James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia, St. Paul's Hospital, Vancouver, British Columbia, Canada
Submitted 1 December 2004 ; accepted in final form 25 April 2005
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ABSTRACT |
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polymolphonuclear neutrophil; flow cytometry; transit time
5'-Bromo-2'-deoxyuridine (BrdU) is a thymidine analog that is incorporated into DNA during DNA synthesis. Because a monoclonal antibody against BrdU has been developed, the immunohistochemical detection of BrdU-labeled nuclei has rapidly become a widely accepted alternative to the conventional radioisotopic approach of monitoring DNA replication (4, 68, 13, 15). In 1994, we developed a method using BrdU instead of the conventional radioisotope method of labeling PMN precursors in the rabbit bone marrow that allowed us to measure their half-lives in the circulation and trace the newly released PMN in the organs (3). In 1996, we modified this method to measure the transit time of PMN through the mitotic and maturation pools in the bone marrow by grading BrdU-labeled PMN (PMNBrdU) into three groups according to their intensity of immunocytochemical staining (Fig. 1) (23). These rabbit models enabled us to monitor and investigate the in vivo bone marrow response and PMN behavior during inflammation without using radioisotopes.
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MATERIALS AND METHODS |
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Experimental protocol. NZW rabbits were given a pulse dosage of 100 mg/kg (15 mg/ml in normal sterile saline) of BrdU (Sigma Chemical, St. Louis, MO) intravenously through the marginal ear vein during a period of 15 min to label dividing leukocyte progenitors in the marrow. Blood samples were obtained from the central ear artery at 6- to 24-h intervals and followed for 10 days. Sedation [fentanyl (20 µg/kg) and droperidol (1 mg/kg)] were administered by subcutaneous injection to facilitate blood collection.
Blood sampling and sample preparation. A total of 1.5 ml of blood was collected at every time point (before and at 24, 30, 36, 48, 72, 96, 120, 144, 168, 192, 216, and 240 h after BrdU administration) from each rabbit. Five hundred microliters of blood were collected in a standard Vacutainer tube containing potassium ethylenediaminetetraacetic acid (Becton Dickinson, Rutherford, NJ) for blood cell and white blood cell differential counts determined on a cell counter (model Cell-Dyn 3700; Abbott, Mississauga, ON, Canada).
Another 1 ml of blood was collected in acid-citrate dextrose (ACD) for preparation of leukocyte-rich plasma (LRP). Erythrocytes in the ACD blood samples were allowed to sediment for 2025 min after the addition of an equal volume of 4% dextran (average mol wt, 162,000; Sigma) in PMN buffer (in mM: 138 NaCl, 27 KCl, 8.1 Na2HPO4, 7 H2O, 1.5 KH2PO4, and 5.5 glucose, pH 7.4), to obtain LRP. LRP was centrifuged to pellet, and the residual red blood cells (RBCs) in the pellet were lysed using sterile water followed by the addition of 2x phosphate-buffered saline (PBS; 27 mM Na2HPO4, 132 mM KH2PO4, and 2.74 M NaCl). PMN were then isolated from the mononuclear cells by centrifugation in Histopaque (Sigma) with a density of 1.077 g/ml at 150 g for 13 min, resuspended in chilled 70% ethanol, and then stored until analyzed. From a total 1 ml of whole blood, we recovered 4.54.8 x 105 PMN. The PMN recovery rate was 1115%, and the PMN purity was 9598%.
Staining procedure for flow cytometric analysis. Ethanol-fixed 4.54.8 x 105 PMN were used for the further staining process. PMN were washed once with PBS and then resuspended in 1 ml of 0.2 mg/ml pepsin in 2 N HCl and incubated for 30 min at room temperature for DNA denaturation (27). This process caused the cells to lose most of their cytoplasm. PMN nuclei were pelleted and neutralized by 1 ml of 0.1 M Na2B4O7 incubated for 10 min. PMN nuclei were repelleted and resuspended in 200 µl of PBS and separated into two tubes. Each 100 µl of PMN nuclei suspension was mixed with 10 µl of FITC-conjugated monoclonal mouse anti-BrdU antibody Bu20a (DakoCytomation, Glostrup, Denmark) and FITC-conjugated mouse IgG (DakoCytomation) as a control, respectively. After a 30-min incubation at 4°C in the dark, PMN nuclei were washed once and resuspended in 300 µl of PBS containing 5 µg/ml propidium iodide (PI).
