Microparticles as mediators of cellular cross-talk in inflammatory disease

Microparticles are a heterogeneous population of membrane-coated vesicles which can be released from virtually all cell types during activation or apoptosis. Release occurs from the cell surface in an exogenous budding process involving local rearrangement of the cytoskeleton. Given their origin, these particles can be identified by staining for cell surface markers and annexin V. As shown in in vitro studies, microparticles may represent a novel subcellular element for intercellular communication in inflammation. Thus, microparticles can transfer chemokine receptors and arachidonic acid between cells, activate complement, promote leukocyte rolling and stimulate the release of pro-inflammatory mediators. Under certain conditions, however, microparticles may also exert anti-inflammatory properties by inducing immune cell apoptosis and the production of anti-inflammatory mediators. Microparticles may play an important role in the pathogenesis of rheumatologic diseases as evidenced by their elevation in diseases such as systemic sclerosis (SSc), systemic vasculitis and antiphospholipid antibody syndrome and correlation with clinical events. A role in inflammatory arthritis is suggested by the finding that leukocyte-derived microparticles induce the production of matrix metalloproteinases and cytokines by synovial fibroblasts. Together, these findings point to novel signaling pathways of cellular cross-talk that may operate along the spectrum of soluble cytokines and mediators of direct cell-cell contact.     

Differential mechanisms of microparticle transfer toB cells and monocytes: anti-inflammatory propertiesof microparticles

Microparticles are small vesicles released from the plasma membrane of various cell types independently of apoptosis or cell death, are transferred between cells, and carry membrane proteins from one cell to another. We have studied the mechanism of uptake of microparticles by monocytes and B cells. The transfer of microparticles to B cells was almost completely dependent on complement. Incubation of microparticles with serum resulted in opsonization of microparticles with the complement cleavage product iC3b. The subsequent transfer to B cells was mediated by the complement receptor CR2. The interaction between iC3b-opsonized microparticles and B cells reduced the activation of B cells as measured by expression of MHC class II, CD86 and CD25. In contrast, transfer of microparticles to monocytes was only partially complement dependent, but involved calcium and annexin V, and was found to change the cytokine profile of monocytes towards a reduced release of the pro-inflammatory cytokines GM-CSF and TNF-alpha and an increased release of the anti-inflammatory cytokine IL-10. These data show that microparticles are taken up by B cells and monocytes by different mechanisms and modulate the activation of monocytes and B cells towards an anti-inflammatory phenotype. Microparticles might be involved in counterbalancing pro-inflammatory signals arising from tissue injury or inflammation.     

The production of anti-inflammatory cytokines in whole blood by physico-chemical induction

Objective and design: Cytokines such as interleukin-1 (IL-1) play an important role in degenerative musculo-skeletal diseases, including osteoarthritis, and a multitude of inflammatory disorders. Agents that inhibit the action of such cytokines have a high therapeutic potential in such diseases. Here we describe a new method for enhancing the production of the interleukin-l receptor antagonist (IL-1Ra) and other anti-inflammatory cytokines in whole blood. Material and methods: Human venous blood was incubated in the presence of CrSO₄-treated glass beads. Serum was recovered and the concentrations of IL-1Ra and other relevant cytokines were measured by ELISA. Results: The interaction of the glass bead surface with cells in whole blood increased production of IL-1Ra and anti-inflammatory cytokines. Removal of the beads and centrifugation generated a serum preparation enriched in anti-inflammatory cytokines. This preparation is of therapeutic value in treating various inflammatory and degenerative disorders. Conclusions: The increased de novo production of anti-inflammatory cytokines by a direct physico-chemical induction of whole blood in the Orthokin system is feasible and offers an alternative, novel approach to treating mild to moderate OA and other orthopaedic conditions such as degenerative spine diseases.Received 20 December 2002; returned for revision 25 March 2003; accepted by J. Skotnicki 1 June 2003     

Mechanisms of brain-mediated systemic anti-inflammatory syndrome causing immunodepression

