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. Author manuscript; available in PMC: 2012 Jun 15.
Published in final edited form as: Stem Cells. 2007 Jun 7;25(9):2245–2256. doi: 10.1634/stemcells.2007-0128

Alteration of Marrow Cell Gene Expression, Protein Production, and Engraftment into Lung by Lung-Derived Microvesicles: A Novel Mechanism for Phenotype Modulation

Jason M Aliotta a, Fermin M Sanchez-Guijo b, Gerri J Dooner a, Kevin W Johnson a, Mark S Dooner a, Kenneth A Greer b, Deborah Greer b, Jeffrey Pimentel b, Luiz M Kolankiewicz b, Napoleon Puente b, Sam Faradyan b, Paulette Ferland c, Elaine L Bearer c, Michael A Passero b, Mehrdad Adedi b, Gerald A Colvin a, Peter J Quesenberry a
PMCID: PMC3376082  NIHMSID: NIHMS241761  PMID: 17556595

Abstract

Numerous animal studies have demonstrated that adult marrow-derived cells can contribute to the cellular component of the lung. Lung injury is a major variable in this process; however, the mechanism remains unknown. We hypothesize that injured lung is capable of inducing epigenetic modifications of marrow cells, influencing them to assume phenotypic characteristics of lung cells. We report that under certain conditions, radiation-injured lung induced expression of pulmonary epithelial cell-specific genes and prosurfactant B protein in cocultured whole bone marrow cells separated by a cell-impermeable membrane. Lung-conditioned media had a similar effect on cocultured whole bone marrow cells and was found to contain pulmonary epithelial cell-specific RNA-filled microvesicles that entered whole bone marrow cells in culture. Also, whole bone marrow cells cocultured with lung had a greater propensity to produce type II pneumocytes after transplantation into irradiated mice. These findings demonstrate alterations of marrow cell phenotype by lung-derived microvesicles and suggest a novel mechanism for marrow cell-directed repair of injured tissue.

Keywords: Adult bone marrow stem cells, Bone marrow transplantation, In vitro differentiation, Irradiation, Microvesicles

INTRODUCTION

The ability of adult bone marrow-derived cells to contribute to the cellular component of nonhematopoietic organs, including the lung, has been demonstrated. Injury is important in this phenomenon. Investigators have used radiation [1–5], bleomycin, [6–8] elastase [9], and monocrotaline [10] injuries to demonstrate this effect. This process may be injury-specific, as endotoxin and inhaled nitric oxide did not significantly affect marrow cell production of lung cells in radiation-injured mice [11]. Donor marrow cell-derived lung cell production has been demonstrated in transgenic mice, including cystic fibrosis trans-membrane conductance regulator gene (CFTR) knockout [12], NOD/SCID [13], and tight skin (emphysematous) mice [14], as well as parabiotic [15] and newborn mice [16]. Different donor populations engraft in injured lung, including whole bone marrow (WBM) [1–5, 8, 11, 12, 14, 17], mesenchymal cells [6, 7, 10, 12, 13], purified hematopoietic stem cells [1, 2, 18], bone marrow side population cells [4, 19], and fetal liver cells [9]. High numbers (20% or more) of type II pneumocytes [18] and pulmonary fibroblasts [8, 15] have been described as marrow-derived, whereas others have reported few (less than 0.1% of all lung cells) marrow cell-derived type I pneumocytes [7, 15], airway epithelial cells [11, 12, 14, 18], endothelial, and smooth muscle cells [10]. It is possible that differences in experimental design, including different transplanted cell populations, mode of lung injury, strain of mouse, and time from transplantation to analysis, could account for these discrepancies.

Investigators have reported that marrow cells are unable to repopulate the injured lung and have attributed the presence of transplanted marrow-derived lung cells to autofluorescent artifact or cell overlap [20, 21]. We have addressed this issue using anti-green fluorescent protein (GFP) antibodies linked to a red fluorochrome in GFP to non-GFP murine transplant experiments and double band-pass filters. Autofluorescence did not create artifacts in these studies, and because deconvolution fluorescent microscopy was used, overlapping cells were not a confounding variable [1]. Thus, marrow cells have the capacity, in vivo, to participate in the production of nonhematopoietic lung cells with epithelial markers.

We hypothesize that injured lung cells are capable of inducing epigenetic modifications of marrow cells, influencing them to assume a lung phenotype. To test this, we used a system in which radiation-injured lung was cultured with marrow cells but separated from them by a 0.4-µm cell-impermeable membrane. The cocultured marrow cells were assessed by immunohistochemical analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) for a variety of lung cell-specific markers. We report that radiation-injured lung is capable of inducing lung cell-specific mRNA and protein production in cocultured marrow cells, a process that may be mediated by the release of RNA-containing microvesicles by lung cells.

