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Sequestration and homing of bone marrow-derived lineage negative progenitor cells in the lung during… Suzuki, Hisashi; Hogg, James C; van Eeden, Stephan F Mar 3, 2008

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ralssBioMed CentRespiratory ResearchOpen AcceResearchSequestration and homing of bone marrow-derived lineage negative progenitor cells in the lung during pneumococcal pneumoniaHisashi Suzuki, James C Hogg and Stephan F van Eeden*Address: The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital, University of British Columbia, Room 166, 1081 Burrard Street, Vancouver, British Columbia, V6Z 1Y6, CanadaEmail: Hisashi Suzuki - hsuzuki-jpn@umin.ac.jp; James C Hogg - JHogg@mrl.ubc.ca; Stephan F van Eeden* - SVaneeden@mrl.ubc.ca* Corresponding author    AbstractBackground: Bone marrow (BM)-derived progenitor cells have been shown to have the potentialto differentiate into a diversity of cell types involved in tissue repair. The characteristics of theseprogenitor cells in pneumonia lung is unknown. We have previously shown that Streptococcuspneumoniae induces a strong stimulus for the release of leukocytes from the BM and theseleukocytes preferentially sequester in the lung capillaries. Here we report the behavior of BM-derived lineage negative progenitor cells (Lin- PCs) during pneumococcal pneumonia usingquantum dots (QDs), nanocrystal fluorescent probes as a cell-tracking technique.Methods: Whole BM cells or purified Lin- PCs, harvested from C57/BL6 mice, were labeled withQDs and intravenously transfused into pneumonia mice infected by intratracheal instillation ofStreptococcus pneumoniae. Saline was instilled for control. The recipients were sacrificed 2 and 24hours following infusion and QD-positive cells retained in the circulation, BM and lungs werequantified.Results: Pneumonia prolonged the clearance of Lin- PCs from the circulation compared withcontrol (21.7 ± 2.7% vs. 7.7 ± 0.9%, at 2 hours, P < 0.01), caused preferential sequestration of Lin-PCs in the lung microvessels (43.3 ± 8.6% vs. 11.2 ± 3.9%, at 2 hours, P < 0.05), and homing of thesecells to both the lung (15.1 ± 3.6% vs. 2.4 ± 1.2%, at 24 hours, P < 0.05) and BM as compared tocontrol (18.5 ± 0.8% vs. 9.5 ± 0.4%, at 24 hours, P < 0.01). Very few Lin- PCs migrated into airspaces.Conclusion: In this study, we demonstrated that BM-derived progenitor cells are preferentiallysequestered and retained in pneumonic mouse lungs. These cells potentially contribute to therepair of damaged lung tissue.BackgroundStreptococcus pneumoniae is the most common cause oflocal inflammatory response in the lung, pneumococcalpneumonia also induces a systemic immune response [4],Published: 3 March 2008Respiratory Research 2008, 9:25 doi:10.1186/1465-9921-9-25Received: 15 October 2007Accepted: 3 March 2008This article is available from: http://respiratory-research.com/content/9/1/25© 2008 Suzuki et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 10(page number not for citation purposes)community acquired pneumonia and is one of the lead-ing causes of death worldwide [1-3]. In addition to thewhich includes stimulation of the bone marrow (BM)with subsequent release of neutrophils and monocytesRespiratory Research 2008, 9:25 http://respiratory-research.com/content/9/1/25that participate in the inflammatory response in the lung.Studies from our laboratory demonstrated that pneumo-coccal pneumonia accelerates the transit time of both neu-trophils and monocytes through the marrow and therelease of these cells into the circulation [5,6]. A signifi-cant fraction of these newly released cells have immaturecharacteristics and preferentially sequester in pneumonicregions of the lung [7].The systemic inflammatory response induced by pneumo-nia is also characterized by an increase in circulating pro-inflammatory mediators [8-10], of which several (such asG-CSF and GM-CSF) are known to release hematopoieticstem cells from the BM [11,12]. Recent studies haveshown that these BM-derived stem cells or progenitor cellshave the