Overwhelming inflammatory immune response can result in systemic inflammation and septic shock. To prevent excessive and deleterious action of proinflammatory cytokines after they have produced their initial beneficial effects, the immune system can release several anti-inflammatory mediators, including interleukin-10, interleukin-1 receptor antagonist, and soluble tumor necrosis factor receptors, thus initiating a compensatory anti-inflammatory response syndrome. However, in vivo the delicate balance between pro- and anti-inflammatory responses is additionally controlled by the central nervous system. Therefore, proinflammatory cytokines stimulate the hypothalamic-pituitary-adrenal axis and enhance sympathetic nerve system activity. The mediators of these neuroimmune pathways can again suppress immune cell functions to control systemic inflammation. The question is, however, what happens if the immunoinhibitory CNS pathways are activated without systemic inflammation? This can result from production of cytokines in the brain following infection, injury, or ischemia or in response to various stressors (e.g., life events, depression, anxiety) or directly from brainstem irritation. The answer is that this may generate a brain-mediated immunodepression. Many animal and clinical studies have demonstrated a stress and brain cytokine mediated decrease in the cellular immune response at the lymphocyte level. More recently, the importance of monocytes in systemic immunocapacity has been shown. Monocytic inactivation with decreased capability of antigen presentation and depressed secretion of proinflammatory cytokines increases the risk of infectious complications. Interestingly, cytokines in the brain and other stressors can also generate systemic immunodepression at the monocyte level. In this scenario the catecholamine-induced release of the potent anti-inflammatory cytokine interleukin-10 is a newly discovered mechanism of the brain-mediated monocyte deactivation in addition to the "well known" immunosuppressive action of glucocorticoids. Furthermore, other neuropeptides such as alpha-melanocyte-stimulating hormone and beta-endorphin which can be released in stressful situations have also inhibitory effects on immune cells. Thus mediators of the CNS are implicated in the regulation of immune functions and may play a role in both conditioning the host's response to endogenous or exogenous stimuli and generating a "brain-mediated" immunodepression.     

微粒在血栓形成中的研究进展

血栓形成是多种疾病的常见并发症,如动脉粥样硬化、自身免疫性疾病、癌症等,是引起患者死亡的重要原因,严重影响了患者的生存质量.因此对于血栓形成的相关研究始终是临床研究的重点.微粒(MPs)是外周血中游离的膜性小囊泡,当细胞应激(活化或凋亡)时,从细胞膜上脱落下来.微粒(MPs)不但携带细胞的表面标志物,而且其携带的细胞表面受体能够调控细胞间的信息传递,从而参与疾病的发生发展.随着对MPs的深入研究,发现MPs在血栓的形成中发挥着重要的生物学作用.因此研究两者之间的关系可能为预防和治疗血栓提供新的思路.     

Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome

Microparticles are membrane vesicles that are released during cell activation and apoptosis. Elevated levels of microparticles occur in many cardiovascular diseases; therefore, we characterized circulating microparticles from both metabolic syndrome (MS) patients and healthy patients. We evaluated microparticle effects on endothelial function; however, links between circulating microparticles and endothelial dysfunction have not yet been demonstrated. Circulating microparticles and their cellular origins were examined by flow cytometry of blood samples from patients and healthy subjects. Microparticles were used either to treat human endothelial cells in vitro or to assess endothelium function in mice after intravenous injection. MS patients had increased circulating levels of microparticles compared with healthy patients, including microparticles from platelet, endothelial, erythrocyte, and procoagulant origins. In vitro treatment of endothelial cells with microparticles from MS patients reduced both nitric oxide (NO) and superoxide anion production, resulting in protein tyrosine nitration. These effects were associated with enhanced phosphorylation of endothelial NO synthase at the site of inhibition. The reduction of O₂⁻ was linked to both reduced expression of p47^phox of NADPH oxidase and overexpression of extracellular superoxide dismutase. The decrease in NO production was triggered by nonplatelet-derived microparticles. In vivo injection of MS microparticles into mice impaired endothelium-dependent relaxation and decreased endothelial NO synthase expression. These data provide evidence that circulating microparticles from MS patients influence endothelial dysfunction.     

