Flow cytometric method for enumeration and characterization of newly released polymorphonuclear leukocytes from the bone marrow using 5'-bromo-2'-deoxyuridine

Chih-Horng Shih, Beth A. Whalen, Yukinobu Goto, James C. Hogg, and Stephan F. van Eeden

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


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Inflammation accelerates polymorphonuclear leukocyte (PMN) release from the bone marrow, and these PMNs are implicated in inappropriate tissue injury. We have previously developed a method using 5'-bromo-2'-deoxyuridine (BrdU) to study PMN kinetics using an immunocytochemical grading system of PMN on cytospin slides. The aim of this study was to develop a flow cytometric method to quantify the number of positively stained PMN and grade the intensity of staining for the transit time calculation of PMN through the marrow. Dividing myeloid progenitors in the marrow of rabbits were labeled with a pulse dosage of intravenous BrdU. BrdU-labeled PMN (PMNBrdU) were detected in the circulation using a FITC-conjugated anti-BrdU monoclonal antibody. The PMNBrdU were assigned to five groups according to their FITC intensity, and the transit times of PMN at different stages of development in the marrow were calculated. Results were compared using parallel immunocytochemical analysis of the same samples. In control animals, PMNBrdU in the circulation peaked at 72 h after BrdU labeling with 36.0% of PMN labeled. In normal rabbits, the transit times of PMN through the mitotic pool (49.5 ± 4.2 h) and maturation pool (65.5 ± 3.1 h) correlated well with immunocytochemical analysis and previously published values. Using this method, we demonstrated that exposure to air pollution particles accelerates the release of PMNBrdU from the marrow. We conclude that a flow cytometric approach for identifying BrdU-labeled leukocytes provides an objective and accurate method for studying leukocyte kinetics and behavior.

polymolphonuclear neutrophil; flow cytometry; transit time


POLYMORPHONUCLEAR LEUKOCYTE (PMN) leukocytosis is a common feature of the inflammatory response. PMNs increase their mobility, deformability, and chemoactivity during the maturation process in the bone marrow (9). The primary granules that contain myeloperoxidase and proteolytic enzymes are formed at the promyelocytic stage, and the number of these granules is reduced by mitosis as the PMN precursors pass through the mitotic pool (28). It has been reported that skipping divisions during the mitotic pool results in the production of PMN with higher granule numbers and greater destructive capabilities (16). Therefore, it is of interest to determine changes in the bone marrow pools of PMN with marrow stimulation, because it may reveal possible mechanisms of inappropriate tissue injury associated with acute and chronic inflammatory conditions (25, 26).

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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Fig. 1. Polymorphonuclear leukocyte (PMN) transit time through the marrow using 5'-bromo-2'-deoxyuridine (BrdU) labeling. Dividing myeloid cells in the marrow are pulse labeled with BrdU. The calculated transit time of PMN through the different pools in the marrow (mitotic pool and maturation pool) was based on the assumption that the levels of BrdU labeling of DNA decreases during the cell division process. Progenitor cells in the last, middle, and first divisions in the marrow mitotic pool, when exposed to BrdU, can be identified as G3 (strongly positive), G2 (moderately positive), or G1 (weakly positive), respectively.

 
This method of identifying PMNBrdU was based on light microscopic enumeration and grading of immunocytochemically stained cells on cytospin slides, a procedure that is time consuming, labor intensive, and relatively subjective. In the present study, we describe a novel method of accomplishing this goal to grade and quantify PMNBrdU using a flow cytometric approach that enables us to monitor the bone marrow response more objectively and with a significant reduction in the labor required.


    MATERIALS AND METHODS
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Experimental animals. Adult female New Zealand White (NZW) rabbits (age 6–8 wk, n = 12) with an average weight of 2.23 ± 0.16 kg (mean ± SD; 2.0–2.5 kg) were used in this study, which was approved by the Animal Experimentation Committee of the University of British Columbia.

