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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Exosomes are membrane vesicles of endosomal origin that are distinct from apoptotic bodies and are thought to represent an acellular mechanism for antigen transfer to classic antigen-presenting cells, as well as for direct antigen presentation with the capacity to induce immune response or tolerance. Nevertheless, it is not known whether exosomes are involved in the induction or regulation of immune responses against intracellular autoantigens that characterize autoimmune diseases. Exosomes have been shown to be secreted by several types of cells, whereas studies of non-neoplastic epithelial cells are lacking. This study was undertaken to investigate the capacity of non-neoplastic salivary gland epithelial cells (SGECs) to release exosomes, and to determine whether epithelial exosomes contain RNPs, which are major autoantigens in systemic rheumatic diseases.

Methods

Exosomes were isolated by ultracentrifugation from culture supernatants of 26 non-neoplastic SGEC lines established from patients with various rheumatic disorders. They were analyzed by electron microscopy, immunoblotting, or immunoprecipitation.

Results

All SGEC lines were found to release comparable and significant amounts of exosomes. Similar to other cell systems, exosome secretion was constitutive and was unrelated to activation or apoptotic processes. SGEC-derived exosomes were found to contain the autoantigenic Ro/SSA, La/SSB, and Sm RNPs, as well as epithelial-specific cytokeratins.

Conclusion

SGECs constitutively secrete exosomes that contain the major autoantigens Ro/SSA, La/SSB, and Sm. This mechanism may represent a pathway whereby intracellular autoantigens are presented to the immune system with an immunogenic or tolerogenic outcome.

Autoimmune diseases are characterized by the development of humoral responses against self antigens, most of which are intracellular proteins. Although thought to involve typical antigen-driven responses, the precise mechanism(s) involved in the presentation of such intracellular autoantigens to the immune system is unclear (1). Apoptosis and the resulting release of autoantigen-containing apoptotic vesicles are considered potential pathways for the generation of autoimmune responses (2).

During the last decade, a novel cell-free mechanism of antigen presentation has been recognized (3) that involves small (30–100 nm) membrane vesicles, which are termed exosomes and are distinct from apoptotic bodies. These are different from the exosome protein complex, which is implicated in RNA processing and degradation processes. Exosomal vesicles are secreted following the fusion of multivesicular late endosome/lysosomes with the plasma membrane. Exosome production has been observed in a variety of cell types, including reticulocytes, platelets, cytotoxic T lymphocytes and B lymphocytes, dendritic cells, and neoplastic intestinal epithelial cells, whereas studies of non-neoplastic epithelial cells are lacking. Several physiologic roles have been assigned to exosomes, including the expulsion of obsolete membrane constituents, the exchange of cellular material, and intercellular communication (4).

In addition, several lines of evidence implicate exosomes in antigen presentation (3, 4). Equipped with functional surface proteins involved in antigen presentation, exosomes secreted from antigen-presenting cells (APCs) have been shown to directly stimulate T cells in an antigen-dependent manner and to induce immunogenic or tolerogenic responses. Furthermore, exosomes may indirectly contribute to the regulation of immune responses by mediating the transfer of antigens to dendritic cells (3). Thus, exosomes may represent a mechanism for the presentation of intracellular autoantigenic proteins to the immune system.

Recently, using long-term cultured non-neoplastic cell lines, we demonstrated that human salivary gland epithelial cells (SGECs) possess immunologic functions (5, 6). One of the aims of the present study was to evaluate the capacity of SGECs to secrete exosomes. We showed that human non-neoplastic epithelial cells, such as SGECs, constitutively secrete exosomes that contain epithelial-specific cytoskeletal proteins. Presuming that exosomes mediate the introduction of intracellular antigens to the immune system, we sought to determine whether SGEC-derived exosomes contain autoantigenic RNPs. This is the first study to show that such epithelial cell–derived exosomes contain the intracellular RNPs Ro/SSA, La/SSB, and Sm, which, in fact, are major autoantigenic targets in patients with systemic rheumatic disorders, such as Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). These findings likely indicate the participation of exosomal vesicles in the regulation of immunologic reactions against intracellular proteins.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Non-neoplastic SGEC lines.

Long-term non-neoplastic SGEC lines were established from minor salivary gland biopsy samples obtained from patients undergoing diagnostic evaluation for possible SS, as previously described (5). Patients gave their informed consent for study. The epithelial origin of cultured SGEC lines was routinely verified by morphology, as well as by the uniform and consistent expression of epithelial-specific markers and the absence of markers indicative of lymphoid/monocytoid cells (5). Exosomes from 26 SGEC lines derived from patients with and without putative SGEC pathology (16 patients with SS and 10 non-SS disease controls, respectively) were studied. SS patients included 10 with primary SS and 6 with secondary SS (associated with SLE in 3 and with vasculitis, rheumatoid arthritis [RA], and mixed connective tissue disease in 1 each). Non-SS disease controls included 10 patients who did not fulfill the American–European classification criteria for SS (6 patients with atypical sicca syndrome, 3 patients with autoimmune thyroiditis, and 1 with RA) (7). Apoptosis, as analyzed by annexin V binding (5), in SGEC cultures was ≤2%.

