Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties

Abstract

Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions. Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions. The emerging notion that cells can communicate by packaged information represents a major shift in our understanding of cell-to-cell interaction in complex settings including inflammation (1Norling L.V. Spite M. Yang R. Flower R.J. Perretti M. Serhan C.N. Cutting edge: Humanized nano-proresolving medicines mimic inflammation-resolution and enhance wound healing.J. Immunol. 2011; 186: 5543-5547Crossref PubMed Scopus (158) Google Scholar). Packaging of mediators (irrespective of their chemical nature) in structures that can be transported through the vascular and lymphatic systems might avoid their rapid dilution and removal by biological fluids and allow the target cell or tissue to receive a biologically relevant amount of a given molecule. As an example, TNF-α produced by mast cells in the mouse paw can reach the lymph nodes unmodified, wrapped up in small structures or vesicles (2Kunder C.A. St John A.L. Li G. Leong K.W. Berwin B. Staats H.F. Abraham S.N. Mast cell-derived particles deliver peripheral signals to remote lymph nodes.J. Exp. Med. 2009; 206: 2455-2467Crossref PubMed Scopus (140) Google Scholar). In this respect, the last few years have witnessed augmented understanding in microparticle function. Described over 50 years ago (reviewed in (3Lynch S.F. Ludlam C.A. Plasma microparticles and vascular disorders.Br J. Haematol. 2007; 137: 36-48PubMed Google Scholar, 4Beyer C. Pisetsky D.S. The role of microparticles in the pathogenesis of rheumatic diseases.Nat. Rev. Rheumatol. 2010; 6: 21-29Crossref PubMed Scopus (214) Google Scholar), microparticles are heterogeneous in nature with their size varying between 0.2 and 1.0 μm, and are characterized by an outer membrane composed of a phospholipid bilayer and cell surface proteins. The mechanism of microparticle production is not fully understood, though it may follow processes not dissimilar from those observed in apoptosis, involving membrane detachment from the anchoring cytoskeleton and loss of membrane symmetry, which leads to exposure of negatively charged phospholipids (5Wang G.J. Liu Y. Qin A. Shah S.V. Deng Z.B. Xiang X. Cheng Z. Liu C. Wang J. Zhang L. Grizzle W.E. Zhang H.G. Thymus exosomes-like particles induce regulatory T cells.J. Immunol. 2008; 181: 5242-5248Crossref PubMed Scopus (118) Google Scholar, 6Montes M. Jaensson E.A. Orozco A.F. Lewis D.E. Corry D.B. A general method for bead-enhanced by Immunol. 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G. of on inflammatory in Immunol. 2009; PubMed Scopus Google Scholar). the of these and information on patients microparticles microparticle volunteer in a from two of were in as described J. L.V. Perretti M. Annexin the rapid of 2008; PubMed Scopus Google and in for of by and were to with an from the were on a and with a in in in and in A in and in The to a R. J. M. A novel method of the 2008; PubMed Scopus Google Scholar). were from a over the on the in the with were by and to human protein and of as and of as The for the and 1.0 for were to the and to and protein to the in the the were and and were be as by the A. R. to the of by and PubMed Scopus Google Scholar). were be A. R. A for proteins by PubMed Scopus Google with two and a of of the for proteins were for and protein with were as of a of proteins by analysis through from microparticles with shock 70 kDa protein or by a with either an or were an and on the of the microparticle microparticles were in containing and and incubated with either mouse or or relevant for and or and either or microparticle cell surface protein a an and of the or these and relevant were in In microparticles were incubated with the or relevant for and analysis with a abundance by the for of the proteins of from the for the relevant In protein levels on neutrophil microparticles in plasma from healthy and septic with exudates from skin were from both and were in the of for to with or and in the of a for and microparticles were in and incubated with or for These were incubated with A in the of a to stimulation with or or microparticles were in to stimulation with with or a levels were a or microparticles were in that through a 0.2 or and the of toward of a of the chemotactic or a were to the of a with and the microparticle containing to the the the of the and the membrane with the number of microparticles in the a as a of the the microparticle were to in and with or microparticles for and an and microparticle were for this determined on of and on the were to the and were by that a on the the is to be a and a by from the the of the were