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Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid

Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present... Copyright ERS Journals Ltd 2003 Eur Respir J 2003; 22: 578–583 European Respiratory Journal DOI: 10.1183/09031936.03.00041703 ISSN 0903-1936 Printed in UK – all rights reserved FRONT ROWS OF SCIENCE Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid # } # # # C. Admyre*, J. Grunewald , J. Thyberg , S. Gripenba ¨ck , G. Tornling , A. Eklund , A. Scheynius*, S. Gabrielsson* *Dept of Medicine, Unit of Clinical Allergy Exosomes with major histocompatibility complex class II and co-stimulatory molecules Research, Division of Respiratory Medicine are present in human BAL fluid. C. Admyre, J. Grunewald, J. Thyberg, S. Gripenba ¨ ck, and Dept of Cell and Molecular Biology, G. Tornling, A. Eklund, A. Scheynius, S. Gabrielsson. #ERS Journals Ltd 2003. Karolinska Hospital and Institutet, Stockholm, ABSTRACT: Exosomes are 30–100 nm diameter vesicles formed by inward budding of Sweden. endosomal compartments and are produced by several cell types, including T-cells, B- cells and dendritic cells (DC)s. Exosomes from DCs express major histocompatibility Correspondence: C. Admyre, Dept of Medicine, complexes (MHC) class I and II, and co-stimulatory molecules on their surface, and Unit of Clinical Allergy Research, Karolinska can induce antigen-specific activation of T-cells. Hospital and Institutet, Stockholm, Sweden. Fax: 46 8335724 The aims of the present study were to investigate for the presence of exosomes in E-mail: [email protected] bronchoalveolar lavage fluid (BALF) from healthy individuals, and to establish if these exosomes bear MHC and co-stimulatory molecules. Keywords: Bronchoalveolar lavage fluid fluid, The authors analysed BALF taken from seven healthy volunteers and used exosomes CD54, CD86, exosomes, human leukocyte from monocyte-derived DC (MDDC) cultures as a reference. After ultracentrifugation, antigen-DR exosomes were bound to anti-MHC class II coated magnetic beads and analysed by flow cytometry and electron microscopy. Received: April 14 2003 The authors report for the first time that exosomes are present in BALF. These Accepted after revision: May 29 2003 exosomes are similar to MDDC derived exosomes as they express MHC class I and II, This work was supported by the Swedish CD54, CD63 and the co-stimulatory molecule CD86. Research Council (grants no. 16x-7924, 74x- The results demonstrate that exosomes are present in the lung, and since they contain 14182, and 6537), the Swedish Council for both major histocompatibility complex and co-stimulatory molecules it is likely that Work Life Sciences, the Swedish Asthma and they are derived from antigen presenting cells and might have a regulatory role in local Allergy Association9s Research Foundation, immune defence. the Hesselman Foundation, Konsul TH Bergs Eur Respir J 2003; 22: 578–583. Foundation, the Swedish Heart-Lung Foun- dation, the Va ˚ rdal Foundation, the Centre for Allergy Research at Karolinska Institutet, the King Oscar II Jubilee Foundation, the King Gustaf V 80th Birthday Fund and the Kar- olinska Institutet. Exosomes are 30–100 nm diameter vesicles produced by on the cell surface, such as the tetraspan proteins CD63 and different cell types, including reticulocytes [1], platelets [2], B- CD82 [7], which interact with membrane proteins, such as lymphocytes [3], T-lymphocytes [4], mast cells [5], epithelial integrins and human leukocyte antigen (HLA)-DR [10]. cells [6] and dendritic cells (DC)s [7]. Exosomes are formed by Exosomes may not only have a role in T-cell activation, but inward budding of the limiting membrane of endosomal may also function as a communicator between cells in the compartments, called multivesicular bodies (MVB)s [8]. In immune system. Intestinal epithelial cells secrete exosome-like antigen presenting cells (APC)s MVBs are the main site for vesicles, which favour cross communication between the loading major histocompatibility complex (MHC) class II and intestinal epithelium and the T-cells in the mucosa or in the recycling MHC class I molecules with exogenous antigens. blood [6]. Upon T-cell receptor (TCR) triggering, T-cells The MHC molecules are located both on the limiting produce exosome-like vesicles expressing TCR and CD3, membrane and on the internal vesicles. When MVBs fuse which may deliver signals to target cells baring the right with the plasma membrane the internal vesicles are released MHC-peptide complex [4]. extracellularly and become exosomes [3]. APCs secrete The lung is constantly exposed to inhaled particles and has exosomes that carry peptide-loaded MHC molecules, which numerous capillaries making its9 mucosa a vulnerable surface can stimulate T-cell proliferation in vitro [3]. T-cell stimula- between the environment and internal tissues. Therefore, it is tion has also been seen when using exosomes from rat mast