ability to differentiate into cells that repopulatedamaged tissues in different organs such as the heart [13],liver [14-16], brain [17] and lungs [18-21]. The majorityof these studies have used models of toxic, traumatic orischemic tissue injury, and showed engraftment of bothtype II [20,21] and type I [19] pneumocytes from BM-derived cells demonstrating the ability of these cells toparticipate in structural repair of the lung followinginjury. Therefore, we postulate that BM-derived progeni-tor cells will preferentially sequester in pneumonia-induced damaged lung tissue.To test this hypothesis, we developed a novel labelingtechnique using quantum dots (QDs), which are fluores-cent nanocrystals, to trace and quantify these progenitorcells in the lung. QDs have previously been used to labellive cells for long-term multicolor in vivo imaging [22].These nanocrystals are taken into cells by endocytoticpathways and the fluorescence of QDs persist intracellu-larly for more than a week [23]. Using this novel cell track-ing technique, we measured clearance of infused wholeBM cells (BMCs) and BM-derived lineage negative progen-itor cells (Lin- PCs) from the circulation, their sequestra-tion in the BM and lung, and their migration intoairspaces in a mouse model of pneumonia.MethodsAnimalsFemale C57BL/6J mice (10–12 weeks old) were used asdonors and recipients. Mice were purchased from JacksonLaboratory (Bar Harbor, ME) and maintained on a stand-ard laboratory diet and housed in a controlled environ-ment with a 12-hour light/dark cycle in the animal carefacility at Jack Bell Research Centre. All animal experi-ments were approved by the Animal Care Committee,University of British Columbia.Pneumonia modelbacteria in saline at a concentration of 2.5 × 109 colony-forming units (CFU)/ml was prepared based on its opticaldensity. Recipients were anesthetized with isoflurane andtheir tracheas were exposed by a small incision in the ven-tral portion of the neck. Bacterial suspension (250 µL/100g body weight) was instilled into the trachea via a 28-gauge needle. An equal amount of sterile saline was usedfor control mice. The incision was sutured after the instil-lation. Following instillation, their weight was dailyrecorded and their behaviors, symptoms, and the condi-tion of the wound were monitored twice a day until theywere sacrificed.Isolation of BM cellsFemurs and tibias were removed from donors (unin-fected, age-matched female C57BL/6J mice) and wholeBM cells (BMCs) were obtained by flushing the marrowcavities with 10 ml phosphate-buffered saline (PBS) witha 25-gauge needle. The BM components were dispersed byrepeated passage through a 10 ml syringe. The cells werewashed twice with PBS+2% fetal bovine serum (FBS) andwere filtered through 70 µm nylon mesh (BD Biosciences,San Jose, CA).Isolation of lineage negative progenitor cellsFor the purified progenitor cell transfusion experiments,lineage negative progenitor cells (Lin- PCs) were separatedfrom whole BMCs using a mouse progenitor cell enrich-ment kit (StemCell Technologies, Vancouver, Canada).Briefly, whole BMCs were incubated with assorted anti-bodies including rat anti-mouse CD5, CD45R, Mac-1, Gr-1, 7-4 and TER-119 that identify differentiated cells. Afterrepeated washings to remove excess antibodies, the cellswere incubated with magnetized microbeads that bindand eliminate the antigen-bound antibodies. UnboundLin- PCs were purified by magnetic separation usingAutoMacs (Miltenyi Biotec Inc, Auburn, CA) as a negativefraction under its "DEPLETES" program. The number ofLin- PCs was counted on the Cell-Dyn system (AbbottLaboratories, North Chicago, Il).Labeling of donor cells with QDsBMCs and Lin- PCs obtained from donor mice werelabeled with QDs (Qtracker 655 Cell Labeling Kit, Quan-tum Dot Corporation, Hayward, CA) by incubating with10 nM QD-labeling solution at 37°C for 60 minutes.Cells were washed twice with PBS to remove the excessQDs.Transfusion of donor cellsForty-eight hours following instillation of S. pneumoniaeor control vehicle into the mouse lung, BMCs (1.0 × 106cells/200 µl) or