Determination of the size distribution of blood microparticles directly in plasma using atomic force microscopy and microfluidics

Microparticles, also known as microvesicles, found in blood plasma, urine, and most other body fluids, may serve as valuable biomarkers of diseases such as cardiovascular diseases, systemic inflammatory disease, thrombosis, and cancer. Unfortunately, the detection and quantification of microparticles are hampered by the microscopic size of these particles and their relatively low abundance in blood plasma. The use of a combination of microfluidics and atomic force microscopy to detect microparticles in blood plasma circumvents both problems. In this study, capture of a specific subset of microparticles directly from blood plasma on antibody-coated mica surface is demonstrated. The described method excludes isolation and washing steps to prepare microparticles, improves the detection sensitivity, and yields the size distribution of the captured particles. The majority of the captured particles have a size ranging from 30 to 90 nm, which is in good agreement with prior results obtained with microparticles immediately isolated from fresh plasma. Furthermore, the qualitative shape of the size distribution of microparticles is shown not to be affected by high-speed centrifugation or the use of the microfluidic circuit, demonstrating the relative stable nature of microparticles ex vivo.     

Dietary microparticles implicated in Crohn’s disease can impair macrophage phagocytic activity and act as adjuvants in the presence of bacterial stimuli

Objective and design: Western diets regularly expose the gastrointestinal tract (GI) to large quantities ( > 10¹²/day) of man-made, submicron-sized, particles derived from food additives and excipients. These are taken up by M cells, accumulate in gut macrophages, and may influence the aetiology of inflammatory bowel diseases (IBD). Materials: We investigated the effects of common dietary microparticles on the function of macrophages from healthy donors or active Crohn’s disease (CD) patients. Methods: Macrophages were incubated for 24 h with microparticles before being assayed for cytokine production and phagocytic activity. Results: Microparticles alone were non-stimulatory but, in the presence of bacterial antigens such as LPS, they could act as adjuvants to induce potent cytokine responses. Uptake of high concentrations of microparticles also impaired macrophage phagocytic capacity – but not their ability – to take up 2μM fluorescent beads. Conclusions: While dietary microparticles alone have limited effects on basic macrophage functions, their ability to act as adjuvants could aggravate ongoing inflammatory responses towards bacterial antigens in the GI tract. Received 26 January 2007; returned for revision 27 March 2007; accepted by I. Ahnfelt-R?nne 19 April 2007     

Negative regulation of TLR signaling in myeloid cells—implications for autoimmune diseases

Toll-like receptors (TLR) are transmembrane pattern recognition receptors that recognize microbial ligands and signal for production of inflammatory cytokines and type I interferon in macrophages and dendritic cells (DC). Whereas TLR-induced inflammatory mediators are required for pathogen clearance, many are toxic to the host and can cause pathological inflammation when over-produced. This is demonstrated by the role of TLR-induced cytokines in autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus. Because of the potent effects of TLR-induced cytokines, we have diverse mechanisms to dampen TLR signaling. Here, we highlight three pathways that participate in inhibition of TLR responses in macrophages and DC, and their implications in autoimmunity; A20, encoded by the TNFAIP3 gene, Lyp encoded by the PTPN22 gene, and the BCAP/PI3K pathway. We present new findings that Lyp promotes TLR responses in primary human monocytes and that the autoimmunity risk Lyp620W variant is more effective at promoting TLR-induced interleukin-6 than the non-risk Lyp620R protein. This suggests that Lyp serves to downregulate a TLR inhibitory pathway in monocytes, and we propose that Lyp inhibits the TREM2/DAP12 inhibitory pathway. Overall, these pathways demonstrate distinct mechanisms of negative regulation of TLR responses, and all impact autoimmune disease pathogenesis and treatment.     

