Microparticles as regulators of inflammation: Novel players of cellular crosstalk in the rheumatic diseases

Authors


Introduction

Microparticles are a heterogeneous population of small, membrane-coated vesicles that represent subcellular elements for cell signaling and intercellular communication in inflammation. Microparticles can be released by virtually all cell types upon activation or apoptosis, although depending upon their origin, they can vary in size, biochemical composition, and biologic effects. Stimuli that trigger the release of microparticles increase the intracellular calcium concentration, which leads to a rearrangement of the cytoskeleton and the budding of microparticles from the plasma membrane. Studies in a variety of in vitro systems have demonstrated that microparticles are potentially important mediators of cellular interactions. Thus, these subcellular structures can transfer bioactive molecules between cells as well as regulate diverse processes, such as inflammation, coagulation, vascular function, apoptosis, and cell proliferation. In view of these activities, microparticles play a role in the pathogenesis of rheumatologic diseases such as rheumatoid arthritis (RA), systemic vasculitis, and antiphospholipid syndrome. This report summarizes the current knowledge about the generation of microparticles, their composition, and their potential as novel effector elements in rheumatologic disease.

Background and definition of microparticles

The first report of microparticles, also referred to as microvesicles, is from 1967, when Wolf described the presence of fragments derived from platelets in human plasma (1). Analysis of these fragments demonstrated that they were small, membrane-coated vesicles. At that time, these vesicles were considered to be a residue or by-product of platelet activation and were therefore named “platelet dust.” Subsequent studies have demonstrated that these particles, like the cells from which they arise, have potent activities in clotting and are not inert. Furthermore, while platelets are an important source of microparticles, microparticles can be released from multiple cell types, including macrophages, monocytes, B and T cells, neutrophils, erythrocytes, endothelial cells, vascular smooth muscle cells, epithelial cells, and tumor cell lines. Their ubiquity has suggested a more general role for microparticles in cellular regulation, beyond mere clotting, with these structures subsuming at least some functions of their cell of origin.

While a precise definition of microparticles is lacking, they are usually defined as a heterogeneous population of small vesicles with a diameter of 0.1–1 μm (Figure 1). These particles are membrane-coated and released from the plasma membrane during cell activation or apoptosis by exocytic budding. During this process, the normal membrane lipid asymmetry is lost. The appearance of phosphatidylserine on the outer leaflet of the microparticle membrane allows binding of annexin V, which is widely used to identify and quantify microparticles. In addition to altered surface lipids, microparticles display cell surface proteins of the cell from which they originate. The identification of microparticles is based on size and membrane marker expression, as assessed by flow cytometry. Isolation can be accomplished by differential sedimentation that separates the particles from cells as well as separating soluble proteins.

Figure 1.

Ultrastructural analysis of microparticles. Microparticles from apoptotic Jurkat cells were isolated by differential centrifugation. Transmission electron microscopy showed a heterogeneous population of membrane-coated circular vesicles ranging in size from 200 to 700 nm. Microparticles contained different amounts of electron-dense material (original magnification × 80,000). Reproduced, with permission, from C. A. Seemayer, Institute for Pathology, University Hospital Basel, Switzerland.

Although arising during apoptosis, microparticles are distinct from apoptotic bodies. After the induction of apoptosis, the cell shrinks and shows chromatin condensation and cellular rearrangement, which leads to formation of blebs. Eventually, the apoptotic cell collapses and fragments, which leads to the formation of membrane-coated structures, the so-called apoptotic bodies. In contrast to microparticles, which are released early during apoptosis, apoptotic bodies form in the final stages of programmed cell death. In addition to the kinetics of their generation, microparticles and apoptotic bodies differ in composition and size, with apoptotic bodies being many times larger in diameter and volume (2).

Microparticles also differ from exosomes, another type of vesicle released from eukaryotic cells either spontaneously or during activation. Unlike microparticles, exosomes are preformed vesicles. Exosomes are stored intracellularly in multivesicular bodies and are secreted when the multivesicular bodies fuse with the cell membrane (3, 4). Although they arise from the inside of cells, exosomes nevertheless can be functionally active. For example, exosomes derived from B lymphocytes can bind to follicular dendritic cells and function in antigen presentation (5), while exosomes from cytotoxic T cells may mediate target cell killing (6). As shown in recent morphologic studies, exosomes range in size from 50 to 100 nm and are coated by a lipid bilayer. Exosomes also display certain surface receptors of the parental cell (7).