MATERIALS AND METHODS

Experimental Animals and Organ Harvest

Six- to 8-week-old female or male C57BL/6 (H2Kb) or C57BL/-TgN (ACTbEGFP)1Osb mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) were bred in our animal facility by mating heterozygous GFP-positive mice to C57BL/6 mice to produce GFP-positive transgenic mice. Animals were given ad libitum access to food and water. All studies were approved by the Institutional Animal Care and Use Committee of Roger Williams Medical Center.

For WBM harvest, mice were anesthetized with inhaled halothane and sacrificed by cervical dislocation. WBM was obtained by flushing the femurs, tibias, and pelvic bones with sterile 1X phosphate-buffered saline (PBS). For lungs used in coculture experiments, the pulmonary vasculature was flushed with 1X PBS. Lungs were minced and placed in coculture. For lungs used in posttransplantation studies, the pulmonary vasculature was flushed with 1X PBS. Lungs were then inflated with 1.5 ml of ice-cold balanced phosphate solution with 2% paraformaldehyde, sodium m-perio-date, and l-lysine (PLP) and transferred to a 7% sucrose solution. The following day, lung pieces were placed into ethanol solutions of increasing concentrations and mineral spirits and embedded in paraffin.

Lung, Marrow Cell Coculture Experiments

Cohorts of C57BL/6 mice were exposed to either 500 or 1,200 cGy total body irradiation (TBI) using a Gammacell 40 Exactor Irradiator at 110 cGy/minute (MDS Nordion, Ottawa, ON, Canada, http://www.mds.nordion.com). Other cohorts received no TBI (n = 3 in each of the three experimental arms). Mice were sacrificed and their lungs extracted 3 hours, 5 days, or 14 days after TBI. Irradiated mice sacrificed 14 days after TBI, as well as the nonirradiated control mice in these experiments, received 5 × 106 WBM cells by tail vein injection to prevent bone marrow failure and death prior to the 14-day time point. Six-well tissue culture plates (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) were filled with 5 ml of Dexter culture medium, consisting of Fischer medium with 100 U/ml penicillin 100 µg/ml streptomycin, 0.0125 µg/ml fungizone, 10−7 M hydrocortisone sodium succinate (all from Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and 20% horse serum (HyClone, Logan, UT, http://www.hyclone.com). Millicell culture plate inserts (Millipore, Billerica, MA, http://www.millipore.com), with a membrane containing 0.4-µm pores, were placed into each well. Minced lung from individual mice was divided into equal portions and placed on top of separate well insert membranes (half of one mouse’s lungs placed in each well). Viable GFP+ WBM cells (1 × 106 cells per cm2; 1 × 107 viable WBM cells total per well) were plated beneath the membrane of well inserts. Alternatively, WBM from C57BL/6 mice was used. Coculture was established using lungs from three individual mice in each treatment arm (no TBI or 500 or 1,200 cGy TBI). In addition, kidneys were removed from the same mice and placed in coculture with WBM. As a control, WBM was cultured in the absence of lung (or kidney, in the case of kidney, WBM coculture experiments). Culture plates were incubated at 33°C in 5% CO2 for 48 hours or 7 days (Fig. 1A). WBM was collected for morphological assessment or for RNA extraction using the RNeasy Mini Total RNA Purification Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com), treated with DNase, and used for real-time RT-PCR.

Figure 1.

Figure 1

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Summary of experimental designs. (A): Lung, WBM coculture (three experiments). (B): LCM, WBM coculture (radiation-injured LCM, five experiments; nonirradiated LCM, three experiments). (C): RNase-treated LCM, WBM coculture (two experiments). (D): Transplantation of WBM cocultured with lung or no lung or uncultured WBM into irradiated mice (one experiment). Abbreviations: LCM, lung-conditioned media; WBM, whole bone marrow.

Prosurfactant B Production by Cocultured Marrow Cells

Lung, marrow cocultures were established, as described above, using lungs harvested from mice exposed to 500 cGy TBI 5 days prior to establishment of coculture or nonirradiated lungs. As a control, WBM was cultured in the absence of lung. After 7 days of coculture, half of the WBM cells in each coculture group were analyzed by standard morphology, immunohistochemistry, and RT-PCR. The remaining WBM cells remained in culture; however, lungs were removed, and WBM from each group was pooled, transferred to separate tissue culture flasks, and cultured in Dexter medium-containing recombinant mouse (rm) interleukin (IL)-3 (50 U/ml), rmIL-6 (50 U/ml), and recombinant human IL-11 (50 ng/ml) or medium-containing no cytokines for 21 days. Fifty percent of media were replaced with fresh media twice each week. Cultured WBM was then analyzed as described above.

Lung-Conditioned Media, Marrow Cell Coculture Experiments

Lungs from nonirradiated mice or mice exposed to 500 cGy TBI 5 days prior to lung harvest were cultured in Dexter medium for 5 days to produce lung-conditioned media (LCM). LCM was confirmed to contain no cells by centrifugation at 300g for 10 minutes, performing Wright-Giemsa staining on the pellet, and evaluating the pellet by light microscopy. LCM was then placed on top of culture well inserts opposite WBM cells in Dexter medium. LCM used in each coculture well was derived from half of one mouse’s lung. Every 12 hours, half of the LCM was replaced with fresh LCM. LCM, WBM cocultures were maintained for 48 hours or 7 days and then analyzed by RT-PCR (Fig. 1B).