Exosome isolation and electron microscopy.

Exosomes were isolated from cell-free, 0.22-μm, prefiltered SGEC culture supernatants by ultracentrifugation, as previously described (8). Collected exosomes were processed for analysis by electron microscopy and Western blotting. To ensure that pelleted exosomes did not originate from the fetal calf serum (FCS) that supplemented the epithelial culture medium (2.5%), fresh complete medium was routinely processed by ultracentrifugation and did not result in exosome pelleting. In addition, the production and RNP content of exosomes were also analyzed in the supernatants of SGEC cultures (10 SGEC lines of the total of 26 studied) where serum-free epithelial medium (KBM; Cambrex, Walkersville, MD) was used (5). Electron microscopic analysis was performed as previously described (8).

Western blotting.

Protein extracts were prepared from exosomes or cells, as previously described (6), and the amount of protein was estimated with the Micro BCA protein assay kit (Pierce, Rockford, IL). Subsequently, the lysates (100 μg of exosomal or cellular lysate/lane) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by immunoblotting. Separation of the 52-kd Ro/SSA proteins from the 48-kd La/SSB proteins was performed in 10% polyacrylamide gels (ratio of 40% acrylamide to 0.232% bisacrylamide). Cytokeratins and autoantigenic RNPs were detected by immunoblotting using a monoclonal antibody against human cytokeratins 5, 6, 8, 17, and 19 (clone MNF116; Dako, Glostrup, Denmark) and several well-characterized prototype human antisera to RNPs, respectively. The latter included bispecific antisera (3 against the Ro/SSA and La/SSB proteins and 2 against the Sm and U1 small nuclear RNP [U1 snRNP] proteins) and monospecific antisera (2 each against La/SSB and against U1 snRNP proteins). In certain experiments, the specificity of La/SSB detection was evaluated by inhibition experiments in which the human prototype anti-La/SSB–positive serum activity was inhibited following overnight incubation with recombinant human La/SSB protein (prepared from a plasmid kindly provided by M. Bachmann, Institute of Immunology, Carl Gustav Carus University, Dresden, Germany).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Exosome secretion by SGECs.

All SGEC lines studied were found to secrete significant amounts of exosomal vesicles (mean ± SD 15.0 ± 4.6 μg protein/106 cells). Electron microscopy revealed that exosomes derived from cultured SGECs were vesicles between 50 and 100 nm in diameter that presented the typical cup-shaped morphology (8) of exosomes (Figure 1A). Similar amounts of exosomes were produced by all SGEC lines tested, as estimated by protein measurements (results not shown). In addition, exosome secretion was constitutive, stable, and unrelated to activation processes, as indicated by similar production in cultures of resting SGECs and cells activated by interferon-γ (IFNγ) (results not shown). Consistent with their epithelial origin, exosomes isolated from SGEC culture supernatants were found to contain epithelial-specific cytoskeletal components, such as cytokeratins (Figure 1B). Culture supernatants obtained from long-term–cultivated non-neoplastic salivary gland fibroblasts or from neoplastic epithelial HeLa cells were not found to contain detectable amounts of exosomes.

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Figure 1. Secretion of exosomes by salivary gland epithelial cells (SGECs). A, Electron microscopy of exosomes (arrowheads) isolated from SGEC culture supernatants reveals the presence of vesicles with the typical cup-shaped morphology of exosomes. Bar = 0.1 μm. B, Detection of cytokeratin expression in SGECs (lane 1) and SGEC-derived exosomal lysates (lane 2), as demonstrated by immunoblotting (IB) using a monoclonal antibody specific for several human cytokeratins. Three distinct cytokeratin protein bands were identified in exosomes, with a molecular mass of ∼62, 54, and 43 kd, respectively. Immunoblotting of exosomal proteins with an isotype-control monoclonal antibody is shown in lane 3.

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Autoantigenic RNPs in SGEC-derived exosomes.