Another term used to characterize vesicles released from cell membranes is ectosomes (4). Stein and Luzio coined the expression “ectocytosis” for the process of releasing vesicles with a “right-side-out” orientation (8). Most microparticles that are released by monocytes, neutrophils, platelets, and fibroblasts correspond to the definition of ectosomes. While the terms microparticles and ectosomes may not be synonymous, there is evidence for a structural and functional similarity of these structures. As these considerations suggest, in studies on the biochemical and functional activities of microparticles, it is important to define preparations by a number of parameters, including size and cell surface molecule display, recognizing that preparations can in fact contain a mixture of different particle types (Table 1).

Table 1. Types of small vesicles other than microparticles
Type of small vesicleRef.
Apoptotic bodies2
Exosomes3–7
Ectosomes4, 8

Role of microparticles in thrombosis

During the last decade, it has become clear that microparticles are much more than debris or inert dust. As shown in provocative experiments conducted primarily on in vitro systems, microparticles appear to be novel subcellular effectors that regulate cellular processes crucial to disease pathogenesis. As such, microparticles exist along a spectrum of regulatory elements ranging from the intact cell to small molecule mediator. In this conceptualization, the activity of microparticles as players in biologic processes reflects the concentration of effector molecules on a compact surface. Furthermore, since these particles originate during apoptosis or activation, they have the potential to induce or amplify disease processes.

Initial studies of thrombosis provided the most compelling evidence for microparticles in cellular crosstalk and their potency as packets for cellular regulation. Thus, as shown in vitro, microparticles can potently promote coagulation. This action reflects the ability of microparticles to provide a negatively charged surface to bind coagulation factors (9–12), to display tissue factor on their surface (13–17), and to induce tissue factor in monocytes (18). Furthermore, microparticles can contribute to blood flow regulation. As shown using preparations from the T cell line CEM, microparticles can modulate the relaxation and dilatation of mouse aortic rings and mesenteric arteries by decreasing expression of nitric oxide synthase and by overexpression of caveolin 1 (19). Similarly, platelet-derived microparticles can stimulate the expression of cyclooxygenase 2 (COX-2) in endothelial cells, thereby inducing vasodilatation via an increased production of prostaglandins (20, 21).

Besides their effects on vascular smooth muscle cells in regulating the luminal diameter, microparticles can stimulate the proliferation of vascular smooth muscle cells by activating mitogen-activated protein kinase signaling pathways (22). Microparticles derived from HT1080 human fibrosarcoma and DU-145 human prostate carcinoma cells can stimulate endothelial cell migration, invasion, and tube formation in vitro; furthermore, microparticles can induce neovascularization in the chorioallantoic membrane assay in vivo (23), which suggests potentially important actions of microparticles on the vasculature, as well as in clotting.

In addition to their role as mediators of thrombosis, microparticles can function as transport vehicles between different cells. In this regard, microparticles can deliver cell surface proteins including receptors from one cell to another. These receptors can be integrated into the membrane of the acceptor cell and show function (24). As conduits of molecules between cells, microparticles can transfer not only proteins, but also lipids such as arachidonic acid (20, 21). This transfer provides a source of bioactive small molecules for promoting thrombosis, including endothelial activation.

As a concentrated source of immunologically active molecules, microparticles can serve as novel mediators of inflammation in conditions such as acute coronary syndromes (25, 26), sepsis (27), systemic inflammatory responses (28), and multiple sclerosis (29, 30). It is important to note that microparticles may play a role in the pathogenesis of rheumatic diseases such as RA (31), systemic vasculitis (32), and the antiphospholipid syndrome (33). The present review summarizes the current knowledge about the release, structure, and activity of microparticles in inflammation and their potential role as disease mediators that operate in the extracellular milieu and represent a middle ground between cell–cell interaction and stimulation by small molecule mediators.

Release of microparticles

While cultures of cells show a constitutive release of microparticles, apoptosis and activation appear to be the major events for the generation of microparticles and their release from cells. An increased release of cellular microparticles has been demonstrated upon stimulation of cells with various proinflammatory agents, such as the cytokines tumor necrosis factor α (TNFα) or interleukin-1 (IL-1); furthermore, stimulation with chemical compounds such as fMLP, phorbol ester, ionomycin, phorbol myristate acetate (PMA), concanavalin A, and the activated complement component C5a also increase the release of cellular microparticles (34–36).

In the formation of microparticles, reorganization of the cytoskeleton appears essential (Figure 2) (36–41). Mills et al demonstrated increased phosphorylation of myosin light-chain kinase (MLCK) during blebbing of apoptotic PC12 cells, a rat pheochromocytoma cell line (38). During this process, the contractile activity of myosin is stimulated through phosphorylation of myosin light-chains by MLCK, with this phosphorylation catalyzing the interaction of the myosin head with actin. This interaction enables the myosin ATPase to produce sliding forces (42). In apoptotic cells, a role of MLCK phosphorylation in the formation of microparticles is supported by the finding that inhibitors of MLCKs such as KT5926, ML-7, and ML-9 decrease the release of microparticles in serum-deprived PC12 cells. Clostridium botulinum toxin C3 transferase, an enzyme that inactivates Rho by ADP ribosylation, can also inhibit this process.