RNase-Treated Lung-Conditioned Media, Marrow Cell Coculture Experiments

Pooled LCM made from mice exposed to 500 cGy TBI 5 days prior to lung harvest was treated with RNase (50 U/ml of RNase A and 2,000 U/ml of RNase T1, final concentration; Ambion, Austin, TX, http://www.ambion.com) for 5 hours at room temperature. The RNase-treated medium or untreated medium was placed on top of culture well inserts opposite WBM for 7 days (Fig. 1C). WBM was analyzed by RT-PCR.

Microvesicle Isolation from Ultracentrifuged LCM

LCM made from GFP+ mice exposed to 500 cGy TBI 5 days prior to lung harvest was ultracentrifuged at 28,000g for 60 minutes at 4°C using a Beckman L8–80 Ultracentrifuge (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). The pellet was resuspended in 1X PBS with HEPES (5 mM; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and centrifuged twice at 18,000g for 30 minutes at 4°C. Half of the washed pellet was lysed for RNA extraction. The other half was labeled with PKH26 dye (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at a 1:250 dilution and incubated for 4 minutes at room temperature. The pellet was then washed three times by centrifugation at 18,000g for 10 minutes at room temperature and resuspended in 1X PBS/5 mM HEPES.

Isolation of GFP+/PHK26+ Lung-Conditioned Media-Derived Particles Using Flow Cytometry

Events were sorted on a MoFlo modular flow high-speed cell sorter (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com). Samples from single-color PKH26-stained and GFP+ ultracentrifuged LCM pellet were used to determine spectral overlap for compensation parameters. GFP+/PKH26+ events from the PKH26-labeled ultracentrifuged pellet derived from GFP+ LCM were then sorted. Particles were either lysed for RNA extraction or cultured with WBM.

GFP+/PKH26+ Lung-Conditioned Media-Derived Particle, Marrow Cell Coculture

Isolated GFP+/PKH+ particles were added to culture wells containing C57BL/6 WBM in Dexter culture medium at a ratio of 100 viable WBM cells to 1 sorted particle. Culture plates were incubated for 48 hours. Cocultured WBM cells were then evaluated by fluorescence microscopy or flow cytometry.

Negative Stain Electron Microscopy of Lung-Conditioned Media-Derived Ultracentrifuged Pellet

Copper grids (200 mesh) were floated on 3–10 µl of a 1X PBS-resuspended ultracentrifuged pellet and transferred sequentially through three 20-µl droplets of 1% aqueous uranyl acetate. Prior to application of the sample, copper grids were prepared by sonication in ethanol, coated with Formvar (Ladd Research, Williston, VT, http://www.laddresearch.com), carbon coated by evaporation, glow-discharged, and deionized. Images were captured on a JEOL CX200 electron microscope (JEOL, Tokyo, http://www.jeol.com) and processed with Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, http://www.adobe.com).

cDNA Amplification and Real-Time RT-PCR

RNA (10–20 ng) extracted from samples (WBM, lung, LCM, the ultracentrifuged pellet from LCM, and sorted particles from the LCM pellet) was used to amplify cDNA with the following reagents (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) in a final volume of 100 µl (per the manufacturer’s instructions): 10X RT buffer, MgCl2, dNTP mix, random hexamers, RNase Inhibitor, and Multiscribe. Amplification reactions consisted of one cycle for 10 minutes at 25°C, one cycle for 60 minutes at 37°C, and one cycle for 5 minutes at 95°C. Gene expression was analyzed by RT-PCR using an ABI Prism 7000 sequence detection system (Applied Biosystems). All 20X assay mixes were purchased from Applied Biosystems; mixes include primers for the housekeeping gene β2 microglobulin (catalog no. Mm00437762_m1) and the following target genes: surfactant protein B (Mm00455681_m1), surfactant protein C (Mm00488144_m1), cytokeratin 5 (Mm00503549_m1), cytokeratin 14 (Mm00516876_m1), Clara cell-specific protein (Mm00442046_m1), Sca-1 (Mm00726565_s1), c-kit (Mm00445212_m1), CD34 (Mm00519283_m1), CD45 (Mm00448463_m1), vascular endothelial growth factor receptor 1 (VEGFR-1) (Mm00438980_m1), VEGFR-2 (Mm00440099_m1), CXCR4 (4329577T), CD44 (Mm01277164_m1), P-selectin (Mm00441295_m1), L-selectin (Mm00441291_m1), platelet/endothelial cell adhesion molecule (PECAM) (Mm00476702_m1), and vascular cell adhesion molecule (Mm00449197_m1). All reactions were performed in 96-well plates with the following reagents in a final volume of 25 µl: 20X assay mix (for either β2 microglobulin or one of the target genes) and 2X TaqMan PCR Master Mix. Ten nanograms of cDNA was added to this mixture. Duplicate reactions of the target and housekeeping genes were performed simultaneously for each cDNA template analyzed. The PCR consisted of an initial enzyme activation step at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. A cycle threshold value (CT) value was obtained for each sample, and duplicate sample values were averaged. The 2−ΔΔCT method was used to calculate relative expression of each target gene [22]. Briefly, mean CT value of target genes in each sample were normalized to its averaged housekeeping gene CT value to give a ΔCT value. This was then normalized to control samples (ΔΔCT), and the 2−ΔΔCT value was obtained. To calculate 2−ΔΔCT for target genes with no expression in the control group, a CT value of 40 was assigned to the control group so that a relative quantity of the target gene could be reported. The control group used for all comparisons was WBM cocultured in the absence of lung unless otherwise specified. The ΔCT values of all above-mentioned lung cell genes (Clara cell-specific protein, surfactants B and C, and cytokeratins 5 and 14) using freshly aspirated WBM as a template were not significantly different from values obtained using WBM cultured without lung as a template.