Allergy therapy: the therapeutic potential of targeting sphingosine kinase signalling in mast cells

Mast cell activation is a central event in allergic diseases, and investigating the signalling pathways triggered during mast cell activation may lead to the discovery of novel therapeutic targets. Mast cells can be activated by a multitude of stimuli including antibodies/antigen, cytokines/chemokines and neuropeptides, resulting in a variety of responses including the immediate release of potent inflammatory mediators. Moreover, recent data suggest that mast cell-mediated responses are also influenced by the differential sphingolipids/sphingosine to sphingosine-1-phosphate ratio. The importance of sphingolipids as potent biological mediators of both intracellular and extracellular responses is being increasingly recognized and accepted; it is now appreciated that activation of mast cells, via the high-affinity IgE-receptor (FcepsilonRI) leads to the activation of sphingosine kinases (SphK), resulting in increased formation of sphingosine-1-phosphate. Furthermore, FcepsilonRI activates SphK-dependent calcium mobilization in mast cells, leading to degranulation, cytokine, and eicosanoid production, and chemotaxis. In the past two years a critical role for SphK in allergic responses in vivo has emerged. In this review, I focus on the current understanding of the role of sphingosine kinases during mast cell signalling in vitro and their role during hypersensitivity responses in vivo, and discuss the potential of these enzymes as novel therapeutic targets to treat allergic diseases.     

Microparticles as a source of extracellular DNA

Microparticles are small membrane-bound vesicles that display pro-inflammatory and pro-thrombotic activities important in the pathogenesis of a wide variety of diseases. These particles are released from activated and dying cells and incorporate nuclear and cytoplasmic molecules for extracellular export. Of these molecules, DNA is a central autoantigen in systemic lupus erythematosus (SLE). As studies in our laboratory show, DNA occurs prominently in microparticles, translocating into these structures during apoptotic cell death. This DNA is antigenically active and can bind to lupus anti-DNA autoantibodies. These findings suggest that microparticles are an important source of extracellular DNA to serve as an autoantigen and autoadjuvant in SLE.     

Rapid shedding of proinflammatory microparticles by human mononuclear cells exposed to cigarette smoke is dependent on Ca2+ mobilization

Objectives

Microparticles are membrane vesicles shed by cells upon activation and apoptosis. Agonists capable of inducing microparticle generation include cytokines, bacterial products, P-selectin, histamine. Cigarette smoke extract has also been recognized as an agonist involved in microparticle generation with an apoptosis-dependent mechanism. We investigated the possibility that cigarette smoke extract induces the rapid generation of proinflammatory microparticles by human mononuclear cells with a calcium-dependent mechanism.

Materials and methods

Human mononuclear cells were exposed to cigarette smoke extract. [Ca²⁺]_i mobilization was assessed with the fluorescent probe Fluo-4 NW. Microparticles were quantified with a prothrombinase assay and by flow cytometry. Normal human bronchial epithelial cells and A549 alveolar cells were incubated with cigarette smoke extract-induced microparticles and the generation of ICAM-1, IL-8, and MCP-1 was assessed by ELISA.

Results

Exposure to cigarette smoke extract induced a rapid increase in [Ca²⁺]_i mobilization. Microparticle generation was also increased. EGTA, verapamil and the calmodulin inhibitor, W-7, inhibited microparticle generation. Incubation of lung epithelial cells with cigarette smoke extract-induced microparticles increased the expression of proinflammatory mediators.

Conclusions

Exposure of mononuclear cells to cigarette smoke extract causes a rapid shedding of microparticles with a proinflammatory potential that might add to the mechanisms of disease from tobacco use.     

Anti-Inflammatory Mechanism of a Folk Herbal Medicine,Duchesnea indica (Andr) Focke at RAW264.7 Cell Line

This study is to explore the anti-inflammatory mechanism of the ethanol extract of Duchesnea indica (Andr) Focke. An inflammatory cellular model was established by addition of lipopolysaccharide (LPS) on RAW264.7 cell line. The cellular secretion of TNF-α, IL-1β, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-α, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, and activation of NF-κB were assayed by ELISA, the Griess method, real-time quantitative PCR, and Western blot and immunocytochemistry method, respectively. The ethanol extract of D. indica not only reduced production of pro-inflammatory cytokines and mediators and blocked NF-κB activation, but also slightly promoted release of the anti-inflammatory mediator HO-1 and suppressed IL-10 secretion. In conclusion, the anti-inflammatory effects of the extract of D. indica are attributed to the suppression of pro-inflammatory cytokines and mediators by blocking NF-κB activation. The extract of D. indica can also slightly promote HO-1 production to reduce inflammation.     