important to have a powerful local immune defence. Whether cells engineered to express mouse or human MHC class II exosomes are part of such a defence in the lung has not molecules [9]. In a mouse model, exosomes have shown a previously been addressed. The authors of the present study good potential for use as a tool in cancer therapies, where investigated if exosomes are present in human broncho- exosomes with tumour-peptide loaded MHC molecules were alveolar lavage fluid (BALF) from healthy individuals, using used for tumour eradication [7]. Exosomes from APCs do not exosomes from monocyte-derived DCs (MDDCs) as a only express MHC molecules, but also co-stimulatory mole- reference. The authors show for the first time, using flow cules, such as CD86. They further express proteins not detected cytometry and electron microscopy (EM), that exosomes are MHC AND CO-STIMULATORY MOLECULES IN BAL FLUID 579 present in BALF. The authors found that these exosomes in PBS and stored at -80uC. Exosome samples were measured selected for MHC class II expression, such as exosomes for protein content using BioRad DC protein assay (Bio-Rad derived from MDDC cultures, express HLA-DR, MHC class Laboratories, Hercules, CA, USA) according to the manu- I, CD54, CD63 and the co-stimulatory molecule CD86. facturer9s instructions. The protein content of the exosome -1 preparations from BALF ranged 0.20–0.51 mg protein?mL , -1 median 0.30 mg protein?mL , of original volume BALF, n 7. Materials and methods Subjects Exosome coating to beads BALF from seven healthy nonsmoking individuals aged Exosomes were adsorbed onto dynabeads precoated with 22–51 yrs (median 25 yrs), two males and five females, were mouse IgM antihuman MHC class II (Dynal1, Oslo, Norway) analysed. None had a history of atopy, and all had normal -1 in PBS containing 0.1% bovine serum albumin (BSA) and serum Immunoglobulin (Ig)E values (v120 kUL , Immuno- 0.01% sodium-azide overnight at RT [15]. Pilot experiments Cap, Pharmacia Diagnostics, Uppsala, Sweden), normal chest with exosomes from MDDC supernatants (MDDC-exosomes) radiographs and were without signs of respiratory infection showed the beads to be saturated with exosomes at a for o1 month prior to the study. The local ethics committee approved the study and informed consent was obtained from concentration of 1 mg protein to 1 ml beads (y4610 beads). all subjects. The authors also tested the saturation of beads with exosomes from BALF (BALF-exosomes). Here, the authors needed up to 10 times more protein to saturate the anti-MHC class II beads. Due to restricted amounts of BALF-exosomes the Bronchoalveolar lavage fluid and handling of cells authors coated the beads with the amount of BALF-exosomes that gave a mean fluorescence intensity (MFI) of o17 for Bronchoscopy with bronchoalveolar lavage (BAL) (five HLA-DR. The MFI for the isotype matched-control antibody aliquots of 50 mL phosphate buffered saline (PBS)) was was normalised to 10 fluorescent units [15]. For EM the performed as described previously [11]. The BALF was then authors used 4–5 mg protein per 1 mL beads for BALF- strained through a double layer of Dacron nets (Millipore, exosomes and 1 mg protein per 1 mL beads for MDDC- Bedford, Ireland), and centrifuged at 4006g for 10 min at exosomes. Due to the limited amount of exosomes the authors 4uC. Cell viability was determined by Trypan blue exclusion could not analyse the exosomes from all individuals for all and was w90%. For differential cell counts, cytospins were molecules with both flow cytometry and EM. prepared at 206g for 3 min and stained in May-Gru ¨ nwald Giemsa. A total of 500 cells were counted. Flow cytometry Generation of monocyte-derived dendritic cells from peripheral blood BALF-exosomes from seven healthy volunteers, MDDC- exosomes and DCs from six blood donors were analysed by Buffy coats from six healthy blood donors at the flow cytometry. The following mouse monoclonal antibodies Karolinska Hospital blood bank (Karolinska, Sweden) were (mAbs) were used: anti-HLA-DR (Fluorescein isothiocyanate used for generation of MDDCs as described previously [12, (FITC)), anti-CD14 FITC and anti-CD54 phycoeythrin (PE) 13]. Peripheral blood mononuclear cells were isolated by (Becton Dickinson, San Jose, CA, USA), anti-CD86 FITC, density centrifugation on Ficoll Paque (Amersham Pharmacia anti-CD80 FITC, anti-CD40 FITC, anti-MHC class I FITC, Biotech AB, Uppsala, Sweden). CD14z monocytes were anti-CD63 PE (PharMingen, San Diego, CA, USA), and anti- positively selected using anti-CD14 microbeads (Miltenyi CD1a PE (Coulter Corporation, Hialeah, FL, USA) and Biotech, Bergisch Gladbach, Germany) according to the compared with isotype-matched controls (Becton Dickinson). manufacturer9s instructions. Separated cells were analysed by Cells or exosomes on beads were incubated with mAbs for flow cytometry for their expression of CD14 and purity 30 min on ice, in the dark, in PBS with 0.05% sodium-azide ranged 