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Adhesion and signaling by B cell-derived exosomes: the role of integrins¹

ALED CLAYTON^*,2, ATILLA TURKES^*, SHARON DEWITT, ROBERT STEADMAN, MALCOLM D. MASON^* and MAURICE B. HALLETT

^* Section of Clinical Oncology, Department of Medicine, University of Wales College of Medicine, Velindre Hospital, Whitchurch, Cardiff, UK;
Neutrophil Signalling Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, UK; and
Institute of Nephrology, University of Wales College of Medicine, Heath Park, Cardiff, UK

²Correspondence: Velindre Hospital, Whitchurch, Cardiff CF14 2TL, UK. E-mail: aled.clayton{at}velindre-tr.wales.nhs.uk

SPECIFIC AIMS

Exosomes are a distinct population of nanovesicles secreted by healthy cells, which have previously been implicated in control of cellular immunity. Their possible interactions with other biological components remain poorly understood. The aim of this study was to investigate the functionality of exosomally expressed integrins with respect to their adhesive and signaling functions.

PRINCIPAL FINDINGS

1. Exosomes express integrins at their outer surface
Expression of ß1 (CD29) and ß2 (CD18) integrins on exosomes of various cellular sources was examined. B-lymphocyte exosomes were purified by ultracentrifugation on a sucrose cushion, and, to allow analysis of surface molecules, were subsequently coupled to beads and analyzed by flow cytometry. Such exosomes express both ß1 and ß2 integrins on their outer surface, at levels below that of MHC molecules, but comparable to expression levels of tetraspanin CD63 (Fig. 1 c). Western blots of ultracentrifugation-isolated fibroblast, epithelial and B cell exosomes demonstrated expression of ß1 integrin, while B cell exosomes alone were positive for ß2 integrin (Fig. 1b ). Affinity isolated B cell exosomes (using class II antibody coated beads) were also examined by Western blot, confirming that 130Kd ß1 integrin and 90Kd ß2 integrin were co-localized with MHC Class II.

View larger version (50K): [in this window] [in a new window]   Figure 1. Integrin expression by exosomes. Exosomes from several B cell lines were immobilized onto anti-MHC Class II coated beads, and analyzed for ß1 and ß2 expression by Western blot (a). Western blot of fibroblast, epithelial and B cell exosomes isolated by ultracentrifugation on a sucrose cushion (b). Flow cytometric analysis of B cell exosome bead complexes, stained with antibodies as indicated (c). Filled histogram, uncoated beads; and unfilled histogram, exosome-coated beads.

The exosome integrin expression pattern is consistent with that of the cell of origin, with integrins having correct orientation (facing extracellular environment), and capable of encountering multiple ligands after their secretion from the cell.

2. Exosomes adhere to extracellular matrix constituents in an integrin dependent manner
Functionality of integrins expressed by B cell exosomes was addressed by examining adhesive interactions with extracellular matrix (ECM) components. Beads saturated with B cell exosomes were added to wells precoated with human collagen-I or fibronectin (from human plasma). After washing, the number of adhered beads was quantified.

Adhesion to collagen and fibronectin was integrin-dependent, being dose- dependently inhibited in presence of blocking antibodies. Adhesion to collagen-I was inhibited by ß1 integrin specific antibodies (80% inhibition at maximum dose), while adhesion to fibronectin was inhibited by antibodies against either ß1 (91% at maximum dose) or 4 (67% at maximum dose). This inhibition was not due to steric hindrance, as ß2 integrin antibody had no effect on matrix adhesion. Binding was dose-dependently inhibited in presence of EDTA, emphasizing the importance of divalent ions in these interactions. Our findings demonstrate the functional capacity of exosome integrins in mediating adhesion to ECM constituents.

3. Exosomes adhere to fibroblasts, but only after cytokine induction of fibroblast adhesion molecules
We used human infant foreskin fibroblasts (HFF), as an in vitro model to study possible integrin involvement in interactions of exosomes with cells. Nonproliferating fibroblasts express low levels of leukocyte adhesion receptors. Under such circumstances there was poor binding between B cell-derived exosome bead complexes and fibroblast monolayers. In contrast, treatment of HFF with TNF, induced surface expression of adhesion molecules including ICAM-1, and binding of exosome beads became significantly elevated. Antibodies against ß1 or ß2 integrins inhibited adhesion to TNF-treated HFF. Low level of binding to untreated fibroblasts was ß2 integrin-independent, but was inhibited by ß1 integrin antibody. These data show that in inflammation, potential cellular targets for exosomes are diverse, due to up-regulation of integrin ligands on cell surfaces.