Data acquisition. Flow cytometric data were acquired on an EPICS XL-MCL (Beckman Coulter, Miami, FL). The cytometer's discriminator was set on integrated red fluorescence (PI staining). Single PMN nuclei were gated using peak vs. integral PI fluorescence. The green fluorescence (BrdU) of single nuclei was measured from 10,000 nuclei, and the data are presented on a logarithmic scale.
Data analysis. List mode data files were analyzed using Summit software (version 3.1; DakoCytomation, Fort Collins, CO). The numbers of BrdU-positive and BrdU-negative PMN nuclei were quantified using Overton's subtraction technique (14). Five regions of BrdU-positive cells, one each for R1, R2, R3, R4, and R5, were positioned depending on the fluorescence intensity corresponding to BrdU-staining levels detected using anti-BrdU-FITC antibodies. The region length position for R1, R2, R3, R4 and R5 was set at a 1:2:4:8:16 ratio of the FITC intensity channel number for the subtracted BrdU-positive curve (Fig. 2). The gate region placement for the BrdU-positive histogram was determined using the mean values of 24-, 30-, and 36-h samples from each rabbit. The quantified results for each region were regrouped as G1 (R1, region of weakest intensity), G2 (R2 + R3 + R4), and G3 (R5, region of strongest intensity) to correspond to the visual grading performed in cytospin slides.
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Transit time of PMNBrdU through the bone marrow.
The number of PMNBrdU was corrected for the disappearance (half-life, t) of cells in the circulation to calculate the transit time of PMN from bone marrow into the circulation. Our previous study using a whole blood transfusion method reported that the t of PMNBrdU in rabbits is 4.5 h (3). We have applied this rate of exponential loss of PMNBrdU from the circulation to calculate the number of PMNBrdU released from the bone marrow and the transit time through the different pools in the marrow in the following manner:
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These calculations were performed for each 6-h interval, and a histogram was drawn to show the distribution of PMNBrdU. The transit times of the different populations of labeled PMN (G1, G2, and G3) were calculated individually in each animal, and the mean transit times were compared among the groups.
Release of PMN from the bone marrow after particulate air pollution exposure. To determine the utility of this method, we used a well-established model of lung inflammation induced by particulate matter air pollution exposure (PM10) (22). Rabbits were anesthetized with 5% halothane, and 1 ml of normal saline (control, n = 3) or PM10 (500 µg of EHC-93 mixed with 1 ml of saline; n = 6) was instilled intrabronchially using fluoroscopic guidance according to a method previously described in detail (22). BrdU was infused intravenously 24 h before the instillation, and blood samples were collected at every 24-h interval (before and at 24, 48, 72, 96, 120, 144, 168 h after BrdU administration) from each rabbit.
Statistical analysis. Values are expressed as means ± SE. We used Pearson's correlation coefficient to determine the correlation between flow cytometric and light microscopic results. Data were analyzed using two-way ANOVA for repeated measures, and the Bonferroni correction was used for multiple comparisons. The transit times of PMNBrdU were compared between the groups using an unpaired Student's t-test. Statistical significance was set at P < 0.05.
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RESULTS |
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Correlation between the flow cytometric and microscopic PMNBrdU counts. The correlation between the flow cytometric and the light microscopic counts for 39 samples (3 rabbits x 13 time points) is shown in Fig. 3. There was a good correlation between the counts using the flow cytometric method (FCM) and light microscopic method (LMM) for each group of labeled cells: all PMNBrdU (R = 0.95; P < 0.0001), G1 (R = 0.64; P < 0.0001), G2 (R = 0.91; P < 0.0001), and G3 (R = 0.80; P < 0.0001).