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 20–25 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 ~4.5–4.8 x 105 PMN. The PMN recovery rate was 11–15%, and the PMN purity was 95–98%.

Staining procedure for flow cytometric analysis. Ethanol-fixed 4.5–4.8 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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Fig. 2. Histogram showing BrdU-labeled PMN (PMNBrdU). The number of BrdU-positive or BrdU-negative PMN was quantified using Overton's subtraction technique. Mouse IgG was used as a negative control. By using the sample 72 h after BrdU administration as an example, we further gated and graded PMNBrdU into five regions, R1–R5, according to FITC intensity. The gated settings for R1–R5 were set at a 1:2:4:8:16 ratio using the channel number (e.g., total channel number = 167 – 74 = 93, so that the channel number for each R1, R2, R3, R4, and R5 was 3, 6, 12, 24, and 48, respectively). The quantified results for each region were then regrouped as G1 (R1), G2 (R2 + R3 +R4), and G3 (R5) for comparison with the light microscopic method. Data are presented on a logarithmic scale.

 
Immunocytochemical detection and evaluation of PMNBrdU on cytospin slides. Cytospin slides were fixed in acetone and stained by the alkaline phosphatase and anti-alkaline phosphatase (APAAP) method using anti-BrdU monoclonal antibody Bu20a to determine the fraction of PMNBrdU. A total of 100 PMN per specimen were morphologically evaluated on a Zeiss Universal light microscope (model 2R; Zeiss, Oberkochen, Germany) in random fields of view and PMN with any nuclear stain for BrdU were counted as positive. Furthermore, PMNBrdU were divided into three groups according to the intensity of nuclear staining, using an arbitrarily designated grading system: weakly positive (staining of <5% of the nucleus, G1), moderately positive (staining of 5–80% of the nucleus, G2), and strongly positive (staining of >80% of the nucleus, G3) as previously described in detail (23).

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:

where {Delta}N is the number of labeled cells released from the marrow in the time interval {Delta}t, ti and tj are the initial and successive time intervals, with t = tjti and k = ln 2/t.

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.


    RESULTS
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FITC intensity for each PMNBrdU region and grade. The mean FITC intensities for the R1, R2, R3, R4, and R5 regions were 17.6 ± 1.9, 20.8 ± 2.3, 29.3 ± 3.2, 59.9 ± 6.6, and 279.1 ± 30.7, respectively. The mean FITC intensities for the G1, G2, and G3 regions were 17.6 ± 2.3, 36.7 ± 4.0, and 279.1 ± 30.7, respectively.

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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Fig. 3. Correlation between flow cytometric (10,000 events analyzed) and light microscopic (100 PMN counted) identification of PMNBrdU. There was a good reciprocal correlation between the flow cytometric and light microscopic count for the labeled cells. All values are expressed as the percentage of labeled cells (n = 39, 3 rabbits x 13 time points). FCM, flow cytometric method; LMM, light microscopic method; solid line, regression line, dashed line, 95% confidence interval.

 
Release of PMNBrdU into the circulation. Typical histograms of flow cytometric analysis of changes in BrdU levels of PMN over time in the circulation are shown in Fig. 4. The changes in these histograms indicate that the PMNBrdU appeared in the circulation at 24 h after BrdU labeling, followed by a rapid increase in the number of PMNBrdU to a peak at 72 h, followed by a decrease during the next week. The mean BrdU fluorescence intensity levels of PMNBrdU in the circulation decreased gradually with time.



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Fig. 4. Temporal changes in circulating PMNBrdU. Typical temporal histogram changes in circulating PMNBrdU from one rabbit are shown. The first PMNBrdU appeared in the circulation 24 h after BrdU labeling, followed by a rapid increase of the population of PMNBrdU to a peak at 72 h (36.0 ± 7.0%; means ± SE from 3 rabbits), and then a gradual decrease during the next 7 days. Both the number of positive PMN in the circulation and the fluorescence intensity of FITC-labeled BrdU decreased over time. The quantified results for each region (R1–R5) were then regrouped as G1 (R1), G2 (R2 + R3 +R4), and G3 (R5).