Transplantation of WBM Cocultured with Lung

Lungs from mice exposed to 500 cGy TBI 5 days prior to lung harvest or nonirradiated lung were cocultured with 1 × 107 GFP+ WBM cells for 7 days. In addition, 1 × 107 GFP+ WBM cells were cultured without lung for 7 days. WBM cells were collected for RT-PCR analysis or for tail vein injection into 1,200 cGy-irradiated mice. Cocultured WBM from individual culture wells were transplanted into irradiated mice, which were sacrificed 6 weeks later for determination of GFP+/prosurfactant C+ lung cells (Fig. 1D).

Determination of Peripheral Blood Chimerism of Transplanted Mice

Peripheral blood was obtained from the tail veins of mice 1 day prior to sacrifice, and GFP+ cells were quantified by fluorescence-activated cell sorting (FACS) analysis. Peripheral blood chimerism was determined by comparing the percentage of GFP+ cells in samples to the percentage of GFP+ cells in a positive control (peripheral blood from a GFP+ mouse) after background subtraction.

Immunohistochemical Labeling

For paraffin-embedded lung samples, 5-µm lung sections were deparaffinized and digested with proteinase K (5 µg/ml) for 3 minutes at 37°C. They were fixed with 10% buffered formalin and blocked with 20% horse serum. Rabbit polyclonal antibody to prosurfactant B or C (Abcam, Cambridge, U.K., http://www.abcam.com) was added to each sample at a 1:1,000 dilution and incubated for 2 hours at room temperature. The secondary antibody, goat anti-rabbit Alexa Fluor-594 (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) was added at a 1:400 dilution and incubated for 30 minutes at room temperature. Vectashield (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) with 0.4 mmol of 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) was added to counterstain nuclei.

Cytospin samples were permeabilized with 0.05% Triton-X for 10 minutes, followed by proteinase K (5 µg/ml) digestion for 1 minute at 37°C. Samples were fixed with 10% buffered formalin and blocked with 20% horse serum. Rabbit polyclonal antibody to prosurfactant B or C was added to each sample (1:1,000 dilution) and incubated for 2 hours at room temperature. The secondary antibody, goat anti-rabbit Alexa Fluor-594 (Molecular Probes), was added at a 1:400 dilution and incubated for 30 minutes at room temperature. Vectashield with 0.4 mmol of DAPI was added to counterstain nuclei.

Fluorescence Microscopy

Samples were visualized using conventional and deconvolution fluorescence microscopy (Zeiss Axioplan 2 microscope; Carl Zeiss, Jena, Germany, http://www.zeiss.com) at room temperature. Eight to 10 random X63 high-power fields from least two sections more than 100 µm apart of each sample were counted. We considered DAPI+ cells that were both GFP+ and prosurfactant C+ to be donor bone marrow-derived type II pneumocytes. Isotype controls for prosurfactant C (1% rabbit serum) and secondary-only controls were performed and were negative. Using deconvolution microscopy, selected sections were photographed at X40 or X63 magnification with the AxioVision software package (Carl Zeiss). Three-dimensional images were created of sample cells from a 25-layer (0.4 µm/layer) z stack to demonstrate colocalization of fluorescent signal. No photosubtraction or processing of the artifact was performed.

Statistics

Data were analyzed using Student’s t test in cases where there were fewer than six measurements within the parent group. The Wilcoxon rank sum test was also performed in cases where there were six or more measurements within the parent group. We considered results to be statistically significant only when p < .05 (two-sided). Data were presented as mean ± 1 SEM.