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DISCUSSION |
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The rabbit model for measuring the bone marrow transit time of neutrophils has been developed in our laboratory for the measurement of neutrophil transit time through both the mitotic and the maturation pools in the marrow. In this method, the BrdU-labeled neutrophils were identified using an immunocytometric method (23). Basu et al. (2) reported a flow cytometric technique to enumerate myeloid cells in mice bone marrow and peripheral blood using the myeloid specific marker Gr-1. We have used a gradient isolation technique to obtain purified PMN, because no neutrophil-specific antibodies are available for rabbits. In contrast to the study by Basu et al., we found that the DNA-denaturing process by HCl changes the cell size and shape so that the conventional gating technique used to identify PMN is not reliable. The main advantage of the rabbit model (compared with the mouse model) to evaluate the bone marrow kinetics is that repeated blood samples can be obtained from the same animal during a 2-wk study period.
The LMM that was developed in our laboratory has the advantage that it does not require the use of radioisotopes (3). This method allowed us to study the kinetics as well as the phenotypic and functional characteristics of different populations of leukocytes with minimal cell manipulation. Using this model, we have demonstrated that acute pneumococcal pneumonia, chronic cigarette smoke, and ambient particulate matter exposure accelerate the transit time of PMN through the marrow and the release of immature PMN into the circulation that are preferentially sequestered in the pulmonary microvessels (11, 20, 21, 23). We have also shown that interleukin (IL)-6 and granulocyte colony-stimulating factor (G-CSF) administration accelerate the release of immature PMN into the circulation, whereas IL-8 administration releases PMN residing in the marrow venous sinusoids without changing the transit time through the marrow (12, 17, 19).
We used Overton's histogram subtraction technique to obtain the number of true PMNBrdU resulting from the overlap between the IgG-negative histogram and the weakly positive PMNBrdU. Identifying the weakly BrdU-positive PMNBrdU is important, because they represent the early myeloid progenitors in the marrow mitotic pool. We used the first three samples of each rabbit, at 24, 30, and 36 h, to set the channel width and determine the total number of positive PMNBrdU cells because these samples contain a mixture of strongly and weakly positive cells. This setting was applied for each subsequent sample and was adjusted to shift the region to the minimum positive channel of PMNBrdU. Applying this method resulted in <2% of PMNBrdU falling out of the positive region. The relationship between the FCM and LMM to enumerate the total number of PMNBrdU was very good (R = 0.90). There was a tendency for the number of PMNBrdU determined using FCM to be less than the number determined using LMM, owing to an overestimation of G1 by the LMM. We suspect that using the APAAP method, designed to amplify the BrdU signal, in the LMM gives more nonspecific staining of weakly positive cells than the direct immunofluorescent labeling used in the FCM, resulting in an overestimation of the weakly stained G1 cells.
The myeloid precursors in the marrow divided an average of five times before entering the postmitotic or maturation pool (1). In the LMM, we used the weakly stained G1 cells to determine the total transit time of PMN through the marrow (mitotic plus maturation pool transits) and the strongly stained G3 cells to determine the transit time of PMN through the maturation pool in the marrow (23). This allowed us to calculate the transit times of PMN through both these pools in the marrow. We have applied this previously established light microscopic grading system of PMN (G1, G2, and G3) to the flow cytometric data. The population of PMNBrdU was divided into five regions according to their fluorescence levels on the basis of the assumption that the BrdU level decreases by a factor of 2 after each cell division process in the mitotic pool. The quantified results for each region were then regrouped into three grades: G1 equals R1, G2 equals R2 + R3 + R4, and G3 equals R5. These region settings showed a correlation between the FCM and LMM counts for G1, G2, and G3 PMNs (R = 0.64, 0.91, and 0.80, respectively; P < 0.0001), supporting the notion that the FCM is a reliable method of quantifying the subpopulations of PMN labeled with BrdU in the mitotic pool. The calculated transit time of all PMN through the bone marrow using the FCM was shorter than that using the LMM, and this discrepancy is attributed to the difference in the calculated total number of PMNBrdU by APAAP (see above). However, the transit time derived by the FCM was still within the previously reported normal range using the LMM. Our previously reported values for the average PMN transit time in normal rabbits were 85.598.1 h in different experiments (19, 22, 23). Similar results in the transit times for each of G1, G2, and G3 with both methods demonstrates that the FCM is a valid method with which to study the kinetics of leukocytes specifically in comparative studies. The added advantage of the FCM is that it allows us to monitor the changes at each cell division stage using the five PMNBrdU subpopulations in regions R1R5. For example, if the transit time of PMN is accelerated through the mitotic pool because of marrow stimulation, it allows us to determine whether cells are released after fewer divisions or whether cell division is accelerated. These measurements and calculations of the transit times of defined subpopulations of myeloid cells are not possible using the method of Basu et al. (2), who classified their PMNBrdU into two arbitrarily designed groups (one group is at least fivefold that of the other, according to its BrdU level).
