On activation, human neutrophils release microparticles, called ectosomes, directly from the cell surface membrane. Microparticles from platelets, endothelial cells, and monocytes were reported to support coagulation or to modulate vascular homeostasis by activating monocytes as well as endothelial cells. We find that neutrophil ectosomes have no proinflammatory activity on human macrophages as assessed by the release of interleukin 8 (IL-8) and tumor necrosis factor ␣ (TNF␣). On the contrary, ectosomes increase the release of transforming growth factor ␤1 (TGF␤1), suggesting that ectosomes down-modulate cellular activation in macrophages. Polymorphonuclear neutrophil (PMN) ectosomes are able to block inflammatory response of macrophages to zymosan and lipopolysaccharide (LPS). We show that an early-phase TGF␤1 secretion and the exposure of phosphatidylserine on the surface of ectosomes independently contribute to this effect. IntroductionPolymorphonuclear neutrophils (PMNs) constitute the bulk of the leukocytes found in our blood. PMNs ingest and eventually kill invading pathogens by means of potent antimicrobial agents released during the process of degranulation. Because this microbicidal weaponry largely lacks specificity, it can lead to severe tissue damage if not controlled or secluded adequately from surrounding tissue. 1 At the time of degranulation, activated PMNs release small microvesicles directly from the cell surface membrane. Many eukaryotic cells, including tumor cells, release vesicles by ectocytosis (ectosomes), either spontaneously or in response to various stimuli. [2][3][4][5][6][7][8][9][10][11][12][13][14] Data on the function(s) of ectosomes have accumulated. 6,7,9,11,[15][16][17] So far, ectosomes have been associated with procoagulant and proinflammatory effects. Ectosomes derived from endothelial cells have been described to induce procoagulant activity in monocytic cells, 18 whereas ectosomes from platelets and monocytes were shown to directly promote hemostasis and induce inflammation by activating endothelial cells. 9,11 MacKenzie et al 6 described monocyte-derived ectosomes as being important proinflammatory agents by mediating the rapid secretion of interleukin 1␤ (IL-1␤).We recently described characteristics and molecular properties of ectosomes released by human PMNs. 19,20 Importantly for the work presented herein, PMN ectosomes expose phosphatidylserine on their outer membrane leaflet, and all ectosome preparations used in our assays are devoid of any cellular debris or apoptotic cells/bodies, as shown by electron microscopy. 20 Here, we show that PMN-derived ectosomes, unexpectedly, feature immunosuppressive/anti-inflammatory functions. Challenging a linear model of induction and resolution of inflammation, our data suggest a more complex balance of proinflammatory and anti-inflammatory events initiated in a parallel manner. Material and methods Antibodies and reagentsZymosan A from Saccharomyces cerevisiae, cytochalasin D, actinomycin D, formyl-methionine-leucine-phenylalan...

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Abstract. Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types, that has been shown to be involved in cell-cell adhesion and expressed during cellular differentiation in vitro. To assess the suggested participation of clusterin in these processes in vivo, we have cloned the cDNA encoding murine clusterin and studied the cellular distribution of clusterin mRNA during murine embryogenesis. Sequence analysis of the cDNA encoding murine clusterin revealed 92 and 75 % sequence identity with the rat and human cDNAs, respectively, and conservation of the predicted structural features which include o~-helical regions and heparin-binding domains. From 12.5 d of development onwards, the clusterin gene is widely expressed in developing epithelia, and selectively localized within the differentiating cell layers of tissues such as the developing skin, tooth, and duodenum where proliferating and differentiating compartments are readily distinguished. In addition, transient and localized clusterin gene expression was detected in certain morphogenetically active epithelia. In the lung, abundant gene transcripts were detected in cuboidal epithelial cells of the terminal lung buds during branching morphogenesis, and in the kidney, clusterin gene expression in the epithelial cells of comma and S-shaped bodies coincided with the process of polarization. Our results demonstrate the in vivo expression of the clusterin gene by differentiating epithelial cells during murine embryogenesis, and provide novel evidence suggesting that clusterin may be involved in the differentiation and morphogenesis of certain epithelia.

SummaryComplement receptor 1 (CR1) is present on erythrocytes (E-CR1), various leucocytes, and renal glomerular epithelial cells (podocytes). In addition, plasma contains a soluble form of CR1 (sCR1) By using a specific ELISA, CR1 was detected in the urine (uCR1) of normal individuals (excretion rate in 12 subjects, 3.12 + 1.15 #g/24 h). Contrary to sCR1, uCR1 was pelleted by centrifugation at 200,000 g for 60 min. Analysis by sucrose density gradient ultracentrifugation showed that uCR1 was sedimenting in fractions larger than 19 S, whereas sCR1 was found as expected in fractions smaller than 19 S. The addition of detergents reduced the apparent size of uCR1 to that of sCR1. After gel filtration on Sephacryl-300 of normal urine, the fractions containing uCR1 were found to be enriched in cholesterol and phospholipids. The membrane-association of uCR1 was demonstrated by analyzing immunoafhnity purified uCR1 by electron microscopy which revealed membrane-bound vesicles. The apparent molecular mass of uCR1 was 15 kD larger than E-Clkl and sCR1 when assessed by SDS-PAGE and immunoblotting. This difference in size could not be explained on the basis of glycosylation only, since pretreatment with N-glycosidase F reduced the size of all forms of CR1; however, the difference in regular molecular mass was not abrogated. The structural alleles described for E-CR1 were also found for uCR1. The urine of patients who had undergone renal transplantation contained alleles of uCR1 which were discordant with E-CR1 in 7 of 11 individuals, indicating that uCR1 originated from the kidney, uCR1 was shown to bind C3b-coated immune complexes, suggesting that the function of CR1 was not destroyed in urine. A decrease in uCR1 excretion was observed in 3 of 10 patients with systemic lupus erythematosus, corresponding to the three who had severe proliferative nephritis, and in three of three patients with focal sclerosis, but not in six other patients with proteinuria. Taken together, these data suggest that glomerular podocytes release CRl-coated vesicles into the urine. The function of this release remains to be defined, but it may be used as a marker for podocyte injury.