RESULTS

Marrow Cells Express Lung Cell-Specific mRNA when Cocultured with Lung

WBM, lung cocultures were established 3 hours, 5 days, or 14 days after radiation injury to lung donor mice, and cocultures were maintained for either 48 hours or 7 days (Fig. 1A). In all groups, expression of various pulmonary epithelial cell-specific genes, including Clara cell-specific protein (CCSP), surfactant protein B (Sp-B), or surfactant protein C (Sp-C), was elevated in WBM cocultured with either irradiated or nonirradiated lung compared with marrow cultured without lung (control) (Wilcoxon, p = .01). This was seen when coculture was maintained for either 48 hours or 7 days. When coculture was established 3 hours or 14 days after irradiation, there was no significant difference in expression of any of these genes in WBM cocultured with irradiated as compared with nonirradiated lung at either 48 hours or 7 days of coculture (Wilcoxon, p > .1). In these groups, peak increases in WBM cocultured with irradiated or nonirradiated lung were 6.5 ± 1.6-fold (SEM), 15.6 ± 5.8-fold, and 19.6 ± 7.4-fold expression of Sp-B, Sp-C, and CCSP, respectively. When WBM, lung cocultures were established 5 days after radiation injury to lung donor mice, there was a statistically significant increase in expression of pulmonary epithelial cell genes in WBM cocultured with radiation-injured lung compared with WBM cocultured with nonirradiated lung (Wilcoxon, p = .01). This was seen in both the 48-hour and 7-day coculture time points (Fig. 2A, 2B). WBM cocultured with 500 cGy-irradiated lung expressed the highest levels of these genes, although increases were also present in the 1,200-cGy groups.

Figure 2.

Figure 2

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Gene expression in WBM cocultured with lung. Pulmonary epithelial cell marker expression in WBM cocultured with lung for 48 Hr (A) or 7 D (B). Pulmonary epithelial cell marker expression in cocultured WBM for 48 Hr (C) or 7 D (D) (coculture established 5 D postirradiation). (E, F): Expression of other genes in cocultured WBM for 7 days. Abbreviations: CCSP, Clara cell-specific protein; D, days; Hr, hours; L-sel, L-selectin; P-sel, P-selectin; Sp-B, surfactant protein B; Sp-C, surfactant protein C; VCAM, vascular cell adhesion molecule; VEGFR, vascular endothelial growth factor receptor; WBM, whole bone marrow.

When WBM, lung coculture was established either 3 hours, 5 days, or 14 days after radiation injury, there was increased expression in a variety of genes expressed by other cells, including hematopoietic cells, such as c-kit (peak increase, 4.0 ± 0.7-fold) or Sca-1 (peak increase, 6.4 ± 1.4-fold), as well as adhesion protein genes, including P-selectin (peak increase, 8.4 ± 2.2-fold) or L-selectin (peak increase, 5.0 ± 0.9-fold) in WBM cocultured with radiation-injured or nonirradiated lung, compared with control (t test, p ≤ .047) (Fig. 2C, 2D). There was no significant difference in the expression of any of these genes when WBM cocultured with radiation-injured lung was compared with nonirradiated lung at either coculture time points (t test, p ≥ .065). In addition, the WBM side of each well insert from all coculture groups was inspected for the presence of GFP+ WBM cells, and no well inserts had adherent cells.

In parallel experiments, kidneys from radiation-injured and nonirradiated mice were cocultured with WBM under identical conditions. WBM cocultured with kidney did not express any pulmonary epithelial cell markers, suggesting that tissue other than lung does not induce pulmonary epithelial cell-specific gene expression in cocultured WBM. In addition, the expression of a variety of other markers, including CD34, CD44, CD45, c-kit, VEGFR-1 and −2, PECAM, and CXCR4, was unaltered or decreased compared with WBM cultured without kidney. There was small increase (as high as 2.5-fold in one experiment) in the expression of Sca-1 in cocultured WBM.

These findings demonstrate that lung induces WBM to express elevated levels of pulmonary epithelial cell-specific genes in coculture. Lung exposed to radiation 5 days prior to initiation of coculture appears to be a more potent stimulus for these elevations.

Prosurfactant Protein B Production by Marrow Cells Cocultured with Lung

WBM was cocultured with 500 cGy-irradiated lung or nonirradiated lung for 7 days. Immunohistochemical labeling of WBM at this time revealed no prosurfactant protein B (pro-Sp-B)-positive cells. Cocultured WBM cells were then maintained in culture for an additional 21 days in the absence of lung but in medium containing cytokines (IL-3, IL-6, and IL-11) or no added cytokines. Cells positive for pro-Sp-B were only seen in WBM maintained in cytokines after exposure to irradiated or nonirradiated lung (0.7% and 0.5% of all nucleated cells, respectively; t test, p = .08) (Fig. 3). Freshly isolated WBM or WBM cultured without lung, with or without cytokines, were all pro-Sp-B-negative. Isotype and secondary-antibody-only controls were also negative. These findings suggest that WBM cells cocultured with lung produce lung cell mRNA and, with time in culture, may be able to produce certain pulmonary epithelial cell proteins.

Figure 3.

