microparticles in Immunity
MP release is one of the early hallmarks of cells undergoing apoptosis, and shedding from senescent cells was shown to be correlated to the degree of apoptosis (4). It is therefore reasonable to consider that systems in charge of the elimination of cell waste products gain information from qualitative or quantitative variations of MPs.
Ectosomes and opsonization
Upon activation, neutrophils release MPs at the site of inflammation in vivo. These MPs, termed ectosomes, bind efficiently to opsonized bacteria and may be designed to focus antimicrobial activity onto opsonized surfaces (25). Ectosomes were also found to become specifically adherent to monocytic and endothelial cells, making them candidates for playing important roles in inflammation and cell signaling (20).
Ectosomes are thought to be released from the plasma membrane through the same mechanisms described above for MPs and also bear a significant proportion of PS. The centrifugation procedure used for their purification probably accounts for a selection of the smallest microvesicles present in the preparation (50–200 nm), thus possibly containing exosomes.
Immune escape mechanisms
MPs are suspected to support a sort of physiological immune escape as described in pregnancy. Since the invading trophoblast represents a semi-allograft, the mother should reject it. A very recent study proposes that secretion of FasL-containing MPs may constitute a mechanism by which trophoblast cells promote a state of immune privilege and, therefore, protect themselves from maternal immune recognition. Abrahams et al. (2) demonstrate that first-trimester trophoblast cells lack membrane-associated FasL but constitutively secrete FasL through the release of MPs, which, following MP disruption, is able to induce Fas-presenting T cell death by apoptosis.
Cancer is another situation in which MPs are implicated in escape from the immune system. A study shows that FasL-bearing MPs are shed in the extracellular medium by tumor cells and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells (3). In another study, epithelial ovarian cancer cells were shown to secrete functional FasL via the release of MPs (1). In contrast, normal ovarian epithelial cells express but do not secrete FasL. Together, these two studies suggest a mechanism by which tumors might neutralize Fas-bearing immune cells, thus facilitating escape and promoting survival.
Microparticles and AIDS
MPs seem to play several roles in AIDS, including implication in HIV infection, as well as in the propagation of the virus and its escape from classical vaccine strategies.
A few years ago, a mechanism allowing HIV to infect cells lacking the chemokine receptor CCR5 was suggested. CCR5 was released through MPs from the surface of CCR5-positive cells and was transferred to deficient peripheral blood mononuclear cells, rendering them CCR5 positive and susceptible to HIV-1 infection (32). A recent study confirms the existence of such a process by showing that platelet- and megakaryocyte-derived MPs can also transfer CXCR4 receptor to CXCR4-null cells (44).
In addition, one cannot exclude a role for CD4+-derived MPs observed in augmented proportions in some HIV-positive patients, especially in a situation in which individuals with high levels of circulating MPs and low circulating CD4+ cell counts seem to be protected from the classical complications of AIDS (4).
Microparticles and cancer
Like AIDS, cancer is a situation in which MPs are involved at different levels. For instance, monocyte-derived MPs were proposed to be a sign of vascular complication in patients with lung cancer, and elevated levels of circulating platelet MPs were observed in gastric cancer, with a possible role for MPs as metastasis predictors in the latter study (30, 31). Furthermore, doxorubicin was reported to accumulate in MPs shed by cancer cells, supporting the hypothesis that membrane vesiculation offers a way for tumors to rid themselves of toxic drugs, accounting in part for resistance to chemotherapy (46).
On the whole, these studies emphasize the increasing significance of MPs in fundamental physiological processes as well as under major pathological situations. They constitute the very first references for the interpretation of the role of MPs in physiology. Recent progress in the understanding of dynamic organization of the plasma membrane and the plasticity of its response should provide new bases for the identification of other biological functions for MPs in multicellular organisms.
We apologize to all colleagues whose work could not be quoted owing to strict space constraints.
Research in our laboratory was supported by institutional funding from INSERM and the Université Louis Pasteur de Strasbourg, and by a grant from the Association “Vaincre la Mucoviscidose” awarded to M. Carmen Martínez.
- © 2005 Int. Union Physiol. Sci./Am. Physiol. Soc.