 
The peak percentage of PMNBrdU was 36.0 ± 7.0%. The rate of increase for G3 was earlier than that of G1 or G2. The calculated percentage of PMNBrdU in the circulation with different BrdU levels shows that the G1 peaked between 120 and 168 h; in contrast, the G3 peaked earlier, at 72 h (Fig. 5).



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Fig. 5. Changes in G1, G2, and G3 cells released from the bone marrow into the circulation. Values are expressed as the calculated absolute number (AC) or percentage (DF) of PMNBrdU (means ± SE from 3 rabbits) in the circulation. G1 cells peaked between 120–168 h; in contrast, the G3 cells peaked earlier, at 72 h. G1 ({circ}, weakly positive; the cells in the early phase of the mitotic pool when exposed to BrdU), G2 ({bullet}, moderately positive; the cells in the middle phase of the mitotic pool when exposed to BrdU), and G3 ({square}, strongly positive; the cells in the last phase of the mitotic pool when exposed to BrdU).

 
Transit time of PMNBrdU through the bone marrow. The calculated transit times for each R1, R2, R3, R4, and R5 were 115.0 ± 1.1 h, 110.3 ± 1.5 h, 99.9 ± 2.8 h, 78.3 ± 0.6 h, and 65.5 ± 3.1 h, respectively (Fig. 6).



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Fig. 6. Transit times of PMN through the bone marrow derived from flow cytometric method. All values are expressed as the calculated hours (means ± SE from 3 rabbits). R1–R5 are different subpopulations of BrdU-labeled PMN and the groups defined by the fluorescence intensity corresponding to BrdU-staining levels. The myeloid precursors in the marrow divided an average of five times before entering the postmitotic or maturation pool. So, 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 intensity of BrdU staining is inversely proportional to the transit time through the bone marrow and results in a reciprocal relationship (Spearman's correlation coefficient by rank). Solid line, regression line; dashed line, 95% confidence interval.

 
Table 1 summarizes the comparison of transit times of PMN through the bone marrow derived from FCM and LMM. The transit time of all PMNBrdU through the bone marrow derived by the flow cytometric method was slightly shorter than the light microscopic method (87.2 ± 0.9 h vs. 97.8 ± 1.9 h, FCM vs. LMM). However, G1, G2, and G3 transit times between the two methods were similar (G1, 115.0 ± 1.1 h vs. 112.5 ± 1.8 h; G2, 88.1 ± 0.4 h vs. 86.9 ± 1.6 h; G3, 65.5 ± 3.1 h vs. 65.6 ± 2.0 h, FCM vs. LMM).


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Table 1. Transit times of PMN through the bone marrow

 
Effect of PM10 on the release of PMNBrdU from the bone marrow. Figure 7 shows the effect of a single exposure of PM10 on the release of PMNBrdU from the marrow into the circulation. The fraction of total PMNBrdU and G2 cells in the circulation increased more rapidly, with a peak at 48 h (P < 0.01), and cleared more gradually (120–144 h; P < 0.05) in the PM10-exposed group (n = 6) compared with the control group (n = 3), which peaked at 72 h. There was no difference in the release of G1 and G3 cells between the groups. Figure 8 shows a representative histogram demonstrating circulating PMNBrdU at 48 h after BrdU administration (24 h after instillation) in rabbits instilled with saline or PM10 intrabronchially.