Lin- PCs (0.5 × 106 cells/200 µl) werePage 2 of 10(page number not for citation purposes)For each experiment, Streptococcus pneumoniae (serotype49619, ATCC, Rockville, MD) was used. A suspension oftransfused into the recipients via tail vein injection.Respiratory Research 2008, 9:25 http://respiratory-research.com/content/9/1/25Blood and tissue collectionRecipients were sacrificed at 2 or 24 hours after the celltransfusion. Blood was collected from the abdominalaorta with a 25-gauge needle. Femurs and tibias wereobtained to isolate BMCs. Lungs were harvested and lungvolume was measured by the water replacement methodafter inflating with 10% neutral-buffered formalin at 20cmH2O. Lungs were then fixed in 10% formalin for morethan 24 hours and each lung was cut into five slices forparaffin embedment.Flow cytometric analysisFlow cytometric analysis was performed to determine theamount of QD-positive donor cells in recipients' periph-eral blood and BM. Mononuclear cells (MNCs) were puri-fied from whole blood by density gradient centrifugationwith Histopaque-1077 (Sigma-Aldrich, St Louis, MO) at400 g for 30 minutes. BMCs were isolated from femursand tibias as described above. Both MNCs and BMCs werewashed twice with PBS+2% FBS and were analyzed by aflow cytometer (Epics XL-MCL, Beckman Coulter Inc.,Fullerton, CA) using the Summit software (Version 3.1,Cytomation Inc., Fort Collins, CO). Typically, 200,000events for MNCs and 400,000 events for BMCs wereacquired and the frequency of QD-positive cells wasmeasured. The numbers of donor cells in circulation andBM were calculated as follows:Number of QD-labeled donor cells in circulation = Frac-tion of donor cells in MNCs × fraction of MNCs × white blood cell count (/ml) × circulating blood volume (7 ml/100 g body weight)Number of QD-labeled donor cells in BM = Fraction of donor cells in BM × total number of BMCsThe number of BMCs harvested from 2 femurs and 2 tib-ias was considered to represent 18.1% of total murinemarrow [24]. The results were shown as percentages oftotal number of transfused donor cells.Histological analysis and detection of donor cellsThin sections (5 µm) of lung tissue were prepared fromparaffin-embedded blocks. Nuclei were stained withHoechst 33342 (Invitrogen, Carlsbad, CA). Briefly, slideswere incubated with diluted Hoechst 33342 solution (2µg/ml) for 10 minutes at room temperature, followed bytwo washes with PBS for 10 minutes. The sections werecoverslipped and examined using confocal microscopy(SP2 AOBS Confocal Microscope, Leica MicrosystemsGmbH, Germany) to detect QD-positive donor cells.Morphometric evaluation of QD-labeled donor cells in lungThe number of QD-labeled donor cells in recipient's lungwas determined using a modification of the sequentiallevel stereologic analysis [25]. A point-counting grid wasplaced over the images of lung slices taken at 4× magnifi-cation. The number of points falling on parenchyma wascounted and its volume fraction (Vv) was estimated as fol-lows:For quantitation of QD-positive cells, 100 randomlyselected fields of lung parenchyma were photodocu-mented from each mouse using confocal microscopy witha 63× objective lens. The Vv of QD-positive cells was cal-culated using a grid of 2025 (45 × 45) points superim-posed onto the captured images. The density of the gridand number of fields counted were determined to main-tain the coefficient of error below 0.2. The number of QD-labeled donor cells sequestered in the recipients' lung wascalculated as follows:Number of QD-labeled donor cells sequestered in lung = Lung volume × Vv lung parenchyma × Vv QD-positive cells × k-1where k is the average volume of a mouse BMC as deter-mined by measurement of 100 BMCs.Localization of the QD-positive cells (either in lungparenchyma or in alveolar airspaces) was also recordedduring the cell counting.Statistical analysesAll results were presented as mean ± standard error (SE).Statistical significance was evaluated using the unpairedStudent's t-test for comparisons between two groups. Mul-tiple comparisons were performed by one-way ANOVAand Tukey's