Figure 2.

Key role of Rho-associated kinase I (ROCK-I) in the release of microparticles from apoptotic cells. Upon induction of apoptosis, Rho becomes activated and exchanges GDP with GTP. GTP/Rho stimulates the activation of ROCK-I by promoting the cleavage of the C-terminal end of ROCK-I. Cleaved ROCK-I in turn activates myosin light-chain kinase (MLCK). Thereafter, MLCKs phosphorylate myosin light-chains (MLC), which stimulate the contractile activity of myosin. The phosphorylation (P) of myosin light-chains triggers the interaction of the myosin head with actin and enables the myosin ATPase to create movement between the actin and myosin filaments. This movement may cause force on the plasma membrane to cause detachment of the cytoskeleton from the membrane. At these sites, local bulges form, finally leading to the release of microparticles.

A study by Coleman et al identified the Rho-associated kinase I (ROCK-I) as a key element in the signaling cascade from Rho, through myosin light-chain phosphorylation, to the release of microparticles (39). In their elegant experiments, Coleman and coworkers demonstrated that ROCK-I is necessary for the release of microparticles from apoptotic cells. ROCK proteins bind to, and are activated by, GTP-bound Rho. Active ROCKs are important for the cytoskeletal arrangement including the phosphorylation of myosin light-chains and coupling of actin-myosin filaments to the plasma membrane. As shown using NIH3T3 fibroblasts, cleavage of the carboxy terminal regions of ROCK-I, leading to an activation of the amino terminal kinase domain, occurs in apoptotic but not in nonapoptotic controls. In these experiments, inhibition of ROCK-I activity by the small molecule inhibitor Y27632 reduced myosin light-chain phosphorylation and formation of microparticles. Furthermore, caspases contribute to the activation of ROCK-I since cleavage of ROCK-I and of microparticle release can be blocked by the caspase inhibitor z-VAD-fmk.

Several studies have now demonstrated that the formation of microparticles is a calcium-dependent process that can be blocked by chelating compounds such as EGTA (40, 43, 44). Interestingly, the calcium concentration is particularly high at sites of vesiculation. The effect of the intracellular calcium increase on the release of microparticles was studied in hepatocytes treated with tert-butyl hydroperoxide (40). In that study, electron microscopy showed a dissociation of the plasma membrane from the cytoskeleton at sites of blebbing. At these sites, no significant disarrangement of actin fibers was found, suggesting that the anchoring proteins connecting the plasma membrane with the cytoskeleton, rather than the cytoskeleton itself, might be modified. Indeed, degradation of talin and α-actinin, 2 proteins that link the cytoskeleton with the plasma membrane, was observed following treatment with tert-butyl hydroperoxide (40).

Calpain μ is a calcium-dependent cytosolic protease that cleaves talin and α-actinin (45). In a study by Miyoshi et al (40), calpain μ was found to be activated within the same time-frame as calcium activation. Furthermore, inhibition of calpain μ by chelation of calcium or, more specifically by calpeptin, prevented the degradation of talin and α-actinin and the release of microparticles. It is unlikely that the calpain pathway is the only mechanism for microparticle release, because calpeptin did not inhibit blebbing to the same extent as did chelation of calcium by EGTA, suggesting a role for other calcium-dependent processes. In platelets, the glycoprotein IIb-IIIa appears to be involved in the release of microparticles (46), since the formation of microparticles is reduced by blocking antibodies to glycoprotein IIb-IIIa and its ligand RGDS.

The formation of microparticles may not be a uniform process, with the release of microparticles differing quantitatively between cell types as well as between apoptotic and activated cells; the extent of release may also vary depending upon the inducing stimulus (35, 47, 48). Furthermore, the release of microparticles may vary qualitatively between apoptotic and activated cells. While blebbing may lead to the release of microparticles, it is possible that other membrane events can generate these structures. Thus, recent studies indicate that endothelial cells release phenotypically different microparticles during stimulation by TNFα as compared with apoptosis due to growth factor starvation (48). Similarly, constitutively expressed endothelial cell markers such as CD31 and CD105 are preferentially expressed on microparticles released from apoptotic cells, whereas microparticles from apoptotic cells stain more strongly for annexin V than do microparticles from TNFα-treated endothelial cells. These findings suggest heterogeneity in the nature of microparticles released from cells, despite overall similarity in size.