and expressed as the were to be the in either of the that for the J. L. A. J. A. R. J. M. J. an for the analysis of and with 2010; PubMed Scopus Google Scholar). are in the with number of the were by with of and the were to the expression levels and and of by including a expression were as as Functional of proteins and the differentially expressed were A. 6: protein and with of biological 2007; PubMed Scopus Google The the or to were The two distinct gene expression in this cells either with or were with the gene expression profiles from small in the of the J. J. A. M. G. R. The to small and PubMed Scopus Google The between the gene expression profiles of and those in the is determined by the from to the that a a gene expression that is to potentially be to novel of we on the analysis of the a The with the were were in and are expressed as were determined analysis of or as A as for of the microparticles can induce distinct on cells by either neutrophils microparticles by PubMed Scopus Google Scholar, M. cell activation by Immunol. PubMed Google or J. L.V. Perretti M. Annexin the rapid of 2008; PubMed Scopus Google Scholar, neutrophils microparticles by PubMed Scopus Google Scholar, M. cell activation by Immunol. PubMed Google Scholar, M. microparticles endothelial cell and tissue in a PubMed Scopus Google Scholar, R. A. G. Z. A. of from human PubMed Scopus Google Scholar). we investigated these actions of neutrophil microparticles from a distinct profile. neutrophils were incubated either in suspension or over a for to to the of microparticle in our the C. L. A. M. and of microparticles in from 2010; PubMed Scopus Google Scholar), and of the were in both with of or endothelial microparticles in these as determined by for and microparticles neutrophils were found to a number of microparticles neutrophils in suspension were in size as determined by the and in between these two subsets These that the microparticle under these with by cell analysis on two distinct microparticle from two of the microparticle in to for in the In this analysis we proteins in the microparticles and proteins in the as determined by in of the microparticle subsets found to be between the two microparticle In were found to be in of the on the microparticle of the proteins were expressed in of the microparticle subsets that might the microparticles are from the cell the abundant proteins in microparticle subset and a can be found in The are in the abundant proteins in both microparticles as or are for this abundant proteins in and microparticles shock 70 kDa protein in a A analysis of on microparticle subset The with both subsets were inflammation by and or and the in their protein this of analysis not between both microparticle it only for the number of proteins in a given of expression and role in the it in both the analysis that both microparticles subsets might have We next the abundance of a of proteins by the in the two microparticle subsets by protein expression in of the subsets we number of microparticles for of the subsets. we found that that and were enriched in the heat shock protein in microparticles Annexin A1 and were expressed levels in the two microparticles subsets. analysis as a the relative protein in these two microparticle subsets assessment of this of proteins demonstrated that and were on the surface of levels were abundant on microparticles whereas expressed on the surface on both microparticle These the finding that distinct neutrophil stimulation microparticles with a profile. the of our to the in we expression of a number of proteins under two distinct inflammatory conditions. The exudates from skin blister which leads to of a response and plasma from patients from or septic shock from these were with plasma neutrophil microparticles from healthy analysis for expression of and on the surface of neutrophil microparticles demonstrated that microparticles from blister exudates levels of and with healthy volunteer plasma neutrophil microparticles the levels of and were observed in plasma neutrophil microparticles from with a in and expression in to and healthy volunteer plasma microparticles These that proteins in our are in and can be expressed by human neutrophil microparticles under both homeostatic and or inflammatory responses. We next investigated these were proteins from to or as in their of the proteome the of of the complex in the microparticles including and and We found for the of and and these were not in the of proteins not in the as in the These findings to investigate microparticles produced reactive oxygen species direct of to microparticles led to as in an that not shared by of production in response to that this a The leukotriene in the proteome of microparticles