post-hoc test. P < 0.05 was considered statis-tically significant. All statistical analyses were performedusing SPSS software (Version 10.1, SPSS Inc., Chicago, IL).ResultsEvaluation of QDs in donor cellsA representative image and an emission scan of QD-labeled donor cells are shown in Figure 1A and 1B. Fluo-rescent particles were detected within the cytoplasm ofdonor cells and the emission spectra of these particlespeaked at 650 nm, thus confirming the identity and pres-ence of QDs in the cells. The labeling efficiency of QDs inwhole BMCs (79.9 ± 3.4%) and Lin- PCs (75.5 ± 2.9%)Vv parenchymaSum of the points on parenchymaSum of the to=tal pointsPage 3 of 10(page number not for citation purposes)was measured by flow cytometry, as depicted in Figure 1C.Respiratory Research 2008, 9:25 http://respiratory-research.com/content/9/1/25Analysis of donor cells in the circulationThe number of QD-labeled donor cells, expressed as aratio of total injected cells, detected in the circulation at 2and 24 hours post-transfusion is shown in Figure 2.In the whole BMC transfusion model (Figure 2A), the pro-portion of QD-labeled donor cells at 2 hours post-transfu-sion was 3.3 ± 0.7% and 4.3 ± 0.7% in control andpneumonia groups, respectively (P = 0.34). However, theratio of labeled cells in the pneumonia group significantlydecreased to 1.8 ± 0.3% at 24 hours, as compared to theprevious timepoint (P = 0.01). In the Lin- PC transfusionmodel (Figure 2B), the amount of QD-labeled donor cellswas significantly higher in the pneumonia group as com-pared to control (21.7 ± 2.7% vs. 7.7 ± 0.9%, P = 0.008)at 2 hours following transfusion. By 24 hours post-trans-fusion, the ratio of labeled cells significantly decreased to3.4 ± 1.1% (P = 0.04) in the control group and to 2.1 ±0.5% (P < 0.001) in the pneumonia group from 2 hours.However, at 24 hours there was no significant differencein the circulating Lin- PCs between the two groups (P =0.29).Homing of donor cells to the BMThe proportion of QD-labeled donor cells (fraction oftotal injected cells) that sequestered and homed to the BMis shown in Figure 3. In the whole BMC transfusion model(Figure 3A), there was a trend towards increased homingof QD-labeled donor cells into the BM in the pneumoniaanimals as compared to control (7.7 ± 0.6% vs. 6.1 ±0.5%, P = 0.09) at 2 hours post-transfusion. At 24 hours,the proportion of QD-labeled cells in both control andpneumonia animals were not different (6.5 ± 0.3% vs. 6.7± 0.6%, P = 0.81). There was also no significant differencebetween 2 and 24 hours post-transfusion in both groups.In the Lin- PC transfusion model (Figure 3B), the propor-tion of QD-labeled donor cells that were sequestered inthe BM at 2 hours post-transfusion was similar betweenthe control and pneumonia groups (10.1 ± 1.5% vs. 12.9± 1.2%, P = 0.19). After 24 hours, the ratio of labeled cellshoming to the BM in the pneumonia group increased sig-nificantly (18.5 ± 0.8%, P = 0.006) as compared to the 2hour timepoint and the fraction of cells was also signifi-cantly higher as compared to control (18.5 ± 0.8% vs. 9.5± 0.4%, P < 0.001).Sequestration of donor cells in recipient lungsFigure 4 shows a QD-labeled donor cell in recipient lungtissue as viewed using confocal microscopy with an emis-sion signal peak of 650 nm.Figure 5 shows the proportion of labeled donor cells,Characteristics of labeled donor cellsFigure 1Characteristics of labeled donor cells. Isolated BM cells harvested from donor mice were labeled with QDs, which have an emission peak at 655 nm. A representative image illustrating a labeled cell emitting red fluorescence as observed under confocal microscopy is shown (A) and the red signal was confirmed as QDs by measuring their emission wavelength (B). Both the whole BM and Lin- progenitor cell populations were labeled with quantum dots and the labeling efficiency was 79.9 ± 3.4% and 75.5 ± 2.9%, respectively (C). Data is shown as mean ± SE, n = 5. Scale bar is 5 µm.Page 4 of 10(page number not for citation purposes)expressed as a fraction of total injected cells, which weresequestered in