88–96% (median 92%, n=6). The cells were diluted to 5 -1 and 0.5% BSA for cells and in PBS with 0.01% sodium-azide 4610 cells?mL and cultured for 7 days in the presence of and 0.1% BSA for exosomes. Samples were analysed by a granulocyte/macrophage colony stimulating factor and inter- Fluorescence-activated cell sorting calibur flow cytometer leukin 4 [13]. The medium was depleted from exosomes as (Becton Dickinson). A minimum of 10 cells and a minimum described previously [14]. On day seven the MDDCs were analysed by flow cytometry and had a typical immature of 5610 beads per sample were examined. The MFI for phenotype [13]. The expression of CD1a ranged 81–91% isotype-matched controls were normalised to 10 fluorescence (median 86%, n 6). = units. The variation between FACS stainings was evaluated with MDDC-exosomes using pentaplicate determinations. The MFI for the isotype control antibody mouse IgG -FITC 2a ¡ ¡ ¡ was 10.0 0.05, 10 0.05 for CD14 and 55.3 1.34 for HLA- Exosome preparations DR. Based on these observations the authors set the end- point for a positive expression at 10.2 (10.0z36SD for isotype BALF stored at -80uC from seven healthy volunteers or control antibody). cell supernatants from MDDCs cultured for 7 days, were used Cells from BALF were analysed by flow cytometry for their for exosome preparations. The exosomes were purified as expression of HLA-DR (DAKO Corporation, Carpinteria, described previously with minor modifications [3]. The super- natants were centrifuged at 3,0006g for 20 min in room CA, USA), CD14 (DAKO Corporation) and CD86 (Serotec, temperature (RT) and ultracentrifuged at 10,0006g for Oxford, UK) using primary antibodies and a secondary PE- 30 min at 4uC to remove cell debris. For the collection of conjugated anti-mouse IgG antibody (DAKO Corporation). exosomes the supernatants were ultracentrifuged at 110,000 A minimum of 10 cells per sample were examined in a 6g for 1 h at 4uC. The exosomes were washed and resuspended FACSCalibur flow cytometer. 580 C. ADMYRE ET AL. Table 1. – Bronchoalveolar lavage fluid (BALF) recovery and Results cell count data from seven healthy individuals Bronchoalveolar lavage fluid cell counts BALF recovery % 78 (67–84) # 6 Total cell count 610 16.1 (10.4–22.3) # 6 -1 BALF cells were retrieved and counted from all seven Total cell concentration 610 L 81.4 (59.3–113.8) individuals (table 1). The results were similar to previous Macrophages % 94.0 (88.4–96.5) reports of BAL cells in healthy nonsmokers [16], with a Lymphocytes % 4.6 (3.4–8.6) majority of the cells characterised as macrophages, and a few Neutrophils % 0.6 (0.4–3.0) as lymphocytes, mast cells or neutrophils. Occasionally, a few Eosinophils % 0.0 (0.0–0.2) epithelial cells could be identified. BALF cells from all seven Basophils % 0.0 (0.0–0.2) Mast cells n 3 (0–9) individuals were analysed by flow cytometry and found to HLA-DR % 81 (66–88) express HLA-DR, CD14 and CD86 (table 1). CD14 % 41 (20–58) CD86 % 66 (50–79) Exosomes are present in bronchoalveolar lavage fluid Data presented as median (range). HLA-DR: human leukocyte antigen- DR; : cell count data from staining with May-Gru ¨ nwald Giemsa; : z Exosomes were detected in BALF from all seven indivi- number of cells in 10 visual fields,616 magnification; : cell count data duals by flow cytometry. The BALF-exosomes expressed from flow cytometry analysis (minimum of 10 cells per sample HLA-DR, CD54, CD63 and CD86 (table 2 and fig. 1). examined). Further, exosomes from two out of three individuals showed expression of MHC class I (table 2). The molecules found on Electron microscopy BALF-exosomes were in agreement with those detected on MDDC-exosomes (fig. 1). In addition, the MDDC-exosomes BALF-exosomes from three healthy volunteers and MDDC- expressed CD40 and CD80, which were not detected on exosomes from one blood donor were coated to beads and BALF exosomes. The level of MHC class I and II expression incubated with a purified mouse IgG anti-CD63 mAb varied between BALF exosome donors, which was also seen (PharMingen) and a purified rat IgG anti-HLA-DR mAb 2a for MDDC-exosomes (table 2). The authors could not detect (Accurate Chemical and Scientific Corporation, Westbury, any expression of CD3 or CD14 (table 2 and fig. 1). NY, USA) overnight at 4uC. After repeated washings the Both BALF exosomes and MDDC exosomes had positive beads were incubated with a 5 nm gold conjugated anti-rat staining for CD63 and HLA-DR when analysed by immuno IgG antibody and a 10 nm gold conjugated anti-mouse IgG EM (fig. 2). The exosomes were 30–100 nm in size, in accord- antibody (Sigma Chemical Company). The exosomes-coated ance with previous findings [17]. BALF-exosomes had a beads were then pelleted (13,400 g, 30 min, at RT) and fixed higher number of anti-CD63 labelling (median, 1.7; range, in 3% glutaraldehyde in 0.1 M sodium cacodylate-HCl buffer 1.2–2.1 antibodies/exosome) than anti-HLA-DR labelling (median, (pH 7.3) with 0.05 M sucrose. The