Composition of microparticles

The membrane of microparticles, which is derived from the plasma membrane of the parental cell, consists primarily of lipids and proteins. Depending on the cell type of origin and the mechanism of microparticle release, the composition of microparticles varies. During the budding process, the phospholipid asymmetry of the plasma membrane is lost, with microparticles exposing phospholipids on their outer membrane leaflet. Analysis of components of microparticles from the blood of healthy donors indicates that phosphatidylcholine comprises almost 60% of the lipids (49). Other quantitatively important lipids are sphingomyelin and phosphatidylethanolamine, which account for another 30% of the lipids. Low amounts of phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylethanolamine are also detected in particle preparations. In the blood of healthy donors, ∼75% of the microparticles are derived from platelets. Nevertheless, the composition of blood microparticles differs from that of platelet plasma membranes. For example, in platelet membranes, phosphatidylcholine and phosphatidylethanolamine each account for 30–35% of total lipids, and lysolipids are not detectable (49).

In a study on inflammatory arthritis, Fourcade and coworkers isolated microparticles from synovial fluids (50) and demonstrated that, in these microparticles, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and lysolipids were present in almost equal amounts (20–25% each), and that phosphatidylserine was detected only in low amounts. In view of the differences observed in these biochemical analyses, one can speculate that the lipid composition of microparticles varies depending on the cell type of origin, as well as the inducing process. In contrast to microparticles isolated from the blood, the majority of microparticles present in synovial fluids from patients with active arthritis are derived from inflammatory cells; in these fluids, platelet microparticles are found in low numbers (31). An alternative explanation for the varying composition of the microparticles may be that the inflammatory milieu in the arthritic joint triggers the release of microparticles with a different lipid composition, despite their origin from the same cell type.

On their surface, microparticles display surface proteins from their parental cells. As such, these antigens can identify the cell type from which the microparticles originate, e.g., CD42a for platelets or CD3 for T cells (31, 32, 47). Microparticles, however, can differ in the expression of cell surface molecules from their parental cells. This phenomenon has been analyzed in the most detail for erythrocytes. Microparticles derived from erythrocytes upon treatment with calcium and ionophore A23187 are enriched for glycosyl phosphatidylinositol–anchored proteins, such as acetylcholinesterase and decay accelerating factor (51). Erythrocyte-derived microparticles are also enriched for the membrane protein stomatin and for the proteins synexin and sorcin, which translocate from the cytosol to the plasma membrane upon calcium binding (52). Furthermore, the expression of surface molecules on microparticles may vary with activation state, with levels of platelet endothelial cell adhesion molecule 1 and E-selectin significantly higher on microparticles from activated endothelial cells than on those from resting cells (53).

In contrast to the characterization of surface molecules, little is known about the intracellular contents of microparticles. Thery et al demonstrated that microparticles from dendritic cells contain histones (54). Microparticles released by THP-1 monocytic cells upon activation of the ATP receptor P2X7 contain IL-1β (55). Based on the mechanisms of microparticle release, it is possible that cytosolic and even nuclear proteins from the parental cells are present within the microparticles. These internal molecules could contribute to biologic activity of the particles, although this has not been investigated extensively.

Microparticles as intercellular transporters

Among their proinflammatory activities, microparticles can transport biologically active compounds such as lipid mediators and cell surface receptors between different cells. Barry and coworkers demonstrated that microparticles can alter cellular functions through transcellular lipid metabolism (20, 21, 34). In those studies, platelet-derived microparticles stimulated the expression of COX-2 and the production of prostaglandins in endothelial cells. Further analyses revealed that the fatty acid fraction was responsible for the stimulatory effects. In particular, the induction was mimicked by arachidonic acid isolated from platelet-derived microparticles.

Recently, it has been shown that microparticles can mediate the intercellular transfer of cell surface receptors. The transferred receptors can be integrated into the plasma membrane of the acceptor cells, rendering them responsive to new stimuli. Horizontal transfer of proteins via microparticles could also play a role in the pathogenesis of human immunodeficiency virus (HIV) infection. Thus, Mack et al demonstrated that CCR5 can be released through microparticles from the surface of CCR5-expressing Chinese hamster ovarian cells and peripheral blood mononuclear cells (PBMCs) (24). After coincubation of CCR5+ microparticles with CCR5− PBMCs, monocytes or T cells, CCR5 was detectable on the surface of these cells as determined by flourescence-activated cell sorting. Stimulation with the CCR5 ligand RANTES (CCL5) induced a down-regulation of the transferred CCR5 in macrophages, suggesting a functional integration into the membrane. In addition, after transfer of CCR5 by microparticles, CCR5− PBMCs could be infected by a macrophage-tropic strain of HIV, which depends on CCR5 as a coreceptor (24). Along this line, microparticles derived from platelets and megakaryocytes can transfer CXCR4, the coreceptor for lymphotropic HIV strains, to CXCR4− cells, making these cells a potential target for the virus (56).