the lung. In the whole BMC transfusionRespiratory Research 2008, 9:25 http://respiratory-research.com/content/9/1/25Page 5 of 10(page number not for citation purposes)Frequency of labeled donor cells in circulationigur  2Frequency of labeled donor cells in circulation. Blood was collected from recipients which were sacrificed at 2 or 24 hours after cell transfusion. The frequency of labeled cells was measured by flow cytometry and the total number of labeled donor cells in recipients' circulation was calculated. The results are shown as percentages of total number of transfused donor cells. In the whole BM cell transfusion experiment (A), the proportion of donor cells at 2 hours post-transfusion was 3.3 ± 0.7% and 4.3 ± 0.7% in control and pneumonia groups, respectively (P = 0.34). However, the ratio of labeled cells in the pneumonia group significantly decreased to 1.8 ± 0.3% at 24 hours (P = 0.01). In the Lin- progenitor cell transfusion model (B), the proportion of labeled progenitor cells in the pneumonia group was signifi-cantly higher than control (21.7 ± 2.7% vs. 7.7 ± 0.9%, P = 0.008) at 2 hours. The percentage of donor cells in the circu-lation in both the control and pneumonia groups decreased after 24 hours and the difference between the two groups was no longer significant at 24 hours post-transfusion. Data is shown as mean ± SE; n = 6 (control group for each time-point) and n = 7 (pneumonia group for each timepoint). *P < 0.05, **P < 0.01.Frequency of labeled donor cells in bone marrowigur  3Frequency of labeled donor cells in bone marrow. BM cells were harvested from recipients which were sacrificed at 2 or 24 hours after the cell transfusion. The frequency of labeled cells was measured by flow cytometry and the total number of labeled donor cells in recipients' bone marrow was calculated. The results are shown as percentages of total number of transfused donor cells. In the whole BM cell trans-fusion experiment (A), there was an upward trend in the pneumonia group as compared to control (7.7 ± 0.6% vs. 6.1 ± 0.5%, P = 0.09) at 2 hours post-transfusion, although by 24 hours, the number of labeled cells equalized and there was no significant difference between the groups (P = 0.81). In the Lin- progenitor cell transfusion model (B), there was no sig-nificant difference in the proportion of labeled cells between the control and pneumonia groups (10.1 ± 1.5% vs. 12.9 ± 1.2%, P = 0.19) at 2 hours. After 24 hours, the ratio of donor cells in the pneumonia group increased significantly as com-pared to control (P < 0.001) and the previous timepoint (P = 0.006). Data is shown as mean ± SE; n = 6 (control group for each timepoint) and n = 7 (pneumonia group for each time-point). **P < 0.01.Respiratory Research 2008, 9:25 http://respiratory-research.com/content/9/1/25model (Figure 5A), significantly more donor cells weresequestered in the pneumonia lungs as compared to con-trol at 2 hours post-transfusion (32.6 ± 4.6% vs. 15.3 ±(4.1 ± 1.7%, P = 0.001) and in the pneumonia group (8.3± 1.1%, P < 0.001) as compared to the previous timepointwith no significant difference between the control andpneumonia groups.In the Lin- PC transfusion model (Figure 5B), significantlymore donor cells were sequestered in the pneumonialungs as compared to control at 2 hours post-transfusion(43.3 ± 8.6% vs. 11.2 ± 3.9%, P = 0.03). After 24 hours, ascompared to 2 hours post-transfusion, the donor cellsremaining in the lung decreased in both the control (2.4± 1.2%) and the pneumonia groups (15.1 ± 3.6%)although the decrease was significant only in the latter (P= 0.04). The percentage of donor cells remaining in thelungs of the pneumonia group was still significantlyhigher than the control group (P = 0.04) at this timepoint.Migration of donor cells into alveolar air space in pneumonia lungsWe next investigated the localization of the labeled donorcells in the pneumonia lungs and detected migration ofthese cells into alveolar air spaces. In the whole BMCtransfusion model (Figure 6A), the percentage of donorcells that migrated into the alveolar air