To determine the utility of the FCM, we used a well-established model of PM10-induced lung inflammation in rabbits (11, 22). We have previously shown that acute exposure to air pollution causes a leukocytosis associated with bone marrow stimulation and the release of immature granulocytes into the circulation (18, 22). Using the FCM, we demonstrated that a single acute exposure to ambient particles accelerated PMN release into the circulation, with transit times similar to those reported previously calculated using the immunocytochemical method (71.0 vs. 85.5 h, colloidal carbon vs. saline) (22). We showed that PM10 exposure shortens the transit time of PMN, especially the PMN progenitors at the early phase in the mitotic pool of the bone marrow. We have previously shown, using the immunocytochemical method, that a single exposure of colloidal carbon (an important component of PM10) or a repeated exposure of ambient particles significantly shortened the transit time of PMN through the bone marrow and accelerated the release of PMN into the circulation (11, 22). Our results from the current study showed that a single PM10 exposure affects mainly the PMN progenitors in the bone marrow mitotic pool.
Several papers have demonstrated that deoxyribonuclease (DNase) is an alternative for DNA denaturation without damaging the cell membrane, and this treatment allows for the double labeling of both leukocyte-specific surface markers and BrdU (5, 10, 24). Phenotypic characteristics of newly released PMN can be determined by double-labeling cells for BrdU and a phenotypic marker such as surface adhesion molecules. The FCM method is also ideally suited for rapid determination of the intravascular behavior of transfused BrdU-labeled cells and their half-lives in the circulation.
We conclude that flow cytometric analysis of BrdU-labeled leukocytes provides several advantages over the previously reported LMM for characterizing leukocyte behavior. It requires only a small volume of blood, is more objective and more precise, and is less labor intensive than immunocytochemical staining and light microscopic analysis of BrdU-labeled leukocytes. It correlates well with the LMM in identifying and grading the intensity of staining for BrdU incorporation into the DNA. This method can be used to measure the transit times of different populations of leukocytes through the bone marrow and characterize their intravascular behavior.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Basu S, Hodgson G, Katz M, and Dunn AR. Evaluation of role of G-CSF in the production, survival, and release of neutrophils from bone marrow into circulation. Blood 100: 854861, 2002.
3. Bicknell S, van Eeden S, Hayashi S, Hards J, English D, and Hogg JC. A non-radioisotopic method for tracing neutrophils in vivo using 5'-bromo-2'-deoxyuridine. Am J Respir Cell Mol Biol 10: 1623, 1994.[Abstract]
4. Dolbeare F, Gratzner H, Pallavicini MG, and Gray JW. Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc Natl Acad Sci USA 80: 55735577, 1983.
5. Gonchoroff NJ, Katzmann JA, Currie RM, Evans EL, Houck DW, Kline BC, Greipp PR, and Loken MR. S-phase detection with an antibody to bromodeoxyuridine: role of DNase pretreatment. J Immunol Methods 93: 97101, 1986.[CrossRef][ISI][Medline]
6. Gratzner HG. Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218: 474475, 1982.[ISI][Medline]
7. Houck DW and Loken MR. Simultaneous analysis of cell surface antigens, bromodeoxyuridine incorporation and DNA content. Cytometry 6: 531538, 1985.[CrossRef][ISI][Medline]
8. Khochbin S, Chabanas A, Albert P, Albert J, and Lawrence JJ. Application of bromodeoxyuridine incorporation measurements to the determination of cell distribution within the S phase of the cell cycle. Cytometry 9: 499503, 1988.[CrossRef][ISI][Medline]
9. Lichtman MA and Weed RI. Alteration of the cell periphery during granulocyte maturation: relationship to cell function. Blood 39: 301316, 1972.[Medline]
10. Lucas B, Vasseur F, and Penit C. Normal sequence of phenotypic transitions in one cohort of 5-bromo-2'-deoxyuridine-pulse-labeled thymocytes: correlation with T cell receptor expression. J Immunol 151: 45744582, 1993.