and and the of in these microparticles we investigated the microparticle subsets herein on both microparticle subsets produced in the of and for and microparticles a that microparticles were incubated in of the the demonstrated of proteins in microparticles and in microparticles including and and we next investigated these microparticles a chemotactic gradient. of microparticles in the of a led to a in the of the with microparticles incubated in the of these that microparticles may to tissue and have been to microparticles their biological actions and processes can with surface on a target cell, can their to a target cell by or or can as we we next investigated a novel mechanism that is microparticles the gene expression of target cells a of their these we to and microparticles subsets the gene expression of endothelial As in microparticles a on the gene expression of and were in endothelial cells with and and A of were in both whereas a number of were only by of the microparticle subsets. 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Ludlam C.A. Plasma microparticles and vascular disorders.Br J. Haematol. 2007; 137: 36-48PubMed Google Scholar, L. A. microparticles in and vascular 2009; PubMed Scopus Google Scholar, microparticles as of endothelial PubMed Scopus Google Scholar, M. endothelial microparticles as a of 2009; PubMed Scopus Google Scholar). that microparticles actions in cell-to-cell in both and have on the of microparticles disease a on endothelial microparticles G. A. A. vascular and 2011; PubMed Scopus Google whereas only information is on the protein of these as well as their actions the by the parent In with with a human cell and endothelial cells D.B. B. analysis of and endothelial 2008; PubMed Scopus Google Scholar, M. M. M. stimulation of cells in distinct of 2009; PubMed Scopus Google we for in neutrophil microparticles produced in response to discrete In with our findings and recently demonstrated that only in response to neutrophils produced microparticles with R. A. G. Z. A. of from human PubMed Scopus Google Scholar). of the in the activation of a neutrophil that leads to to the of inflammation is interaction with to the of A. C. in the activation and of and Rev. Immunol. 2011; PubMed Scopus Google Scholar, M. The neutrophil in vascular Med. 2011; PubMed Scopus Google Scholar). for microparticle neutrophil the endothelial recently been R. M. is a in through Exp. Med. PubMed Scopus Google Scholar), the of the of these from in a number of that neutrophil microparticles may be in a number of inflammatory For example, in as and plasma microparticle are L. L. J. L. L. of neutrophil and microparticles and PubMed Scopus Google Scholar, Y. Y. production of microparticles with expression of from in PubMed Scopus Google Scholar). it is to the that microparticles be exerting their For these herein we on the neutrophil microparticle proteome two distinct with microparticles with as by analysis of these microparticle subsets demonstrated that distinct profiles with of the proteins for the two subsets being expressed in of the microparticle subsets. expression profiles by for a of proteins the the by both with to their and between the two microparticle The of these proteins in of the samples by the of of the are in have been for in over distinct of neutrophil microparticles not In we that both and may be as in these microparticle subsets were expressed in both microparticle subsets and found to be levels These findings those in our that on microparticles in not J. L.V. Perretti M. Annexin the rapid of 2008; PubMed Scopus Google that we found to in the by this neutrophil microparticle subset D.B. B. analysis of and endothelial 2008; PubMed Scopus Google Scholar). The that neutrophils have the to to a by microparticles with a distinct the notion that microparticle production is a and that might be with very discrete as herein and The is by the finding that neutrophil microparticles R. A. G. Z. A. of from human PubMed Scopus Google Scholar). is that proteins to be expressed on neutrophil microparticles as and were not in our may be to a expression of these proteins in the two microparticle subsets investigated herein with the proteins. In these proteins are to a that not only their to in the the for the of our findings we next investigated the proteins herein were expressed on neutrophil microparticles from three distinct human we microparticles from skin blister exudates this characterized by a inflammation The inflammatory of on in 2008; PubMed Scopus Google Scholar), it might the microparticle We plasma microparticles from patients from as a to in of the role of endothelial activation in this disease and Google Scholar). The to a this the of of neutrophil