Anti-inflammatory Mechanism of Rungia pectinata (Linn.) Nees

This study is to explore the inner anti-inflammatory mechanism of the ethanol extract of Rungia pectinata (Linn.) Nees. As a result, the ethanol extract of Rungia pectinata (Linn.) Nees could not only strongly reduce production of pro-inflammatory cytokines and mediators via blocking NF-κB activation but slightly promote release of anti-inflammatory mediator HO-1 and suppress IL-10 secretion. In conclusion, compared to Dexamethasone, Rungia pectinata (Linn.) Nees has not only similar effects on antagonizing pro-inflammatory mediators and cytokines but also mild effects on promoting production of anti-inflammatory mediators.     

Microparticles and exosomes: impact on normal and complicated pregnancy

Eukaryotic cells release vesicles into their environment by membrane shedding (ectosomes or microparticles) and secretion (exosomes). Microparticles and exosomes occur commonly in vitro and in vivo. The occurrence, composition and function(s) of these vesicles change during disease (progression). During the last decade, the scientific and clinical interest increased tremendously. Evidence is accumulating that microparticles and exosomes may be of pathophysiological relevance in autoimmune, cardiovascular and thromboembolic diseases, as well as inflammatory and infectious disorders. In this review, we will summarize the discovery, biology, structure and function of microparticles and exosomes, and discuss their (patho-) physiological role during normal and complicated pregnancy.     

The immune response to Prevotella bacteria in chronic inflammatory disease

The microbiota plays a central role in human health and disease by shaping immune development, immune responses and metabolism, and by protecting from invading pathogens. Technical advances that allow comprehensive characterization of microbial communities by genetic sequencing have sparked the hunt for disease‐modulating bacteria. Emerging studies in humans have linked the increased abundance of Prevotella species at mucosal sites to localized and systemic disease, including periodontitis, bacterial vaginosis, rheumatoid arthritis, metabolic disorders and low‐grade systemic inflammation. Intriguingly, Prevotella abundance is reduced within the lung microbiota of patients with asthma and chronic obstructive pulmonary disease. Increased Prevotella abundance is associated with augmented T helper type 17 (Th17) ‐mediated mucosal inflammation, which is in line with the marked capacity of Prevotella in driving Th17 immune responses in vitro. Studies indicate that Prevotella predominantly activate Toll‐like receptor 2, leading to production of Th17‐polarizing cytokines by antigen‐presenting cells, including interleukin‐23 (IL‐23) and IL‐1. Furthermore, Prevotella stimulate epithelial cells to produce IL‐8, IL‐6 and CCL20, which can promote mucosal Th17 immune responses and neutrophil recruitment. Prevotella‐mediated mucosal inflammation leads to systemic dissemination of inflammatory mediators, bacteria and bacterial products, which in turn may affect systemic disease outcomes. Studies in mice support a causal role of Prevotella as colonization experiments promote clinical and inflammatory features of human disease. When compared with strict commensal bacteria, Prevotella exhibit increased inflammatory properties, as demonstrated by augmented release of inflammatory mediators from immune cells and various stromal cells. These findings indicate that some Prevotella strains may be clinically important pathobionts that can participate in human disease by promoting chronic inflammation.     

循环微粒(细胞外囊泡)在心血管外科领域的研究进展

循环微粒(细胞外囊泡)在血管调节、炎症、凝血、细胞增殖与凋亡等生物过程中具有细胞间传递信息的功能。所含成分复杂多样,与心血管疾病的发生发展过程联系紧密。循环微粒(细胞外囊泡)未来可以作为反映凝血功能、炎症反应、组织器官损伤的临床标志物,或者是调控血管稳态、纠正凝血、改善内环境、保护组织器官功能的临床治疗靶点,具有重要的研究意义。本文总结循环微粒(细胞外囊泡)在心血管外科领域的相关研究进展,讨论循环微粒(细胞外囊泡)在相关疾病过程中所起的作用,及其在相关疾病诊断与治疗当中的研究及应用前景。     

Anti-inflammatory Effects of Interleukin-4, Interleukin-10, and Transforming Growth Factor-β on Human Placental Cells In Vitro