While receptor transfer provides a novel regulatory mechanism, the integration of receptors from microparticles into the plasma membrane of the acceptor cells may not lead to new function. For example, down-regulation of CCR5 was not observed in T cells exposed to microparticles upon stimulation with RANTES. This finding suggests that the functional integration of chemokine receptors may be limited to certain cell types. In addition, the transfer of cell surface molecules by microparticles has been demonstrated only for chemokine receptors. It remains unknown whether other cell surface proteins can be exchanged between different cells through this mechanism.

Microparticles and inflammation

As shown in in vitro studies, microparticles display a variety of proinflammatory activities that could contribute to the pathogenesis of rheumatic disease. Thus, these particles can promote adhesion and rolling of leukocytes, contain proinflammatory cytokines, and trigger the release of microparticles from various cell types in vitro. Further evidence for a potential role in disease pathogenesis comes from observations of increased numbers of microparticles during inflammatory states in vivo (27, 30, 32, 33, 57, 58).

Like damaged cells, microparticles could trigger inflammation by activating the complement cascade. The recognition unit of the classical complement pathway, C1q, binds to microparticles released from apoptotic Jurkat cells (59). C1q bound to microparticles can activate the classical complement pathway, as demonstrated by deposition of C3 and C4 on the surface of microparticles. The in vivo relevance of these results has been confirmed by the detection of a population of C1q-positive microparticles in human plasma. The deposition of early components of the classical complement pathway could trigger the typical proinflammatory effects of the complement pathway.

In their interactions with cells, platelet-derived microparticles can enhance the binding of neutrophils to other neutrophils prebound to the surface of a flow chamber (60). In these experiments, even when L-selectin is blocked, platelet-derived microparticles allow neutrophils to aggregate and adhere to prebound cells. The molecular mechanism for this phenomenon appears to be an interaction between P-selectin on platelet-derived microparticles and P-selectin–glycoprotein ligand 1 on neutrophils, since administration of blocking antibodies against these surface molecules abrogates the effects of platelet-derived microparticles. Furthermore, platelet-derived microparticles can promote cell–cell contact by inducing adhesion molecules.

Among the mechanisms for stimulating inflammation, microparticles can deliver arachidonic acid to other cells. Arachidonic acid from platelet-derived microparticles can increase the adhesion of monocytes to endothelial cells. The increased adhesion is due to stimulated expression of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells and of lymphocyte function–associated antigen 1 and macrophage antigen 1 in monocytes (21). Furthermore, microparticles derived from platelets or arachidonic acid isolated from these microparticles can increase chemotaxis of U937 monocytic cells. Both effects, the increased adhesion and chemotaxis, appear to be mediated by protein kinase C–dependent pathways, as demonstrated by the effects of blocking protein kinase C signaling by the inhibitor GF 109203X (21). The transfer of arachidonic acid also induces the expression of COX-2, but not COX-1, in endothelial cells and monocytes, thereby stimulating the production of prostaglandins (20, 34).

In addition to platelet-derived microparticles, microparticles released from other cell types can exert proinflammatory properties. Microparticles derived from fMLP-stimulated polymononuclear cells can induce the expression of IL-6 and monocyte chemotactic protein 1 (MCP-1) in endothelial cells (61, 62). In contrast, in those experiments, levels of TNFα, IL-1β, and platelet-derived growth factor remained unchanged, providing evidence against a nonspecific activation. Analyzing the mechanism for this stimulation, Mesri et al observed that blocking antibodies to the adhesion molecules β2 integrin and ICAM-1 or to TNFα did not reduce the induction of IL-6 and MCP-1. Microparticles also did not activate NF-κB or ERK-1 signaling pathways in endothelial cells. However, a sustained up-regulation of tyrosine phosphorylation of JNK1 was noted after stimulation with microparticles from polymononuclear cells (62).