11. Mukae H, Vincent R, Quinlan K, English D, Hards J, Hogg JC, and van Eeden SF. The effect of repeated exposure to particulate air pollution (PM10) on the bone marrow. Am J Respir Crit Care Med 163: 201209, 2001.
12. Mukae H, Zamfir D, English D, Hogg JC, and van Eeden SF. Polymorphonuclear leukocytes released from the bone marrow by granulocyte colony-stimulating factor: intravascular behavior. Hematol J 1: 159171, 2000.[CrossRef][Medline]
13. Nusse M, Julch M, Geido E, Bruno S, Di Vinci A, Giaretti W, and Ruoss K. Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements. Cytometry 10: 312319, 1989.[CrossRef][ISI][Medline]
14. Overton WR. Modified histogram subtraction technique for analysis of flow cytometry data. Cytometry 9: 619626, 1988.[ISI][Medline]
15. Sasaki K, Murakami T, Ogino T, and Takahashi M. Flow cytometric dual-parameter analysis of human leukemic cells using monoclonal anti-BrdUrd antibody [Article in Japanese]. Gan No Rinsho 31: 549551, 1985.[Medline]
16. Sibille Y and Reynolds HY. Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am Rev Respir Dis 141: 471501, 1990.[ISI][Medline]
17. Suwa T, Hogg JC, Klut ME, Hards J, and van Eeden SF. Interleukin-6 changes deformability of neutrophils and induces their sequestration in the lung. Am J Respir Crit Care Med 163: 970976, 2001.
18. Tan WC, Qiu D, Liam BL, Ng TP, Lee SH, van Eeden SF, D'Yachkova Y, and Hogg JC. The human bone marrow response to acute air pollution caused by forest fires. Am J Respir Crit Care Med 161: 12131217, 2000.
19. Terashima T, English D, Hogg JC, and van Eeden SF. Release of polymorphonuclear leukocytes from the bone marrow by interleukin-8. Blood 92: 10621069, 1998.
20. Terashima T, Klut ME, English D, Hards J, Hogg JC, and van Eeden SF. Cigarette smoking causes sequestration of polymorphonuclear leukocytes released from the bone marrow in lung microvessels. Am J Respir Cell Mol Biol 20: 171177, 1999.
21. Terashima T, Wiggs B, English D, Hogg JC, and van Eeden SF. The effect of cigarette smoking on the bone marrow. Am J Respir Crit Care Med 155: 10211026, 1997.[Abstract]
22. Terashima T, Wiggs B, English D, Hogg JC, and van Eeden SF. Phagocytosis of small carbon particles (PM10) by alveolar macrophages stimulates the release of polymorphonuclear leukocytes from bone marrow. Am J Respir Crit Care Med 155: 14411447, 1997.[Abstract]
23. Terashima T, Wiggs B, English D, Hogg JC, and van Eeden SF. Polymorphonuclear leukocyte transit times in bone marrow during streptococcal pneumonia. Am J Physiol Lung Cell Mol Physiol 271: L587L592, 1996.
24. Tough DF and Sprent J. Turnover of naive- and memory-phenotype T cells. J Exp Med 179: 11271135, 1994.
25. Van Eeden SF and Hogg JC. The response of human bone marrow to chronic cigarette smoking. Eur Respir J 15: 915921, 2000.
26. Van Eeden SF, Kitagawa Y, Sato Y, and Hogg JC. Polymorphonuclear leukocytes released from the bone marrow and acute lung injury. Chest 116: 43S46S, 1999.
27. Van Erp PE, Brons PP, Boezeman JB, de Jongh GJ, and Bauer FW. A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation. Cytometry 9: 627630, 1988.[CrossRef][ISI][Medline]
28. Wiedermann CJ, Schmalzl F, and Braunsteiner H. Investigation of granulocytopoietic kinetics by microdensitometric evaluation of primary granule naphthol-AS-D-chloroacetate esterase activity. Blut 47: 271277, 1983.[CrossRef][ISI][Medline]
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