Exosomes obtained from 26 SGEC cultures were studied for autoantigenic RNP content. All of them were found to contain readily detectable amounts of the Ro/SSA, La/SSB, and Sm autoantigens, but not U1 snRNP proteins (Figure 2A–D). Prototype human bispecific and monospecific antisera to Ro/SSA and/or La/SSB reacted with 3 distinct protein bands in the lysates of SGEC exosomes. These corresponded to the full-length and degradation products of the La/SSB protein, as confirmed by inhibition experiments using recombinant human La/SSB (Figure 2A). The presence of Sm RNP in exosomal extracts was indicated by reactivity with the typical SmB′/B protein bands (Figure 2C). There were no quantitative or qualitative differences between patients in the occurrence of RNPs in exosomal preparations or with regard to the presence of primary and/or secondary SS or the serologic autoantibody status.

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Figure 2. Ro/SSA, La/SSB, and Sm RNPs in salivary gland epithelial cell (SGEC)–derived exosomes. Representative immunoblotting (IB) analyses of protein extracts of SGEC-derived exosomes (exos) were used to detect the presence of autoantigenic RNPs, using human specific prototype antisera to La/SSB (a-La/SSB), Ro/SSA and La/SSB (a-Ro/SSA and a-La/SSB), and Sm/U1 small nuclear RNPs (a-Sm/U1 snRNP). No reactivity was observed with normal human serum (negative control). Exosomal extracts derived from a patient with Sjögren's syndrome (SS) and from a non-SS disease control (Ct), as well as a total cell extract from HeLa cells (H; positive control), are shown. A, Detection of the La/SSB RNP (47 kd) and the degradation products of the La/SSB protein (45- and 43-kd bands) in SGEC exosomes using a prototype human monospecific antiserum to La/SSB. Preincubation of antiserum to anti-La/SSB with recombinant human La/SSB protein (a-La/SSB + rhLa/SSB) results in the abolition of all 3 La/SSB protein bands. B, Detection of the Ro/SSA RNPs (60 and 52 kd) in SGEC exosomes using a prototype human antiserum to both anti-Ro/SSA and anti-La/SSB antigens. The 47-kd band corresponds to the La/SSB protein. C, Detection of the SmB′/B protein bands of ∼28 kd (lower panel), but not of the 70-kd U1 snRNP protein in exosomal lysates, using a prototype human antiserum to Sm/U1 snRNP proteins (a-Sm/U1 snRNP). The 70-kd U1 snRNP was detected in HeLa cell protein extracts (positive control). D, Detection of RNP species in cell lysates from SGEC and HeLa, including Ro/SSA (60- and 52-kd proteins) and La/SSB (47 kd, and the 45- and 43-kd degradation products) (left panel), as well as U1 snRNP (70 kd) and SmB′/B (right panel).

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The possible occurrence of exosomes in the FCS used to supplement culture media has been considered (3). However, as mentioned above, we could not confirm this using fibroblast or HeLa cell culture supernatants that contained 10% FCS. In addition, no visible exosome precipitation was observed in routine analyses of fresh complete SGEC medium (containing 2.5% FCS). Furthermore, in experiments with paired samples of FCS-supplemented (2.5%) and serum-free culture supernatants of the same SGEC lines, no qualitative differences were observed in the detection of autoantigenic proteins in exosomes. This indicates that the RNPs detected in SGEC exosomal preparations are not derived from putative FCS-derived exosomes (results not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Herein, we demonstrated that non-neoplastic SGECs constitutively secrete significant amounts of exosomes. Similar to other cell systems, the release of exosomes by SGECs appears to be a constitutive and stable process that is likely unaffected by cell activation or apoptosis-related processes. Comparable production of exosomes was observed in cultures of resting SGECs and following activation by IFNγ. In addition, similar amounts of exosomal vesicles were found to be released by SGEC lines derived from SS patients, which are intrinsically activated (1), and by those from non-SS disease controls. Moreover, exosome secretion by SGECs is unrelated to cellular destruction processes, since negligible apoptosis or other types of SGEC death occur in our culture system (5). Constitutive exosome secretion has also been observed in cultures of neoplastic intestinal epithelial cells, dendritic cells, and B cells transformed by Epstein-Barr virus, whereas it is regulated by activation in T cells and mast cells (3).

Exosomes have previously been shown to contain several types of proteins, including cytoskeletal components, such as actin or tubulin (3). In this study, we demonstrated that SGEC-derived exosomes contain significant amounts of cytokeratins that are epithelial-specific markers. The occurrence of these cell type–specific cytoskeletal proteins indicates that the cellular origin of exosomes may be specifically identified by their content.