With certain stimuli, cytokines might accumulate within microparticles as demonstrated for IL-1β (55). The secretion of IL-1β occurs via nonclassical pathways. First, Toll-like receptor (TLR) agonists such as endotoxins initiate the synthesis of the IL-1β precursor on microtubules rather than the endoplasmic reticulum. The colocalization of procaspase 1 with the IL-1β precursor into specialized secretory lysosomes is essential for the release of IL-1β, since agents that inhibit the accumulation of hydrolases into lysosomes also inhibit IL-1β secretion (63). The next step leading to secretion of IL-1β is conversion of the inactive procaspase 1 to active caspase 1 by a complex of proteins termed the “IL-1β inflammasome” (64). Active caspase 1 in turn converts the IL-1β precursor to biologically active IL-1β. Finally, lysosomal exocytosis and release of IL-1β are triggered by activation of P2X7 receptors either by ATP or by the small peptide LL37, which is released from activated neutrophils and epithelial cells (65–67). Stimulation of P2X7 receptors increases the intracellular calcium concentration, thereby activating phosphatidylcholine-specific phospholipase C (68).

MacKenzie et al demonstrated that the activation of P2X7 receptors by extracellular ATP also stimulates shedding of microparticles from THP-1 monocytic cells (55). Interestingly, IL-1β released upon the activation of the P2X7 receptors under these conditions preferentially appears within the microparticle fraction. Within the first 30 minutes after stimulation, levels of IL-1β were significantly higher in the microparticle fraction than in the microparticle-free supernatant. In addition, IL-1β appeared in the microparticle-free supernatant with a time lag of 2–10 minutes. Within the microparticle fraction, IL-1β was detectable only after treatment with the detergent triton, and its levels were not reduced by digestion of microparticles with trypsin, suggesting that IL-1β is inside the microparticles. These results suggest that upon activation of P2X7 receptors, active IL-1β can accumulate in microparticles for release (55). It is also unknown whether other cytokines utilize this mechanism.

Interestingly, a recent study suggests that microparticles can, under certain circumstances, exert antiinflammatory effects. In these experiments, microparticles derived from fMLP-stimulated polymononuclear cells up-regulated the expression of the antiinflammatory cytokine transforming growth factor β1 (TGFβ1) in macrophages (69). In contrast, the release of the proinflammatory cytokines TNFα, IL-8, and IL-10 induced by zymosan and lipopolysaccharide was reduced by the microparticles in a dose-dependent manner. Blocking the binding of phosphatidylserine exposed on the surface of microparticles to its receptors on macrophages inhibited the release of TNFα, but not of the other cytokines. In contrast, neutralizing antibodies to TGFβ blocked the release of TNFα, IL-8, and IL-10, suggesting that the antiinflammatory effects of microparticles are mediated by TGFβ as a second mediator.

There are several methodologic differences between the above-described study and others that might have influenced the results. Thus, in the study by Gasser and Schifferli (69), polymorphonuclear cells were stimulated for only 20 minutes, whereas in other studies, stimulation periods of several hours were used. By using the short stimulation period, Gasser and Schifferli may have selected for microparticles released early after stimulation, which might differ in composition from microparticles released at later time points. Furthermore, filter devices, instead of differential centrifugation, were used for microparticle isolation in their study. Because the morphology of the microparticles was analyzed by nonspecific staining with the amphiphilic linker dye PKH67, but not by antibody staining or by electron microscopy, the microparticles in that study may have differed from those used for other studies.

Microparticles may also exert antiinflammatory effects by the induction of apoptosis in immunocompetent cells. Jodo and colleagues showed that cells can release microparticles that bear Fas ligand (FasL) on their surface (70). Unlike soluble FasL, which is a relatively poor mediator of cytotoxicity, the efficacy of microparticle-associated FasL was similar to that of cellular FasL in killing B and T cell lines. In similar experiments, we demonstrated that T cell microparticles can induce apoptosis in RAW 264.7 macrophages, thereby triggering the release of microparticles from dying macrophages (47). It is possible that in those experiments the microparticle preparations used contained exosomes that bear FasL and promote cytotoxicity. In this regard, the proapoptotic effects of microparticles could provide a mechanism to control overwhelming inflammatory responses, although in the setting of malignancy, induction of apoptosis in killer T cells by FasL-bearing microparticles derived from tumor cells could lead to tumor escape (71).

Evidence for the proinflammatory effects of microparticles is bolstered by the detection of increased numbers of microparticles in inflammatory conditions in vivo. For instance, in type 1 diabetes, the number of endothelial cell microparticles is elevated (18). High levels of endothelial cell– and platelet-derived microparticles are also found in sera of patients with multiple sclerosis (30). Furthermore, an increase of microparticles derived from granulocytes occurs in patients with meningococcal sepsis (15), and microparticles derived from T helper cells are elevated in HIV patients (57). Microparticles appear to play an important role in the pathogenesis of coronary artery disease. Microparticles derived from monocytes and lymphocytes are found in atherosclerotic plaques (25), and endothelial cell– and platelet-derived microparticles are significantly increased in coronary artery disease, especially in patients with acute events (26).