microparticles under disease conditions. For instance, expression of the proteins or in neutrophil microparticles may be of a neutrophil as observed in microparticles from skin blister a inflammatory be that microparticles of these can rapid and homeostatic J. L.V. Perretti M. Annexin the rapid of 2008; PubMed Scopus Google Scholar). the microparticles from septic patients levels and a in and reflecting a neutrophil activation with the role for these microparticles in the of of and to be in we determined microparticles distinct biological actions in to the In this we microparticles be to The and of of of J. PubMed Scopus Google Scholar). These chemical an role in production can be and the of this complex are to the cell membrane or surface only stimulation of the kDa in the of J. PubMed Scopus Google Scholar). a role been to distinct from their levels of induce by of G. B. Wang Zhang by in 2007; PubMed Scopus Google Scholar). Stimulation of microparticles not with led levels for these microparticles were those produced by an number of neutrophils not that from microparticles might this to be investigated in we found that microparticles in response to a chemotactic in with the of proteins in the microparticle proteome that microparticles may be to to inflammatory target cells to inflammatory In our that of neutrophils with leads to the of to of by their may to the may either be for or to the cell surface R.J. of PubMed Scopus Google Scholar). finding that may be to the cell surface in it may be these that in microparticles distinct to those found in the parent cells which to be in is a the of by the actions of two and leukotriene is a neutrophil to neutrophil and the to the of The of both microparticle subsets to on stimulation is in the of an inflammatory that microparticles can the production of is that the of these microparticles the or a given tissue to be and to disease L. L. J. L. L. of neutrophil and microparticles and PubMed Scopus Google Scholar, J. inflammation in microparticle 2010; PubMed Scopus Google Scholar). We the by the of the two microparticle be the by cells the for actions of these microparticle subsets that are with protein and of with endothelial cells produced the with to the number of differentially expressed and the biological processes These demonstrate that microparticles with from the cell for the can in gene the of this response we a recently the that the gene expression profiles to those by demonstrated that microparticles a gene that is of with an not the with In of endothelial cells with microparticles a gene that the gene of and to assessment of the proteins in microparticles the notion that these microparticles may R. A. G. Z. A. of from human PubMed Scopus Google Scholar). These proteins and the proteins and that were or in and the proteins or which expression in can a of that well their of through and These cells can and mediators to by cells and A. C. in the activation and of and Rev. Immunol. 2011; PubMed Scopus Google for a In neutrophil microparticles and cell in the of the and C. M. by neutrophils induce a in 2010; PubMed Scopus Google Scholar). microparticles the of cells (1Norling L.V. Spite M. Yang R. Flower R.J. Perretti M. Serhan C.N. Cutting edge: Humanized nano-proresolving medicines mimic inflammation-resolution and enhance wound healing.J. Immunol. 2011; 186: 5543-5547Crossref PubMed Scopus (158) Google Scholar, J. Serhan C.N. of human microparticles and PubMed Scopus Google and the of mediators by J. Serhan C.N. of human microparticles and PubMed Scopus Google Scholar). demonstrated that neutrophil microparticles the for the of mediators (1Norling L.V. Spite M. Yang R. Flower R.J. Perretti M. Serhan C.N. Cutting edge: Humanized nano-proresolving medicines mimic inflammation-resolution and enhance wound healing.J. Immunol. 2011; 186: 5543-5547Crossref PubMed Scopus (158) Google Scholar, J. Serhan C.N. of human microparticles and PubMed Scopus Google that can be to are to mediators J. Serhan C.N. of human microparticles and PubMed Scopus Google Scholar). microparticles are of of these and the gene modulation we for this In we novel of neutrophil We that their production is a with their varying on the nature of the we that these distinct with an to inflammatory mediators as and to to the through production and novel roles of microparticles in inflammatory responses. In we that the two microparticle subsets investigated this and distinct in gene expression profiles in These shed light on an of to been very the of a analysis of neutrophil in an analysis with in disease the to the of the for the of We and and the in for with the samples and for in the blister We to for with the Microarray analysis and of and for with the with