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Introduction: Red blood cell (RBC) transfusion may affect the recipient immune system. During RBC storage in an unphysiological environment, RBC quality and function are impaired, the cells bleb extracellular vesicles (EVs), and other bioactive substances accumulate in the storage medium. EVs can carry reactive biomolecules and mediate cell-cell interactions. Thus, EVs could explain RBC transfusion related immunomodulation, particularly after prolonged storage. Methods: We exposed peripheral blood mononuclear cells (PBMCs) to allogeneic RBC supernatant (SN) and EVs from fresh and longer-stored RBC units, diluted plasma, and storage solution SAGM, and studied activation and proliferation of T-cells by flow cytometry, and cytokine secretion of LPS-stimulated PBMCs by enzyme-linked immunosorbent assay (ELISA). Results: Both fresh and longer-stored RBC SN but not EVs induced immunomodulation in recipient cells. RBC SN and diluted plasma augmented the proliferation of particularly CD8+ T-cells in a 4-day proliferation assay. T-cell activation by SN was evident already after 5 h as shown by upregulation of CD69. SN suppressed monocyte TNF-α and increased IL-10 secretion while diluted plasma increased secretion of both cytokines. Conclusion: This in vitro study demonstrates that stored RBC SN will have mixed immunomodulatory effects depending on responder cells and conditions, independent of RBC storage age. Fresh RBCs containing relatively few EVs can induce immune responses. Residual plasma in the products may contribute to these effects.
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BACKGROUND: Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus. METHODS: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays. RESULTS: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations. CONCLUSIONS: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.
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Membrane type 1 matrix metalloproteinase (MT1-MMP, MMP14) is an efficient extracellular matrix (ECM) degrading enzyme that plays important roles in tissue homeostasis and cell invasion. Like a number of type I membrane proteins, MT1-MMP can be internalized from the cell surface through early and recycling endosomes to late endosomes, and recycled to the plasma membrane. Late endosomes participate in the biogenesis of small (30-100 nm) vesicles, exosomes, which redirect plasma membrane proteins for extracellular secretion. We hypothesized that some of the endosomal MT1-MMP could be directed to exosomes for extracellular release. Using cultured human fibrosarcoma (HT-1080) and melanoma (G361) cells we provide evidence that both the full-length 60 kDa and the proteolytically processed 43 kDa forms of MT1-MMP are secreted in exosomes. The isolated exosomes were identified by their vesicular structure in electron microscopy and by exosomal marker proteins CD9 and tumor susceptibility gene (TSG101). Furthermore, exosomes contained beta1-integrin (CD29). The exosomes were able to activate pro-MMP-2 and degrade type 1 collagen and gelatin, suggesting that the exosomal MT1-MMP was functionally active. The targeting of MT1-MMP in exosomes represents a novel mechanism for cancer cells to secrete membrane type metalloproteolytic activity into the extracellular space.