specimens were postfixed 0.1; range, 0.1–0.2 antibodies/exosome). In contrast, MDDC in 1.5% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.3) exosomes had higher HLA-DR labelling (2.5 antibodies/ with 0.5% potassium ferrocyanate for 90 min at 4uC, dehy- exosome) than CD63 labelling (0.6 antibodies/exosome). This drated in ethanol, stained with 2% uranyl acetate in ethanol, was also evident when the exosomes were analysed by flow and embedded in epoxy resin. Sections of 50–70 nm thickness cytometry (table 2 and fig. 1). were cut with a Leica Ultracut (Leica/Reichert, Vienna, Austria), picked up on grids, stained with lead citrate, and examined in a Philips CM120 EM (Philips, Eindhoven, The Discussion Netherlands). The number of gold particles were counted on 50 exosomes from each preparation and compared with isotype- The authors showed for the first time that exosomes are matched controls, mouse IgG (Dako, Glostrup, Denmark) present in BALF obtained from healthy individuals, and that and rat IgG (PharMingen), and the mean number of gold they are similar to MDDC exosomes in that they express 2a particles per exosome was calculated. HLA-DR, MHC class I, CD63, as well as CD86 and CD54. Table 2. – Characterisation of surface molecules on exosome-coated beads # # BALF exosomes MDDC exosomes z } z } MFI Experiments n MFI Experiments n HLA-DR 7 63.6 (49.3–117.3) 6 20.6 (17.0–33.4) MHC class I 3 18.2 (10.2–34.8) 3 12.7 (9.8–14.3) CD3 3 10.1 (9.9–10.1) 4 10.1 (10.0–10.2) CD14 3 9.9 (9.7–10.3) 5 9.5 (9.4–9.9) CD40 3 12.8 (11.9–16.9) 5 10.2 (10.1–10.2) CD54 3 16.0 (13.6–16.2) 3 12.0 (11.5–12.3) CD63 3 17.5 (13.4–39.6) 6 31.6 (26.8–38.0) CD80 2 10.8 (10.5–12.4) 5 9.9, 10.1 CD86 3 18.7 (15.1–21.3) 5 12.1 (11.7–13.0) All data presented as median (range) unless otherwise stated. Valuesw10.2 were considered positive. BALF: bronchoalveolar lavage fluid; MFI: mean fluorescence intensity; MDDC: monocyte-derived dendritic cell; HLA-DR: human leukocyte antigen-DR; MHC: major histocompatibility complexes; : exosomes from BALF taken from healthy individuals and exosomes from monocyte-derived dendritic cells cultured for 7 days from healthy blood donors were coated to anti-MHC class II magnetic beads and analysed by flow cytometric analysis for the expression of surface 3 } molecules. A minimum of 5610 beads per sample were examined; : number of independent experiments using exosomes from different donors; : the MFI for isotype-matched controls were normalised to 10 fluorescence units [15]. MHC AND CO-STIMULATORY MOLECULES IN BAL FLUID 581 & & & & & & & & & & & & "  # & & & & $  % Fig. 1. – Expression of surface molecules exosomes coated beads, with exosomes from bronchoalveolar lavage fluid (a), c), e), g), i), k), m), o), q)) and monocyte derived dendritic cells supernatants (b), d), f), h), j), l), n), p), r)), with controls shown in. All exosomes were coated to antimajor histocompatibility complex (MHC) class II magnetic beads and analysed by flow cytometry. All antibodies used were compared with isotype-matched controls. A minimum of 5610 beads per sample were examined. Results shown are from one experiment taken from the 2–7 experiments performed. HLA-DR: human leukocyte antigen-DR. The protein levels needed to saturate the beads with CD54 found on exosomes from DCs is in accordance with MDDC exosomes were highly reproducible, supporting the previous studies [7, 14, 15], as is also the absence of CD14 and theory that the MDDC supernatant is a reliable source of the poor expression of CD80 [15]. However, the detection of exosomes. Higher levels of protein were needed to saturate CD40 on MDDC exosomes is contradictory to the findings anti-MHC class II magnetic beads with BALF exosomes, of CLAYTON et al. [15] who were unable to detect CD40. probably because exosomes from BALF come from different MHC and co-stimulatory molecules are not only present on cellular sources, including MHC class II negative cells. exosomes from DCs, therefore the exosomes detected in Another reason why higher protein levels are needed for BALF might also be derived from other cell types. Exosomes saturation of anti-MHC class II magnetic beads with BALF from intestinal epithelial cells bear both MHC class I and II exosomes compared with MDDC exosomes could be that molecules, besides CD63 [6]. CD54, CD86, CD40 and MHC the purification of exosomes from BALF might lead to class II have been found on mast cell-derived exosomes [19]. co-purification of surfactant, which is a complex mixture of Moreover, CD54, CD86, CD63 and MHC class I and II have lipids and proteins of vital importance for lung function [18]. been detected on exosomes derived from B-cells [3, 20, 15]. Therefore, surfactant could contribute to the total protein Exosomes from T-cells have been shown to bear CD63 and content in the exosome preparations from BALF. The reason MHC class I and II molecules [4]. 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Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid

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Copyright ERS Journals Ltd 2003 Eur Respir J 2003; 22: 578–583 European Respiratory Journal DOI: 10.1183/09031936.03.00041703 ISSN 0903-1936 Printed in UK – all rights reserved FRONT ROWS OF SCIENCE Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid # } # # # C. Admyre*, J. Grunewald , J. Thyberg , S. Gripenba ¨ck , G. Tornling , A. Eklund , A. Scheynius*, S. Gabrielsson* *Dept of Medicine, Unit of Clinical Allergy Exosomes with major histocompatibility complex class II and co-stimulatory molecules Research, Division of Respiratory Medicine are present in human BAL fluid. C. Admyre, J. Grunewald, J. Thyberg, S. Gripenba ¨ ck, and Dept of Cell and Molecular Biology, G. Tornling, A. Eklund, A. Scheynius, S. Gabrielsson. #ERS Journals Ltd 2003. Karolinska Hospital and Institutet, Stockholm, ABSTRACT: Exosomes are 30–100 nm diameter vesicles formed by inward budding of Sweden. endosomal compartments and are produced by several cell types, including T-cells, B- cells and dendritic cells (DC)s. Exosomes from DCs express major histocompatibility Correspondence: C. Admyre, Dept of Medicine, complexes (MHC) class I and II, and co-stimulatory molecules on their surface, and Unit of Clinical Allergy Research, Karolinska can induce antigen-specific activation of T-cells. Hospital and Institutet, Stockholm, Sweden. Fax: 46 8335724 The aims of the present study were to investigate for the presence of exosomes in E-mail: [email protected] bronchoalveolar lavage fluid (BALF) from healthy individuals, and to establish if these exosomes bear MHC and co-stimulatory molecules. Keywords: Bronchoalveolar lavage fluid fluid, The authors analysed BALF taken from seven healthy volunteers and used exosomes CD54, CD86, exosomes, human leukocyte from monocyte-derived DC (MDDC) cultures as a reference. After ultracentrifugation, antigen-DR exosomes were bound to anti-MHC class II coated magnetic beads and analysed by flow cytometry and electron microscopy. Received: April 14 2003 The authors report for the first time that exosomes are present in BALF. These Accepted after revision: May 29 2003 exosomes are similar to MDDC derived exosomes as they express MHC class I and II, This work was supported by the Swedish CD54, CD63 and the co-stimulatory molecule CD86. Research Council (grants no. 16x-7924, 74x- The results demonstrate that exosomes are present in the lung, and since they contain 14182, and 6537), the Swedish Council for both major histocompatibility complex and co-stimulatory molecules it is likely that Work Life Sciences, the Swedish Asthma and they are derived from antigen presenting cells and might have a regulatory role in local Allergy Association9s Research Foundation, immune defence. the Hesselman Foundation, Konsul TH Bergs Eur Respir J 2003; 22: 578–583. Foundation, the Swedish Heart-Lung Foun- dation, the Va ˚ rdal Foundation, the Centre for Allergy Research at Karolinska Institutet, the King Oscar II Jubilee Foundation, the King Gustaf V 80th Birthday Fund and the Kar- olinska Institutet. Exosomes are 30–100 nm diameter vesicles produced by on the cell surface, such as the tetraspan proteins CD63 and different cell types, including reticulocytes [1], platelets [2], B- CD82 [7], which interact with membrane proteins, such as lymphocytes [3], T-lymphocytes [4], mast cells [5], epithelial integrins and human leukocyte antigen (HLA)-DR [10]. cells [6] and dendritic cells (DC)s [7]. Exosomes are formed by Exosomes may not only have a role in T-cell activation, but inward budding of the limiting membrane of endosomal may also function as a communicator between cells in the compartments, called multivesicular bodies (MVB)s [8]. In immune system. Intestinal epithelial cells secrete exosome-like antigen presenting cells (APC)s MVBs are the main site for vesicles, which favour cross communication between the loading major histocompatibility complex (MHC) class II and intestinal epithelium and the T-cells in the mucosa or in the recycling MHC class I molecules with exogenous antigens. blood [6]. Upon T-cell receptor (TCR) triggering, T-cells The MHC molecules are located both on the limiting produce exosome-like vesicles expressing TCR and CD3, membrane and on the internal vesicles. When MVBs fuse which may deliver signals to target cells baring the right with the plasma membrane the internal vesicles are released MHC-peptide complex [4]. extracellularly and become exosomes [3]. APCs secrete The lung is constantly exposed to inhaled particles and has exosomes that carry peptide-loaded MHC molecules, which numerous capillaries making its9 mucosa a vulnerable surface can stimulate T-cell proliferation in vitro [3]. T-cell stimula- between the environment and internal tissues. Therefore, it is tion has also been seen when using exosomes from rat mast important to have a powerful local immune defence. Whether cells engineered to express mouse or