4. Adhesion of exosomes to cytokine-treated fibroblasts triggers Ca²⁺ signals
Single cell imaging of fibroblasts loaded with a fluorescent calcium indicator (Fura-2) was used to examine cytosolic Ca²⁺ levels after exosome adhesion. Depositing a single exosome bead complex at the cell surface using a micropipette resulted in a rapid transient rise in cytosolic Ca²⁺ to 800 nM in 5–30 s (Fig. 2 A). This calcium spike resolved to basal levels (100 nM) by 120 s. Multiple Ca²⁺ oscillations were observed after this interaction, while neighboring cells that had not had contact with exosome beads remained at basal levels (Fig. 2B ). Exosome-fibroblast adhesion and signaling required both ß1 and ß2 integrins, presence of Mg²⁺, and pretreatment of fibroblasts with TNF.

View larger version (34K): [in this window] [in a new window]   Figure 2. TNF treated fibroblasts loaded with fluorescent calcium indicator Fura-2, were used to visualize changes in cytosolic calcium after adhesion of an exosome-coated bead. A series of images (A) shows the approach of an exosome bead, contact with the fibroblast surface, and an immediate increase in cytosolic calcium which resolves to basal levels. Images are pseudocolored according to the scale shown (B). The entire time course of this experiment (B) demonstrates multiple oscillations in the cell which has had contact with the exosome bead (cell 1), while a neighboring cell has not encountered exosomes and does not respond with calcium oscillations (cell 2).

Our findings present the first direct evidence that exosomes can elicit signaling responses in target cells through an adhesion molecule dependent mechanism. These observations highlight a novel role for exosomes during inflammatory responses.

CONCLUSIONS

Gaining insight into interactions between exosomes and various components in their surrounding microenvironment is important for understanding their complex physiological functions. Although believed by some to be nothing more than a mode for purging cellular waste, it is increasingly apparent that the molecular complexity of secreted exosomes suggests more wide-ranging biological functions. We present a novel role for exosomes in inflammation, with exosome integrins playing a key function in adhesion and signaling.

True physiological targets of exosomes are not yet known. Although much current evidence points to dendritic cells as target cells, an exosome-specific receptor on such cells has yet to be identified. Our current findings highlight that integrin expression by exosomes is a general phenomenon found on exosomes from diverse cell types, which, if functional, raises the number of potential noncellular and cellular exosome targets. Until now, it was unknown whether such integrins were effective as molecular anchors and signal initiating molecules. We demonstrate that exosome integrins are expressed in a functional conformation, capable of adhesion to ECM, and under conditions mimicking inflammation, to fibroblasts.

We propose a new model for the role of exosomes during inflammatory responses. Beta1 integrin-positive exosomes, secreted by tissue resident cells (e.g., fibroblasts), are likely capable of interactions with surrounding extracellular matrix (ECM). Such adhesive interactions may limit diffusion of exosomes from the site of secretion, leading to high local concentrations in situ (Fig. 3 a). In the event of injury, disruption of ECM may lead to release of ECM-bound exosomes, freeing them to interact with infiltrating inflammatory cells (Fig. 3b ). In such an environment, proinflammatory cytokines act to facilitate invasion of tissue with leukocytes by mechanisms that include adhesion molecule induction in local cells. Exosomes secreted by cellular infiltrate, therefore, may also be free to interact with tissue resident cells such as fibroblasts (Fig. 3c ). The effect of such an interaction is generation of calcium signaling (Fig. 2) .

View larger version (18K): [in this window] [in a new window]   Figure 3. A model for exosome integrins in steady-state conditions and in inflammation. Exosomes secreted by diverse cell types express integrins which likely mediate adhesive interactions with extracellular milieu, limiting their diffusion from the site of secretion, forming extracellular pools of exosomes in situ (a). During tissue injury, proinflammatory cytokines induce adhesion molecule expression of local cells, while leukocytes infiltrate the interstitium. Dynamic reorganization of ECM may liberate bound exosomes (b), which may in turn interact with resident/infiltrating cells. Exosomes secreted by infiltrating leukocytes bind to resident cells through their up-regulated adhesion molecules, triggering a calcium signaling response. Exosomes deliver adhesion-like communication without actual cell–cell contact taking place, and may act to amplify delivery of adhesion signals to a variety of target cells during inflammation (c).

Physical contact between two cells is an important form of communication, providing early signaling events (such as Ca²⁺ oscillations), followed by behavioral changes in one or both cells. During inflammation, adhesion may be one of multiple communication signals, acting in concert to coordinate inflammatory response and its resolution. We have not yet defined behavioral modifications that occur after exosome-mediated signaling in fibroblasts, but the existence of this phenomenon demonstrates an activating role for exosomes in cell-to-cell communication, rather than a role in preventing delivery of adhesion-like signals by sequestering cell-surface adhesion molecules in a nonsignaling manner.

Our report supports a hitherto unknown role for exosomes in inflammation. B cell exosomes bearing functional integrins, are capable of high affinity adhesive interactions with cytokine-activated cells (fibroblasts), acting not only as a mode of physical association, but also as a mechanism for communication between cells. In this way, adhesion signal delivery capacity of B cells (and probably other infiltrating cells) may be drastically amplified, through secretion of many exosomes, allowing contact-like influence over the behavior of resident cells, without actual cell–cell contact taking place.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1094fje;

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