space in pneumo-nia lungs was 2.2 ± 1.2% and 18.7 ± 3.7% at 2 and 24hours post-transfusion, respectively, and the differencebetween the two timepoints was significant (P = 0.001).In the Lin- PC transfusion model (Figure 6B), 1.7 ± 1.1%and 3.1 ± 4.3% of donor cells were found in alveolar airspace of pneumonia lungs at 2 and 24 hours, respectively.No significant difference was observed between the twotimepoints (P = 0.60).DiscussionIn this study, we demonstrated that both whole BMCs andBM-derived Lin- PCs were sequestered (2 hours) in thelung while the Lin- PCs were preferentially retained (24hours) there during pneumococcal pneumonia. Further-more, these progenitor cells also remained in the lung tis-sues and rarely migrated into the alveolar air spaces,suggesting that BM-derived progenitor cells sequester andhome to lung tissues. Several studies have shownincreased circulating progenitor cells in lung injury mod-els [26-28], as well as the participation of progenitor cellsin the repair response of the lung [18-21]. We proposethat during infectious inflammation of the lung, theseBM-derived progenitor cells could contribute to eitherlung inflammation and/or repair following lung infec-tion.We used QDs to label and trace donor cells in recipients.QDs are highly luminescent semiconductor nanocrystalsLabeled donor cells in recipient lungFigure 4Labeled donor cells in recipient lung. A representative image of a QD-labeled donor cell (arrow) as detected in recipient's lung tissue under confocal microscopy (A). Blue denotes nuclei stained with Hoechst 33342, green is autoflu-orescence from lung tissue, and red is emission signal from quantum dots. The graph (B) shows the wavelength and fluo-rescence level of QDs on the labeled cell (circle) and lung tis-sue (square). The sharp peak at 655 nm indicates that the emission from QDs is very distinct from the autofluores-cence of lung tissue. Scale bar is 10 µm.Page 6 of 10(page number not for citation purposes)1.8%, P = 0.007). By 24 hours, the number of labeled cellsremaining in the lung significantly decreased in controls(CdSe/ZnS-core/shell) and their surface chemistry is mod-ified with peptides so that they can be delivered into cyto-Respiratory Research 2008, 9:25 http://respiratory-research.com/content/9/1/25Page 7 of 10(page number not for citation purposes)Frequency of labeled donor cells in lungigur  5Frequency of labeled donor cells in lung. The total number of QD-positive donor cells sequestered in the whole lung was calculated by morphometric analysis. The results are shown as the donor cell proportion of total injected cells that were sequestered in the lung. In the whole BM cell transfusion model (A), significantly more donor cells were sequestered in the pneumonia lungs as compared to control (32.6 ± 4.6% vs. 15.3 ± 1.8%, P = 0.007) at 2 hours post-transfusion. After 24 hours, the number of donor cells in lung significantly decreased in both groups, and the difference between the two groups was no longer significant. In the Lin- progenitor cell transfusion model (B), there was a signifi-cantly higher number of donor cells sequestered in the pneu-monia lungs as compared to control (43.3 ± 8.6% vs. 11.2 ± 3.9%, P = 0.03) at 2 hours post-transfusion. After 24 hours, although the percentage of donor cells decreased in both groups, the ratio of donor cells in the pneumonia group was still significantly higher than control (15.1 ± 3.6% vs. 2.4 ± 1.2%, P = 0.04). Data is shown as mean ± SE; n = 6 (control group for each timepoint) and n = 7 (pneumonia group for each timepoint). *P < 0.05, **P < 0.01.Migration of labeled donor cells into alveolar air spaceFi ure 6Migration of labeled donor cells into alveolar air space. In pneumonia lungs, the ratio of cells that migrated into alveolar air space was measured. The results are shown as the percentage of donor cells that migrated into air space out of the total number of donor cells sequestered in pneu-monia lung. In the whole BM cell transfusion model (A), a sig-nificantly higher number of donor cells migrated into the air space