Microparticles as novel players in the pathogenesis of rheumatic diseases

A potential role of microparticles in rheumatologic diseases was first suggested by Combes and colleagues (33) in the context of thrombosis. Those investigators showed that microparticles derived from TNFα-stimulated human vascular endothelial cells (HUVECs) decreased the clotting time up to 70% in a dose-dependent manner. In contrast, the clotting time was reduced maximally by only 20% with microparticles from unstimulated HUVEC cells. When factor VII–deficient plasma was used, there was only a small decrease in the clotting time after addition of HUVEC-derived microparticles, suggesting that the procoagulant activity of endothelial cell–derived microparticles is primarily mediated via the extrinsic coagulation pathway.

In view of the procoagulant properties of endothelial cell microparticles, Combes et al (33) hypothesized that the number of microparticles would be increased in patients at increased risk of thrombosis. Indeed, the levels of endothelial cell microparticles in patients with lupus anticoagulant were increased compared with those of healthy controls. Moreover, the microparticle count was higher in patients with thrombotic complications than in those without. Together, these findings are consistent with an effector role for microparticles in the disease setting.

In RA, microparticles released from different cell types have been implicated in pathogenesis. Knijff-Dutmer and colleagues (58) found a significant increase of platelet microparticles in the blood of patients with RA. In addition, the platelet-derived microparticle count was higher in patients with active disease, with the number of platelet-derived microparticles correlated with the RA disease activity as measured by the disease activity score in 28 joints. However, no correlation was observed between microparticle counts and the levels of C-reactive protein or the erythrocyte sedimentation rate, suggesting the contributions of other factors in determining cell numbers. These factors could relate to microparticle clearance as well as their origin from processes other than clearance.

The correlation between microparticle numbers and disease activity, however, was not confirmed by a study by Berckmans et al (31). Thus, those investigators failed to detect an increased number of microparticles derived from platelets in RA patients compared with controls. In fact, there was a trend toward lower numbers in arthritis patients. Although both Knijff-Dutmer et al (58) and Berckmans et al (31) used the same method of isolating microparticles and the same surface antigen (glyocoprotein IIIa) for identification, differences between the patient groups in these studies could explain the discordant result. These differences include the disease duration in the 2 studies. Furthermore, the mean erythrocyte sedimentation rate and the mean number of swollen and tender joints was higher, and the patients received fewer disease-modifying antirheumatic drugs in the study by Knijff-Dutmer and coworkers, suggesting that arthritis was more active.

In a study of microparticles from other cell types, Berckmans et al (31) did not find differences in the plasma levels of microparticles derived from T cells, B cells, monocytes, granulocytes, or erythrocytes between RA patients, patients with other forms of arthritis, and members of the healthy control group. Nevertheless, Berckmans et al found high numbers of microparticles in the synovial fluid of arthritis patients. In these experiments, most of the microparticles (>40%) were derived from macrophages, while smaller amounts were derived from T cells and granulocytes (20–25% each). Microparticles from B cells, platelets, and erythrocytes were present only in low numbers (<10% total). The numbers and ratios of microparticles were highly variable between individual patients. Tissue factor was detected on microparticles isolated from synovial fluid, and microparticles induced thrombin generation by a factor VII–dependent mechanism in some patients. On the basis of these findings, the authors speculated that microparticles could contribute to fibrin deposition in the joint, although levels in the periphery may not be elevated.

Another facet of microparticle activity in RA concerns the stimulatory activity of microparticles on fibroblasts. Recently, we studied the effects of microparticles derived from monocytes and T cells on synovial fibroblasts (35). Microparticles from both cell types induced the synthesis of matrix metalloproteinase 1 (MMP-1), MMP-3, MMP-9, and MMP-13 messenger RNA and protein in a dose-dependent manner in synovial fibroblasts, with increases up to 80-fold (Figure 3). This up-regulation was not due to a nonspecific activation, because no induction of MMP-2, MMP-14, cathepsin K, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, or TIMP-3 was observed. Microparticles stimulated the synthesis of not only MMPs but also the proinflammatory cytokines IL-6, IL-8, MCP-1, and MCP-2. Similar results were obtained with microparticles isolated from synovial fluid of patients with RA (72). These cytokines might attract more inflammatory cells to the joint and activate them, resulting in the release of more microparticles.

Figure 3.

Dose-dependent induction of matrix metalloproteinase 1 (MMP-1) by microparticles (MPs) from Jurkat cells. Cultured rheumatoid arthritis synovial fibroblasts (RASF), osteoarthritis synovial fibroblasts (OASF), normal synovial fibroblasts (NSF), and healthy dermal fibroblasts (HDF) were stimulated with increasing amounts of microparticles isolated from Jurkat cells. Strong induction of MMP-1 was observed in all types of fibroblasts. Values are the mean and SD. Co = control; SNCo = supernatant control. Adapted, with permission, from ref. 35.