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Fig. 7. Effect of particulate matter air pollution exposure (PM10) on the release of total PMNBrdU (A), G1 (B), G2 (C), and G3 cells (D) from the bone marrow into the circulation. PMNBrdU and G2 cells in the circulation increased more rapidly, with a peak at 48 h, and decreased more gradually in the PM10-exposed group (n = 6) compared with the saline-exposed control group (n = 3), which peaked at 72 h (A and C). G1, G2, and G3 cells represent the myeloid cells that were in the first, middle, and last divisions in the mitotic pool, respectively, when exposed to BrdU. There was no difference between the groups regarding the release of G1 and G3 cells (B and D). Values are expressed as the calculated percentage of PMNBrdU (means ± SE). PM10-exposed ({bullet}), saline-exposed ({circ}), and control. *P < 0.01, {dagger}P < 0.05 vs. saline-exposed control.

 


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Fig. 8. Histogram comparing circulating PMNBrdU between saline and PM10 exposure. Representative histogram showing circulating PMNBrdU at 48 h after BrdU administration (24 h after instillation) in rabbits instilled with saline or PM10 intrabronchially. Note the marked difference in the total number of PMNBrdU (5,632/12,223 vs. 608/11,250, PM10-exposed vs. saline-exposed control). The count for each R1, R2, R3, R4, and R5 is 13, 108, 487, 2,287, and 2,737, respectively, in PM10-exposed rabbits vs. 9, 43, 131, 351, and 74, respectively, in saline-exposed rabbits. R1–R5 are different subpopulations of BrdU-labeled PMN, and the groups are defined by the fluorescence intensity corresponding to BrdU-staining levels. Data are presented on a logarithmic scale.

 
A single PM10 exposure shortened the transit time of all PMNBrdU through the bone marrow (79.8 ± 0.9 h vs. 83.1 ± 0.5 h, PM10-exposed vs. saline-exposed control; P < 0.05) and R2 (PMN progenitors at early phase in the bone marrow mitotic pool, 98.1 ± 1.6 h vs. 104.3 ± 1.8 h, PM10-exposed vs. saline-exposed control; P < 0.05). The transit times of other subpopulations of PMNBrdU (R1, R3, R4, and R5) were not significantly shortened by a single PM10 exposure [R1, 99.4 ± 2.8 h vs. 105.4 ± 2.4 h; R3, 92.3 ± 1.0 h vs. 95.8 ± 2.6 h; R4, 75.2 ± 1.6 h vs. 75.9 ± 0.6 h; R5, 63.1 ± 4.2 h vs. 62.0 ± 3.5 h; R1–R5 (transit time through the mitotic pool), 36.3 ± 8.3 h vs. 43.4 ± 5.8 h, PM10-exposed vs. saline-exposed control] (Table 2).


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Table 2. Effect of PM10 on the transit times of PMN through the bone marrow

 

    DISCUSSION
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Flow cytometry provides a more objective and faster method with which to quantify and characterize BrdU-labeled leukocytes in the circulation and is a useful technique to study the bone marrow response induced by inflammatory conditions. The FCM of measuring PMN transit time reported here provides several improvements over the current LMM. The FCM analyzes a larger number of cells in each blood sample (>104 cells vs. 102 cells, FCM vs. LMM), evaluates the intensity of BrdU more objectively, enumerates the BrdU-labeled cells more precisely, and is less labor intensive.

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 85.5–98.1 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 R1–R5. 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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This work was supported by a grant from the Heart and Stroke Foundation of Canada, the Wolfe and Gita Churg Foundation, and National Heart, Lung, and Blood Institute Grant HL-407201. Dr. S. F. van Eeden is an American Lung Association Career Investigator and William Thurlbeck Distinguished Researcher.


    ACKNOWLEDGMENTS
 
We thank Dr. Mark Elliott for critical reading of the manuscript. We also acknowledge Diane Minshall, Karen Groden, Lynne Carter, and Anna Meredith for technical support, and Health Canada for making the EHC-93 air particles available.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. F. van Eeden, James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital-University of British Columbia, Rm. 166, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6 (e-mail: svaneeden{at}mrl.ubc.ca)

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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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