of pneumonia lungs at 24 hours post-transfusion as compared to 2 hours (18.7 ± 3.7% vs. 2.2 ± 1.2%, P = 0.001). In the Lin- progenitor cell transfusion model (B), no signifi-cant difference was observed between the 2 and 24 hour timepoints (1.7 ± 1.1% vs. 3.1 ± 4.3%, P = 0.60). Data is shown as mean ± SE; n = 7. **P < 0.01.Respiratory Research 2008, 9:25 http://respiratory-research.com/content/9/1/25plasm of live cells by endocytosis. The advantages of QDsover conventional organic fluorophores are their high lev-els of brightness, resistance to photobleaching, wide rangeof excitement wavelengths, and tunable fluorescent wave-lengths depending on the size of the particles [29,30].QDs are now widely used for cellular imaging and manystudies have shown the ability to trace diverse types ofcells following cell proliferation and differentiation for upto a week without any effects on cell activation or cellfunction [22,23,31]. We used whole BMCs and BM-derived Lin- PCs and the labeling efficiency of QDs wasapproximately 80% for both cell types (Figure 1C).Although QDs may be cytotoxic to live cells due to theirreactive metal core [23], we used low QD concentrationsthat have been shown to be noncytotoxic and have noimpact on cell function, such as cell proliferation and dif-ferentiation [23]. Due to the brightness and high emissionsignals of QDs, resistance to photobleaching and reten-tion in labeled cells, we were able to clearly identifylabeled cells in the blood, BM and the lung using a com-bination of flow cytometry and confocal microscopy.Plett and colleagues have shown that BM-derived progen-itor cells are rapidly cleared from the circulation aftertransfusion [32]. In our study 92.3% of the transfused Lin-PCs were cleared from recipient's circulation within 2hours after the injection in control group. Interestingly,this clearance from the circulation occurred much morerapidly than those shown for transfusion of labeled neu-trophils [33] or labeled monocytes [5]. Cell size anddeformability have been shown to be important in deter-mining removal of infused cells from the circulation [34].Immature BM-derived granulocytes are larger and lessdeformable than mature granulocytes [35], and wehypothesize that cell immaturity is responsible for therapid clearance of Lin- PCs from the circulation.In the pneumonia model, however, the clearance of Lin-PCs from the circulation at 2 hours following infusiondecreased (Figure 2). Interestingly, these findings are dif-ferent from the results we have shown previously on neu-trophils and monocytes. In bacterial infection,neutrophils and monocytes are more rapidly cleared fromthe circulation to be sequestered into the infected lungs[6,36]. A unique and independent mechanism might existfor the mobilization and sequestration of progenitor cells.Increased number of circulating progenitor cells has beenshown in tissue injury animal models [26,27] as well as inclinical settings including burn, post-cardiac surgery [37],and pneumonia [28] patients. These findings suggest thatprogenitor cells acquire the capability to remain in the cir-culation during systemic inflammation for subsequentsequestration into target organs upon demand. The rea-do not respond to chemo-attractants in the acute inflam-matory milieu but are recruited later when cells for resolu-tion and repair are required. Alternatively, these cells mayremain in the less hostile intravascular milieu to prolifer-ate and mature, to home and migrate into the inflamma-tory site at a stage when the acute inflammatory responsehas been dampened.Several studies have reported that BM-derived progenitorcells accumulate in lung tissue and have the ability toreplace damaged cells following injury [18-21]. We showhere that significant sequestration of transfused Lin- PCsoccurred in pneumonic lungs at 2 hours as compared tocontrol lungs, suggesting preferential sequestration ofthese cells in inflamed lung tissues. To determine homingof BM-derived cells to inflamed lung tissues, we investi-gated the localization of these cells at 24 hours after trans-fusion. Using