human MHC class II exosomes are part of such a defence in the lung has not molecules [9]. In a mouse model, exosomes have shown a previously been addressed. The authors of the present study good potential for use as a tool in cancer therapies, where investigated if exosomes are present in human broncho- exosomes with tumour-peptide loaded MHC molecules were alveolar lavage fluid (BALF) from healthy individuals, using used for tumour eradication [7]. Exosomes from APCs do not exosomes from monocyte-derived DCs (MDDCs) as a only express MHC molecules, but also co-stimulatory mole- reference. The authors show for the first time, using flow cules, such as CD86. They further express proteins not detected cytometry and electron microscopy (EM), that exosomes are MHC AND CO-STIMULATORY MOLECULES IN BAL FLUID 579 present in BALF. The authors found that these exosomes in PBS and stored at -80uC. Exosome samples were measured selected for MHC class II expression, such as exosomes for protein content using BioRad DC protein assay (Bio-Rad derived from MDDC cultures, express HLA-DR, MHC class Laboratories, Hercules, CA, USA) according to the manu- I, CD54, CD63 and the co-stimulatory molecule CD86. facturer9s instructions. The protein content of the exosome -1 preparations from BALF ranged 0.20–0.51 mg protein?mL , -1 median 0.30 mg protein?mL , of original volume BALF, n 7. Materials and methods Subjects Exosome coating to beads BALF from seven healthy nonsmoking individuals aged Exosomes were adsorbed onto dynabeads precoated with 22–51 yrs (median 25 yrs), two males and five females, were mouse IgM antihuman MHC class II (Dynal1, Oslo, Norway) analysed. None had a history of atopy, and all had normal -1 in PBS containing 0.1% bovine serum albumin (BSA) and serum Immunoglobulin (Ig)E values (v120 kUL , Immuno- 0.01% sodium-azide overnight at RT [15]. Pilot experiments Cap, Pharmacia Diagnostics, Uppsala, Sweden), normal chest with exosomes from MDDC supernatants (MDDC-exosomes) radiographs and were without signs of respiratory infection showed the beads to be saturated with exosomes at a for o1 month prior to the study. The local ethics committee approved the study and informed consent was obtained from concentration of 1 mg protein to 1 ml beads (y4610 beads). all subjects. The authors also tested the saturation of beads with exosomes from BALF (BALF-exosomes). Here, the authors needed up to 10 times more protein to saturate the anti-MHC class II beads. Due to restricted amounts of BALF-exosomes the Bronchoalveolar lavage fluid and handling of cells authors coated the beads with the amount of BALF-exosomes that gave a mean fluorescence intensity (MFI) of o17 for Bronchoscopy with bronchoalveolar lavage (BAL) (five HLA-DR. The MFI for the isotype matched-control antibody aliquots of 50 mL phosphate buffered saline (PBS)) was was normalised to 10 fluorescent units [15]. For EM the performed as described previously [11]. The BALF was then authors used 4–5 mg protein per 1 mL beads for BALF- strained through a double layer of Dacron nets (Millipore, exosomes and 1 mg protein per 1 mL beads for MDDC- Bedford, Ireland), and centrifuged at 4006g for 10 min at exosomes. Due to the limited amount of exosomes the authors 4uC. Cell viability was determined by Trypan blue exclusion could not analyse the exosomes from all individuals for all and was w90%. For differential cell counts, cytospins were molecules with both flow cytometry and EM. prepared at 206g for 3 min and stained in May-Gru ¨ nwald Giemsa. A total of 500 cells were counted. Flow cytometry Generation of monocyte-derived dendritic cells from peripheral blood BALF-exosomes from seven healthy volunteers, MDDC- exosomes and DCs from six blood donors were analysed by Buffy coats from six healthy blood donors at the flow cytometry. The following mouse monoclonal antibodies Karolinska Hospital blood bank (Karolinska, Sweden) were (mAbs) were used: anti-HLA-DR (Fluorescein isothiocyanate used for generation of MDDCs as described previously [12, (FITC)), anti-CD14 FITC and anti-CD54 phycoeythrin (PE) 13]. Peripheral blood mononuclear cells were isolated by (Becton Dickinson, San Jose, CA, USA), anti-CD86 FITC, density centrifugation on Ficoll Paque (Amersham Pharmacia anti-CD80 FITC, anti-CD40 FITC, anti-MHC class I FITC, Biotech AB, Uppsala, Sweden). CD14z monocytes were anti-CD63 PE (PharMingen, San Diego, CA, USA), and anti- positively selected using anti-CD14 microbeads (Miltenyi CD1a PE (Coulter Corporation, Hialeah, FL, USA) and Biotech, Bergisch Gladbach, Germany) according to the compared with isotype-matched controls (Becton Dickinson). manufacturer9s instructions. Separated cells were analysed by Cells or exosomes on beads were incubated with mAbs for flow cytometry for their expression of CD14 and purity 30 min on ice, in the dark, in PBS with 0.05% sodium-azide ranged 88–96% (median 92%, n=6). The cells were diluted to 5 -1 and 0.5% BSA for cells and in PBS