Figure 3

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Cocultured marrow cell expressing prosurfactant B. (A, C): A prosurfactant protein B (Sp-B)+ (red) WBM cell (solid white arrow); Texas Red/4,6-diamidino-2-phenylindole (DAPI) filters (A) and Texas Red (C). (B): Same cell (solid red arrow) stained with Wright-Giemsa stain. Mouse lung cells were pro-Sp-B+ (open arrows) or – (asterisks). (D): Texas Red/DAPI. (E): Texas Red. Scale bar = 10 µm (two experiments).

Lung-Conditioned Media Induce Marrow Cells to Express Lung Cell-Specific mRNA

WBM was cocultured for 7 days with LCM made from lung 5 days after radiation injury (500 cGy) to lung donor mice or made from nonirradiated lung (Fig. 1B). There was a significant increase in Sp-B expression in WBM cocultured with 500 cGy LCM compared with control (52.9 ± 12.4; t test, p = .014), whereas Sp-B expression in WBM cocultured with nonirradiated lung was not different from control (13.8 ± 9.4; t test, p = .062) (Fig. 4A). There were lesser elevations of CCSP and Sp-C in both coculture groups. WBM cocultured with 500 cGy LCM also had increased levels of other genes compared with control (Fig. 4B). These findings suggest that a substance released by the lung that is smaller than the 0.4-µm well insert pores may induce changes in gene expression in cocultured WBM.

Figure 4.

Figure 4

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Gene expression of WBM cocultured with LCM with/without RNase. (A): Sp-B expression in WBM cocultured with LCM from irradiated or nonirradiated lung for 7 days. (B): Expression of Sp-B and other genes in WBM cocultured with LCM from irradiated lung with or without RNase for 7 days (mean values of two Exps). Abbreviations: Exp, experiment; LCM, lung-conditioned media; PECAM, platelet/endothelial cell adhesion molecule; Sp-B, surfactant protein B; VCAM, vascular cell adhesion molecule; WBM, whole bone marrow.

RNase Treatment of LCM Attenuates Gene Expression Changes Seen in Cocultured Marrow

Pulmonary epithelial cell-specific RNA was found to be contained within LCM. In one experiment, there was a 4.35 × 106-fold, 9.4 × 104-fold, and 2.59 × 105-fold increase in CCSP, Sp-B, and Sp-C expression, respectively, in LCM compared with WBM cultured without lung. No pulmonary epithelial cell RNA was found in unconditioned media. To determine whether gene expression changes seen in WBM cocultured with LCM was due to RNA transfer, LCM was treated with RNase prior to coculture with WBM (Fig. 1C). Incubation with RNase markedly reduced the presence of the above-mentioned pulmonary epithelial cell RNA populations in the LCM prior to coculture with WBM. Expression of Sp-B in WBM cells cocultured with LCM treated with RNase was 70% less than the level of expression in WBM cocultured with untreated LCM (Fig. 4B). Similarly, RNase treatment of LCM reduced expression of other RNAs (Fig. 4B). These data suggest that transfer of RNA may, in part, induce changes in WBM gene expression when cocultured with lung.

Microvesicles Containing Pulmonary Epithelial Cell-Derived RNA Are Found in Lung-Conditioned Media

Ultracentrifugation of LCM (from 500 cGy-irradiated lung) produced a small pellet that was found to contain RNA. A sample of this pellet was visualized using electron microscopy (Fig. 5A). The LCM pellet derived from GFP+ mice was then labeled with the membrane dye PKH26. A population of GFP+/PHK26+ events (0.13% of all events) was isolated using flow cytometry (Fig. 5B, R2). These particles are presumed to contain both cell membrane (PKH26+) as well as cytoplasm (GFP+) and were also found to contain RNA. RT-PCR was performed on cDNA samples made from equal amounts of RNA extracted from 500 cGy-irradiated lung, LCM made from irradiated lung, the ultracentrifuged pellet derived from LCM made from irradiated lung, and GFP+/PKH26+ particles sorted from the LCM pellet made from irradiated lung (Fig. 5C). Higher levels of pulmonary epithelial cell gene expression were found in LCM and its derived components (pellet and particle) compared with irradiated lung. The highest levels were found in the LCM pellet, where there was a 5.24 × 106-fold, 1.50 × 105-fold, and 6.27 × 106-fold increase in CCSP, Sp-B, and Sp-C expression, respectively, compared with control. These particles also expressed increased levels of other genes compared with control (Sca-1 and PECAM, 70- and 50-fold increases, respectively).

Figure 5.

Figure 5

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Isolation and imaging of lung-derived microvesicles. (A): Electron microscopy of the ultracentrifuged lung-conditioned media (LCM) pellet demonstrates numerous 100–250-nm membrane-bound vesicles (top, scale bar = 300 nm; bottom, panels of individual vesicles, scale bar = 100 nm). (B): Fluorescence-activated cell sorting-separated green fluorescent protein+/PKH26+ events (R2). (C): Pulmonary epithelial cell marker expression in LCM and its derived components (one experiment). Abbreviations: Comp, compensated; UCF, ultracentrifuge.