purified Lin- PCs, approximately 15.1% oflabeled cells were still in the lung at 24 hours while 2.1%remained in the circulation, showing a 7 times enrich-ment of Lin- PCs in the pneumonia lungs. Few of theseLin- PCs migrated into the airspaces (approximately 3.1%of all the cells remaining in the lung), therefore approxi-mately 14.6% of the initially infused Lin- PCs homed tolung tissues and were potentially available for lung tissueregeneration and repair. These findings showed firstly thatvery few progenitor cells migrate into the airspaces to par-ticipate in airspace inflammation but the majority of cellsremain in the lung tissues where they have the potential,via proliferation and differentiation, to participate in lungstructural repair. Secondly, a significant fraction of BM-derived progenitor cells homed to the injured lung ascompared to the control lung. If this fraction of progenitorcells that home to damaged lung regions remain constant(as shown in the BM homing study by Szilvassy and col-leagues [38]), increasing the number of infused progeni-tor cells will increase the number of these cells that hometo injured lung tissues and become available for tissueregeneration and repair.Interestingly, in the present study, approximately 10–13%of injected Lin- PCs, both in the control and pneumoniagroups, returned to the marrow at 2 hours, which sup-ports previous work by other authors [32,38]. However,more Lin-PCs homed back to the marrow at 24 hours inpneumonia group (Figure 3B). This could represent themarrow's ability to recruit progenitors from the circula-tion pool during infection, in an attempt to produce addi-tional inflammatory cells demanded from the BM by theinfection.ConclusionOur study showed that during pneumonia, BM-derivedPage 8 of 10(page number not for citation purposes)son for this prolonged stay in the circulation is unclearand needs further investigation. It could be that these cellslineage negative progenitor cells remain in the intravascu-lar space for a prolonged time, preferentially sequester inRespiratory Research 2008, 9:25 http://respiratory-research.com/content/9/1/25the inflamed lung tissues and are enriched in the lungover a short period of time (24 hours). These cells mayplay an important role in inflammatory responses againstlung infection as well as contributing in tissue repair proc-esses following the infection. Further studies will berequired to elucidate the mechanism of behavior of theseprogenitor cells and to determine the phenotypic charac-teristics of the cell type responsible for the tissue repara-tion. Together these concepts may pave the way for futurenovel cell-based therapy for lung tissue repair followinginjury.Competing interestsThe author(s) declare that they have no competing inter-ests.Authors' contributionsHS carried out the experiments, performed data analysesand drafted the manuscript. JH participated in interpreta-tion and critical review of data as well as the revision ofthe manuscript for important intellectual content. SvEconceived the study and made substantial contributionsto conception, design and drafting of the manuscript. Allauthors read and approved the final manuscript.AcknowledgementsWe would like to thank Beth Whalen, Anna Meredith, Amrit Samra, Joanna Marier and Kris Gillespie for their technical help and Sze-Yin Yuen for pro-viding the bacteria.This study was supported by BC Lung Association and the Wolfe & Gina Churg Foundation.References1. Bartlett JG, Dowell SF, Mandell LA, File Jr TM, Musher DM, Fine MJ:Practice guidelines for the management of community-acquired pneumonia in adults. Infectious Diseases Society ofAmerica.  Clin Infect Dis 2000, 31(2):347-382.2. Mandell LA, Marrie TJ, Grossman RF, Chow AW, Hyland RH: Cana-dian guidelines for the initial management of community-acquired pneumonia: an evidence-based update by the Cana-dian Infectious Diseases Society and the Canadian ThoracicSociety. The Canadian Community-Acquired PneumoniaWorking Group.  Clin Infect Dis 2000, 31(2):383-421.3. 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