with 0.01% sodium-azide 4610 cells?mL and cultured for 7 days in the presence of and 0.1% BSA for exosomes. Samples were analysed by a granulocyte/macrophage colony stimulating factor and inter- Fluorescence-activated cell sorting calibur flow cytometer leukin 4 [13]. The medium was depleted from exosomes as (Becton Dickinson). A minimum of 10 cells and a minimum described previously [14]. On day seven the MDDCs were analysed by flow cytometry and had a typical immature of 5610 beads per sample were examined. The MFI for phenotype [13]. The expression of CD1a ranged 81–91% isotype-matched controls were normalised to 10 fluorescence (median 86%, n 6). = units. The variation between FACS stainings was evaluated with MDDC-exosomes using pentaplicate determinations. The MFI for the isotype control antibody mouse IgG -FITC 2a ¡ ¡ ¡ was 10.0 0.05, 10 0.05 for CD14 and 55.3 1.34 for HLA- Exosome preparations DR. Based on these observations the authors set the end- point for a positive expression at 10.2 (10.0z36SD for isotype BALF stored at -80uC from seven healthy volunteers or control antibody). cell supernatants from MDDCs cultured for 7 days, were used Cells from BALF were analysed by flow cytometry for their for exosome preparations. The exosomes were purified as expression of HLA-DR (DAKO Corporation, Carpinteria, described previously with minor modifications [3]. The super- natants were centrifuged at 3,0006g for 20 min in room CA, USA), CD14 (DAKO Corporation) and CD86 (Serotec, temperature (RT) and ultracentrifuged at 10,0006g for Oxford, UK) using primary antibodies and a secondary PE- 30 min at 4uC to remove cell debris. For the collection of conjugated anti-mouse IgG antibody (DAKO Corporation). exosomes the supernatants were ultracentrifuged at 110,000 A minimum of 10 cells per sample were examined in a 6g for 1 h at 4uC. The exosomes were washed and resuspended FACSCalibur flow cytometer. 580 C. ADMYRE ET AL. Table 1. – Bronchoalveolar lavage fluid (BALF) recovery and Results cell count data from seven healthy individuals Bronchoalveolar lavage fluid cell counts BALF recovery % 78 (67–84) # 6 Total cell count 610 16.1 (10.4–22.3) # 6 -1 BALF cells were retrieved and counted from all seven Total cell concentration 610 L 81.4 (59.3–113.8) individuals (table 1). The results were similar to previous Macrophages % 94.0 (88.4–96.5) reports of BAL cells in healthy nonsmokers [16], with a Lymphocytes % 4.6 (3.4–8.6) majority of the cells characterised as macrophages, and a few Neutrophils % 0.6 (0.4–3.0) as lymphocytes, mast cells or neutrophils. Occasionally, a few Eosinophils % 0.0 (0.0–0.2) epithelial cells could be identified. BALF cells from all seven Basophils % 0.0 (0.0–0.2) Mast cells n 3 (0–9) individuals were analysed by flow cytometry and found to HLA-DR % 81 (66–88) express HLA-DR, CD14 and CD86 (table 1). CD14 % 41 (20–58) CD86 % 66 (50–79) Exosomes are present in bronchoalveolar lavage fluid Data presented as median (range). HLA-DR: human leukocyte antigen- DR; : cell count data from staining with May-Gru ¨ nwald Giemsa; : z Exosomes were detected in BALF from all seven indivi- number of cells in 10 visual fields,616 magnification; : cell count data duals by flow cytometry. The BALF-exosomes expressed from flow cytometry analysis (minimum of 10 cells per sample HLA-DR, CD54, CD63 and CD86 (table 2 and fig. 1). examined). Further, exosomes from two out of three individuals showed expression of MHC class I (table 2). The molecules found on Electron microscopy BALF-exosomes were in agreement with those detected on MDDC-exosomes (fig. 1). In addition, the MDDC-exosomes BALF-exosomes from three healthy volunteers and MDDC- expressed CD40 and CD80, which were not detected on exosomes from one blood donor were coated to beads and BALF exosomes. The level of MHC class I and II expression incubated with a purified mouse IgG anti-CD63 mAb varied between BALF exosome donors, which was also seen (PharMingen) and a purified rat IgG anti-HLA-DR mAb 2a for MDDC-exosomes (table 2). The authors could not detect (Accurate Chemical and Scientific Corporation, Westbury, any expression of CD3 or CD14 (table 2 and fig. 1). NY, USA) overnight at 4uC. After repeated washings the Both BALF exosomes and MDDC exosomes had positive beads were incubated with a 5 nm gold conjugated anti-rat staining for CD63 and HLA-DR when analysed by immuno IgG antibody and a 10 nm gold conjugated anti-mouse IgG EM (fig. 2). The exosomes were 30–100 nm in size, in accord- antibody (Sigma Chemical Company). The exosomes-coated ance with previous findings [17]. BALF-exosomes had a beads were then pelleted (13,400 g, 30 min, at RT) and fixed higher number of anti-CD63 labelling (median, 1.7; range, in 3% glutaraldehyde in 0.1 M sodium cacodylate-HCl buffer 1.2–2.1 antibodies/exosome) than anti-HLA-DR labelling (median, (pH 7.3) with 0.05 M sucrose. The specimens were postfixed 0.1; range, 0.1–0.2 antibodies/exosome). In contrast, MDDC in 