According to this scenario in the pathogenesis of RA, microparticles might trigger a vicious circle of self-amplifying inflammation and destruction of cartilage and bone (Figure 4). Insights into the mechanism of this stimulation were provided by analysis of NF-kB. Using IκB–transfected fibroblasts and electromobility shift assays, we could demonstrate that NF-κB–dependent pathways contribute to the induction of MMPs by microparticles. Interestingly, neutralizing antibodies against TNFα, soluble TNF receptor, and IL-1 receptor antagonists did not reduce the induction of MMPs by microparticles, suggesting that the activation of synovial fibroblasts by microparticles was independent of TNFα and IL-1. If microparticles function as effectors in the joint, the induction of MMPs and cytokines by microparticles could contribute to the resistance of patients to biologics targeting TNFα and IL-1.

Figure 4.

Potential role of microparticles in the pathogenesis of rheumatoid arthritis. Microparticles derived from T cells and macrophages stimulate the release of proinflammatory and chemotactic cytokines from rheumatoid arthritis synovial fibroblasts in a dose-dependent manner. These cytokines attract even more mononuclear cells to the joint, thereby contributing to their activation and stimulating the shedding of more microparticles. The latter might further enhance the synthesis of cytokines by synovial fibroblasts. Microparticles derived from T cells and macrophages also stimulate the expression of matrix metalloproteinases in synovial fibroblasts in a dose-dependent manner. Therefore, microparticles could trigger a vicious circle of self-amplifying inflammation and destruction of cartilage and bone in rheumatoid arthritis.

These studies, however, do not define the mechanisms for stimulation by microparticles. While microparticles from 1 cell type can bind to another, studies have not yet defined whether activation is mediated by receptors at the surface, transfer of bioactive molecules into the membrane, or following internalization into the cell. Since some microparticles contain nucleic acids and others contain nuclear molecules, it is possible that TLRs are involved in stimulation events. The activation of NF-κB is consistent with this mechanism, although future experiments are needed to determine how microparticles activate fibroblasts, as well as other target cell populations.

For another group of immune-mediated diseases, Brogan et al (32) compared the number of microparticles in the blood of young patients with systemic vasculitis with the number of microparticles in the blood of patients with febrile diseases and healthy controls. In the majority of patients with vasculitis, Kawasaki disease or panarteritis nodosa was diagnosed; only a few patients with microscopic polyangitis and Wegener's granulomatosis were included in the study. The levels of endothelial cell microparticles positive for E-selectin or endoglin, but not endothelial cell microparticles positive for ICAM-1, were significantly increased in vasculitis patients compared with controls. No differences were observed between the different vasculitis subgroups. Besides microparticles derived from endothelial cells, the number of platelet-derived microparticles and the total number of microparticles were also higher. The number of endothelial cell–derived microparticles correlated positively with the Birmingham Vasculitis Activity Score. A significant positive correlation was also noted between the total number of microparticles, endothelial cell microparticles, and conventional acute-phase reactant markers such as the erythrocyte sedimentation rate and the C-reactive protein level.

Together, these results suggest that increased levels of microparticles are present in an increased number in patients with rheumatic disease, and that microparticles could function in disease pathogenesis, acting systemically on the vasculature as well as locally in the joint. Since cell death and activation are ubiquitous events at sites of inflammation, microparticle release could be a common occurrence, with these structures further stimulating inflammation and incorporating the roles of other cell types (e.g., T cell activation leading to fibroblast activation via transferred particles). Immunosupressive effects are also possible, since at least some types of microparticles can induce apoptosis. Similar to the effects of cytokines, the activity of microparticles may depend upon the milieu as well as the balance of activity for microparticles arising from different cells or cell types.

Summary and conclusion

As illustrated in Figure 5, microparticles represent novel signaling structures that can be generated during cell activation and apoptosis, 2 events fundamental to inflammation. These particles may serve to both induce and amplify inflammation and potentiate tissue damage in rheumatic disease. Furthermore, microparticles represent potentially useful biomarkers, since key cells in inflammation can be sampled from the blood, using cell surface markers to allow identification of individual cell types. Future studies will define the cell processes generating microparticles, the signaling pathways they induce, and their potential as targets for new therapies, based on either their formation or their triggering of downstream effects in inflammation. Once viewed as by-products of other cellular processes, microparticles are emerging as an exciting new element for information exchange during inflammation, with rheumatic disease as an important setting for their expression.

Figure 5.

Properties of microparticles.

Ancillary