Autoimmune responses to RNPs characterize certain systemic rheumatic diseases, such as SS and SLE. However, the precise mechanism whereby these intracellular antigens gain access to and stimulate the immune system of patients remains unclear. To date, apoptosis has been thought to represent the major pathway for the generation of autoimmune responses via the capture of autoantigen-containing apoptotic vesicles by professional APCs (2). This is the first study to demonstrate that the intracellular autoantigenic Ro/SSA, La/SSB, and Sm RNPs are present in vesicles that are constitutively released by resting and viable cells. Significant expression of these RNPs was invariably observed in exosomes derived from SS patients and non-SS disease controls, a fact that likely points toward the constant targeting of these RNPs in SGEC exosomes. It is unclear whether the absence of U1 snRNP proteins in these vesicles is due to specialized properties of protein transfer mechanisms in exosomal vesicles (3) or to low concentrations. However, it is notable that, despite extensive proteomic analyses in previous studies, the presence of RNPs has not been reported in exosomes derived from B cells, dendritic cells, mast cells, and intestinal epithelial cells (8–11). In this laboratory, proteomic studies are currently in progress to determine the protein content of SGEC-derived exosomes, as well as the occurrence of autoantigenic RNPs in exosomes released by cells other than SGECs.

Exosomes have been considered to mediate antigen presentation, either directly by antigen presentation via their surface receptors (major histocompatibility complex–antigenic peptide complexes and costimulatory molecules) or indirectly by loading of APCs (3, 12, 13). Nevertheless, despite intensive studies, the in vivo physiologic significance of exosomes remains unclear. Further studies are needed to investigate the immunologic role of SGEC-derived exosomes in vivo and, in particular, to define the possible participation of these vesicles in the regulation of autoimmune responses. In this context, it is tempting to hypothesize that exosomes participate in the presentation of intracellular autoantigens, such as RNPs, to the immune system. In fact, loading of professional APCs by RNP-containing exosomes appears to be a plausible mechanism for the transfer and efficient antigen presentation to autoantigen-specific T cells. As previously demonstrated (3), such an antigen presentation process involving exosomes could lead to the induction of immunogenic or tolerogenic immune responses, depending on the involvement of mature or immature APCs, respectively.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We would like to acknowledge Professor L. Margaritis for his assistance in electron microscopy analysis and Dr. S. Paikos for performing the minor salivary gland biopsies.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  • 1
    Manoussakis MN, Moutsopoulos HM. Sjogren's syndrome: autoimmune epithelitis. Baillieres Best Pract Res Clin Rheumatol 2000; 14: 7395.
  • 2
    Rosen A, Casciola-Rosen L, Ahearn J. Novel packages of viral and self-antigens are generated during apoptosis. J Exp Med 1995; 181: 155761.
  • 3
    Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002; 2: 56979.
  • 4
    Fevrier B, Raposo G. Exosomes: endosomal-derived vesicles shipping extracellular messages. Curr Opin Cell Biol 2004; 16: 41521.
  • 5
    Dimitriou ID, Kapsogeorgou EK, Abu-Helu RF, Moutsopoulos HM, Manoussakis MN. Establishment of a convenient system for the long-term culture and study of non-neoplastic human salivary gland epithelial cells. Eur J Oral Sci 2002; 110: 2130.
  • 6
    Kapsogeorgou EK, Moutsopoulos HM, Manoussakis MN. Functional expression of a costimulatory B7.2 (CD86) protein on human salivary gland epithelial cells that interacts with the CD28 receptor, but has reduced binding to CTLA4. J Immunol 2001; 166: 310713.
  • 7
    Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, et al, and the European Study Group on Classification Criteria for Sjögren's Syndrome. Classification criteria for Sjögren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002; 61: 5548.
  • 8
    Thery C, Boussac M, Veron P, Ricciardi-Castagnoli P, Raposo G, Garin J, et al. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 2001; 166: 730918.
  • 9
    Thery C, Regnault A, Garin J, Wolfers J, Zitvogel L, Ricciardi-Castagnoli P, et al. Molecular characterization of dendritic cell-derived exosomes: selective accumulation of the heat shock protein hsc73. J Cell Biol 1999; 147: 599610.
  • 10
    Wubbolts R, Leckie RS, Veenhuizen PT, Schwarzmann G, Mobius W, Hoernschemeyer J, et al. Proteomic and biochemical analyses of human B cell-derived exosomes: potential implications for their function and multivesicular body formation. J Biol Chem 2003; 278: 1096372.
  • 11
    Skokos D, Le Panse S, Villa I, Rousselle JC, Peronet R, David B, et al. Mast cell-dependent B and T lymphocyte activation is mediated by the secretion of immunologically active exosomes. J Immunol 2001; 166: 86876.
  • 12
    Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med 1996; 183: 116172.
  • 13
    Thery C, Duban L, Segura E, Veron P, Lantz O, Amigorena S. Indirect activation of naive CD4+ T cells by dendritic cell–derived exosomes. Nat Immunol 2002; 3: 115662.