1.5% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.3) exosomes had higher HLA-DR labelling (2.5 antibodies/ with 0.5% potassium ferrocyanate for 90 min at 4uC, dehy- exosome) than CD63 labelling (0.6 antibodies/exosome). This drated in ethanol, stained with 2% uranyl acetate in ethanol, was also evident when the exosomes were analysed by flow and embedded in epoxy resin. Sections of 50–70 nm thickness cytometry (table 2 and fig. 1). were cut with a Leica Ultracut (Leica/Reichert, Vienna, Austria), picked up on grids, stained with lead citrate, and examined in a Philips CM120 EM (Philips, Eindhoven, The Discussion Netherlands). The number of gold particles were counted on 50 exosomes from each preparation and compared with isotype- The authors showed for the first time that exosomes are matched controls, mouse IgG (Dako, Glostrup, Denmark) present in BALF obtained from healthy individuals, and that and rat IgG (PharMingen), and the mean number of gold they are similar to MDDC exosomes in that they express 2a particles per exosome was calculated. HLA-DR, MHC class I, CD63, as well as CD86 and CD54. Table 2. – Characterisation of surface molecules on exosome-coated beads # # BALF exosomes MDDC exosomes z } z } MFI Experiments n MFI Experiments n HLA-DR 7 63.6 (49.3–117.3) 6 20.6 (17.0–33.4) MHC class I 3 18.2 (10.2–34.8) 3 12.7 (9.8–14.3) CD3 3 10.1 (9.9–10.1) 4 10.1 (10.0–10.2) CD14 3 9.9 (9.7–10.3) 5 9.5 (9.4–9.9) CD40 3 12.8 (11.9–16.9) 5 10.2 (10.1–10.2) CD54 3 16.0 (13.6–16.2) 3 12.0 (11.5–12.3) CD63 3 17.5 (13.4–39.6) 6 31.6 (26.8–38.0) CD80 2 10.8 (10.5–12.4) 5 9.9, 10.1 CD86 3 18.7 (15.1–21.3) 5 12.1 (11.7–13.0) All data presented as median (range) unless otherwise stated. Valuesw10.2 were considered positive. BALF: bronchoalveolar lavage fluid; MFI: mean fluorescence intensity; MDDC: monocyte-derived dendritic cell; HLA-DR: human leukocyte antigen-DR; MHC: major histocompatibility complexes; : exosomes from BALF taken from healthy individuals and exosomes from monocyte-derived dendritic cells cultured for 7 days from healthy blood donors were coated to anti-MHC class II magnetic beads and analysed by flow cytometric analysis for the expression of surface 3 } molecules. A minimum of 5610 beads per sample were examined; : number of independent experiments using exosomes from different donors; : the MFI for isotype-matched controls were normalised to 10 fluorescence units [15]. MHC AND CO-STIMULATORY MOLECULES IN BAL FLUID 581 & & & & & & & & & & & & "  # & & & & $  % Fig. 1. – Expression of surface molecules exosomes coated beads, with exosomes from bronchoalveolar lavage fluid (a), c), e), g), i), k), m), o), q)) and monocyte derived dendritic cells supernatants (b), d), f), h), j), l), n), p), r)), with controls shown in. All exosomes were coated to antimajor histocompatibility complex (MHC) class II magnetic beads and analysed by flow cytometry. All antibodies used were compared with isotype-matched controls. A minimum of 5610 beads per sample were examined. Results shown are from one experiment taken from the 2–7 experiments performed. HLA-DR: human leukocyte antigen-DR. The protein levels needed to saturate the beads with CD54 found on exosomes from DCs is in accordance with MDDC exosomes were highly reproducible, supporting the previous studies [7, 14, 15], as is also the absence of CD14 and theory that the MDDC supernatant is a reliable source of the poor expression of CD80 [15]. However, the detection of exosomes. Higher levels of protein were needed to saturate CD40 on MDDC exosomes is contradictory to the findings anti-MHC class II magnetic beads with BALF exosomes, of CLAYTON et al. [15] who were unable to detect CD40. probably because exosomes from BALF come from different MHC and co-stimulatory molecules are not only present on cellular sources, including MHC class II negative cells. exosomes from DCs, therefore the exosomes detected in Another reason why higher protein levels are needed for BALF might also be derived from other cell types. Exosomes saturation of anti-MHC class II magnetic beads with BALF from intestinal epithelial cells bear both MHC class I and II exosomes compared with MDDC exosomes could be that molecules, besides CD63 [6]. CD54, CD86, CD40 and MHC the purification of exosomes from BALF might lead to class II have been found on mast cell-derived exosomes [19]. co-purification of surfactant, which is a complex mixture of Moreover, CD54, CD86, CD63 and MHC class I and II have lipids and proteins of vital importance for lung function [18]. been detected on exosomes derived from B-cells [3, 20, 15]. Therefore, surfactant could contribute to the total protein Exosomes from T-cells have been shown to bear CD63 and content in the exosome preparations from BALF. The reason MHC class I and II molecules [4]. 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European Respiratory Journal – Unpaywall

Published: Oct 1, 2003

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