GFP+/PKH26+ particles that were cocultured with WBM entered 0.1% of all nucleated (DAPI+) cells, and these particles could be visualized by fluorescence microscopy (Fig. 6B–6H). In parallel experiments, GFP+/PKH26+ particles derived from nonirradiated lung were also observed to enter WBM cells. WBM isolated from GFP+ and GFP− mice cultured without particles did not contain similar fluorescent events. To determine the phenotype of the accepting marrow cell, WBM cocultured with GFP+/PKH26+ particles was sorted by flow cytometry based on the WBM cell’s GFP and PKH26 positivity (Fig. 6A, R2). Wright-Giemsa staining performed on these cells revealed that the predominant cell type containing fluorescent particles appeared to be differentiated granulocytes (74% of all nucleated cells), whereas 26% were of indeterminate mononuclear morphology, some having characteristics of blasts, lymphocytes, or monocytes.

Figure 6.

Figure 6

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WBM cells cultured with lung-derived microvesicles. (A): Fluorescence-activated cell sorting-separated WBM that consumed GFP+/ PKH26+ particles in culture (R2). Particles were visualized in WBM with fluorescein isothiocyanate (FITC) (C), Texas red (D), 4,6-diamidino-2-phenylindole (E), and all filters (B). Three-dimensional view reveals colocalization of GFP (G) and PKH26 (H). (F): FITC/Texas red filters. Scale bar = 10 µm (one experiment). Abbreviations: CCSP, Clara cell-specific protein; Comp, compensated; GFP, green fluorescent protein; LCM, lung-conditioned media; Sp-B, surfactant protein B; Sp-C, surfactant protein C.

Transplanted Cocultured Marrow Cells Have a Greater Propensity to Engraft the Radiation-Injured Lung

Cohorts of lethally irradiated mice received GFP+ WBM that was cocultured for 7 days with 500 cGy-irradiated lung, nonirradiated lung, or no lung (Fig. 1D). Alternatively, mice received a similar number of uncultured GFP+ WBM cells (Fig. 7A). All mice (six of six) that were transplanted with WBM cultured in the absence lung died 11–12 days post-TBI and transplant, suggesting bone marrow engraftment failure. Immediately prior to sacrifice, the average level of peripheral blood chimerism was more than 90% in all living cohorts and not significantly different from each other (t test, p ≥ .14). Lungs from mice that received WBM cocultured with radiation-injured or nonirradiated lung had a higher number prosurfactant C+ (pro-Sp-C) cells that were donor (GFP+) WBM-derived (1.55% ± 0.07% and 2.01% ± 0.22% of all nucleated cells, respectively) compared with those that received uncultured WBM cells (1.05% ± 0.12%; t test, p = .02 and .003, respectively, vs. uncultured WBM cohort; Fig. 7L). There was no significant difference in the number of GFP+/pro-Sp-C+ cells in mice that received WBM cocultured with radiation-injured or nonirradiated lung (t test, p = .08). GFP+/pro-Sp-C+ cells had morphological features consistent with type II pneumocytes (Fig. 7B–7K). These findings suggest that transplanted WBM was cocultured with lung have a greater tendency to participate in the production of type II pneumocytes, in vivo, in the radiation-injured lung than transplanted uncultured WBM cells.

Figure 7.

Figure 7

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Transplantation results for WBM cocultured with lung. (A): WBM baseline characteristics, transplantation results. (L): GFP+/ prosurfactant protein C (Sp-C)+ cells, percentage of 4,6-diamidino-2-phenylindole (DAPI)+ cells. GFP+/pro-Sp-C+ (solid and dashed white arrows), GFP+/ pro-Sp-C– (asterisk) and GFP–/pro-Sp-C+ (open arrow) cells. (D, G): Fluorescein isothiocyanate (FITC). (C, F): Texas Red. (B, E): Both filters/DAPI. (K): H&E. Three-dimensional view (H–J) reveals colocalization of GFP (J) and pro-Sp-C (I). (H): FITC/Texas Red filters. Scale bar = 20 µm. Abbreviations: GFP, green fluorescent protein; WBM, whole bone marrow.

DISCUSSION

These studies show that marrow cells cocultured with lung change phenotype. Cocultured marrow had increased expression of a variety of markers, including those for pulmonary epithelial cells. When coculture was established 5 days after radiation injury to the lung, the expression of pulmonary epithelial cell markers was markedly increased in cocultured marrow. This effect appears to be organ-specific, as WBM cocultured with kidney expressed no pulmonary epithelial cell markers. Expression of markers found on other cells, including hematopoietic cells, was unchanged or decreased compared with control. This may be a reflection of fewer resident hematopoietic cells being found in the kidney compared with the lung and thus less influence on cocultured cells to produce hematopoietic cell markers. LCM induced pulmonary epithelial cell-specific mRNA production in cocultured marrow and contained microvesicles with high levels of this RNA. Furthermore, microvesicles isolated from LCM could be separated by FACS and were shown to enter a minority of marrow cells. This suggests that radiation-injured lung releases microvesicles containing pulmonary epithelial cell-specific RNA, which then enter marrow cells, inducing them to express lung-specific mRNA. RNase treatment of LCM reduced subsequent expression of pulmonary epithelial cell-specific mRNA in cocultured marrow cells, further supporting the idea that mRNA expression is due to transfer of RNA from microvesicles shed by the lung.