PROBLEM: To determine the effects of anti-inflammatory cytokines on the production of inflammatory mediators by placental cells. METHOD OF STUDY: Cells from term human placentas were isolated and cultured in vitro in the presence of anti-inflammatory cytokines interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-β. Their effects on the production of IL-8 and prostaglandin E₂ (PGE₂) were investigated under basal conditions and after stimulation with IL-1β and tumor necrosis factor (TNF)-α. RESULTS: Both IL-10 and IL-4 inhibited IL-1β- and TNF-α-induced PGE₂ production but had no significant effects on the production of PGE₂ under basal conditions. TGF-β₁ was without effect in stimulated cells, whereas under basal conditions TGF-β₁ stimulated PGE₂ production. Similar trends were seen for IL-8 production, with the exceptions that TGF-β₁ decreased the TNF-α-induced production and IL-4 decreased basal IL-8 production. CONCLUSIONS: The anti-inflammatory effects shown by IL-4, IL-10, and (to lesser extent) TGF-β may play a role in ameliorating the potentially harmful effects of pro-inflammatory mediators in the feto-placental unit.     

Pivotal role of PGE2 and IL-10 in the cross-regulation of dendritic cell-derived inflammatory mediators

Exposure to pathogens induces antigen-presenting cells （APC） such as macrophages and dendritic cells （DC） to produce various endogenous mediators, including arachidonic acid （AA）-derived eicosanoids, cytokines, and nitric oxide （NO）. Many secreted products of activated APC can act by themselves in an autocrine manner and modulate their function. Moreover, the cross-interaction between endogenous bioactive molecules regulates the function of professional APC with important consequences for their ability to activate and sustain immune and inflammatory responses, and to regulate immune homeostasis. Although neglected for many years when compared to their role in cardiovascular homeostasis, cancer and inflammation, the importance of eicosanoids in immunology is becoming more defined. The role of prostaglandin （PG） E2 （PGE2）, one of the best known and most well studied eicosanoids, is of particular interest. It modulates the activities of professional DC by acting on their differentiation, maturation and their ability to secrete cytokines. Uniquely among haematopoietic cytokines, interleukin-10 （IL-10） is a pleiotropic molecule that displays both immunostimulatory and immunoregulatory activities. IL-10 has attached much attention because of its anti-inflammatory properties. It modulates expression of cytokines, soluble mediators and cell surface molecules by cells of myeloid origin, particularly macrophages and DC. We previously reported that PGE2 is a potent inducer of IL-10 in bone marrow-derived DC （BM-DC）, and PGE2-induced IL-10 is a key regulator of the BM-DC pro-inflammatory phenotype. BM-DC may be considered as an important model to study complex interactions between endogenous mediators, and autocrine IL-10 plays a pivotal role in the crossregulation of AA-derived lipid mediators, cytokines, and NO, with critical effects on immune and inflammatory responses.     

T helper type 2-polarized invariant natural killer T cells reduce disease severity in acute intra-abdominal sepsis

Sepsis is characterized by a severe systemic inflammatory response to infection that is associated with high morbidity and mortality despite optimal care. Invariant natural killer T (iNK T) cells are potent regulatory lymphocytes that can produce pro- and/or anti-inflammatory cytokines, thus shaping the course and nature of immune responses; however, little is known about their role in sepsis. We demonstrate here that patients with sepsis/severe sepsis have significantly elevated proportions of iNK T cells in their peripheral blood (as a percentage of their circulating T cells) compared to non-septic patients. We therefore investigated the role of iNK T cells in a mouse model of intra-abdominal sepsis (IAS). Our data show that iNK T cells are pathogenic in IAS, and that T helper type 2 (Th2) polarization of iNK T cells using the synthetic glycolipid OCH significantly reduces mortality from IAS. This reduction in mortality is associated with the systemic elevation of the anti-inflammatory cytokine interleukin (IL)-13 and reduction of several proinflammatory cytokines within the spleen, notably interleukin (IL)-17. Finally, we show that treatment of sepsis with OCH in mice is accompanied by significantly reduced apoptosis of splenic T and B lymphocytes and macrophages, but not natural killer cells. We propose that modulation of iNK T cell responses towards a Th2 phenotype may be an effective therapeutic strategy in early sepsis.