The functional phenotype of cocultured marrow may also altered by microvesicular RNA transfer, as we have observed that marrow cells express pro-Sp-B 3 weeks after lung exposure. The cell type affected remains unclear. Granulocytic cells took up microvesicles, as did lesser defined mononuclear cells. Whether these latter include monocytes or stem cells remains to be established; both are candidate cells that may mediate the marrow to nonmarrow plasticity [18, 23, 24].

Evidence of alterations in cellular phenotype is provided by the experiments showing that marrow cells cocultured with irradiated or nonirradiated lung had an increased capacity to produce donor-derived type II pneumocytes, in vivo, in lethally irradiated mice. Marrow cocultured with irradiated lung and nonirradiated lung appeared to exert equal effects. In contrast, marrow cocultured with irradiated lung 5 days after irradiation showed higher pulmonary epithelial cell-specific mRNA levels than marrow cocultured with nonirradiated lung. In previous studies, 1,200 cGy of host irradiation provided more marrow-derived lung cells than 500 cGy irradiation [1]. There are qualitative differences between these injuries that may result in release of intrinsically different microvesicles; some may be superior at mediating engraftment, whereas others selectively induce increased lung-specific mRNA expression. Others [3], using different indices of injury, after 600 cGy, have shown little evidence of lung injury and no conversion events, whereas we showed increased alveolar wall thickening and cellularity after 500 cGy. These differences probably relate to differences in experimental detail.

The timing of lung harvest after irradiation may be an important variable. In these experiments, 5-day-postirradiation lung had the greatest influence on pulmonary epithelial cell-specific mRNA expression in cocultured marrow, corresponding to a time of acute radiation injury in the lung [2]. We have previously shown that the timing of transplant and injury affected the degree of marrow cell production of skeletal muscle [25].

Vesicular transfer of information may be an important form of cell-to-cell communication in activated normal cells [26–28]. Microvesicles can derive from platelets, lymphocytes, monocytes, endothelial cells, embryonic stem cells, and cancer cell lines [29–35]. Shed microvesicles may also increase in number under certain conditions, including irradiation, hypoxia, oxidative injury, shearing stress, and exposure to activated complement [27–29, 34, 36]. Increased numbers of microvesicles have been reported in plasma of humans with systemic lupus erythematosus, diabetes, unstable angina, heparin-induced thrombocytopenia, sepsis, thromboembolic disease, transient ischemic attacks, and cancer. Under different circumstances, microvesicles have been reported to contain DNA, mRNA, proteins, and bioactive lipids [37–44]. They have been found to transfer CD41, integrin or CXCR4 [33, 45], and HIV and prions [46, 47] between cells. Embryonic stem cell-derived microvesicles have been reported to reprogram hematopoietic stem/progenitor cells via the horizontal transfer of mRNA and protein [35]. Similarly, tumor-derived microvesicles transfer several surface determinants and mRNA to monocytes [34]. Apoptotic bodies from irradiated Epstein-Barr virus (EBV)-carrying cell lines transfer DNA to a variety of cocultured cells and integrated copies of EBV, result in high expression of the EBV-encoded genes EBER and EBNAI in recipient cells [48]. Extracts from T lymphocytes containing transcription factor complexes induce fibroblasts to express lymphoid genes [49]. Most recently, it was reported that apoptotic, stimulated T cells released microvesicles, which then induced differentiation of human K562 cells toward the megakaryocyte lineage [29]. In addition, cultures of murine hematopoietic stem cells exposed to damaged liver cells across a cell-impermeable membrane have been shown to induce marrow stem cells to express genes specific for hepatocytes such as albumin [50]. This was interpreted as a humoral induction of differentiation, but it could be due to microvesicular transfer of information.

In summary, these studies are the first to demonstrate that a lung phenotype can be transferred to marrow cells from injured lung cells through lung cell-derived microvesicles. In addition, they suggest a mechanism for the transfer of information from injured cells to healthy cells and may provide a mechanism for some forms of phenotypic modulation of stem cells and tissue repair.

ACKNOWLEDGMENTS

This publication was made possible by support from the following grants: NCRR P20RR018757-04, NIDDK 1R01DK61858-03, NHLBI 1R01HL73747-02, NHLBI 1KO8 HL072332-01, NIDDK 5K08 DK064980, NIGMS GM47368 (to E.L.B.).

Footnotes

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

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