McDonald Research Laboratories and iCAPTURE Centre, University of British Columbia, Saint Paul's Hospital, Vancouver, British Columbia, Canada V6Z1Y6
Submitted 23 January 2003 ; accepted in final form 25 March 2003
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ABSTRACT |
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leukocytes; transit time; half life; pneumonia; rabbits
Monocytes recruited into the lung originate from either the intravascular
pool of monocytes (21) or from
precursors in the marrow (38).
In the marrow, the monocytes originate from stem cells that undergo three
divisions (monoblast, promonocyte, and monocyte) before their release into the
circulation. Within the vascular space, the monocytes reside in a marginated
pool that can be twice as large as the number in the monocytes that are
circulating in the blood (37).
The circulating half life of monocytes is 71 h in humans
(43),
42 h in rats
(41), and 17.4 h in mice
(37). In the tissues,
monocytes differentiate into tissue macrophages that have a life span
estimated to be 25 wk
(39). The increase in tissue
macrophages at the site of infection is accompanied by an increase in
monocytes in the circulating blood
(37,
38), and the majority of the
tissue macrophages are derived from this circulating pool of monocytes. These
kinetic studies have all been performed using radioisotopes
(14,
3739,
41).
Inflammatory mediators released from the inflamed lung have the ability to
induce the marrow to release monocytes and stimulate monocytopoiesis. The
hematopoietic growth factors, granulocyte/macrophage colony-stimulating factor
(GM-CSF) and M-CSF, interleukin-6 (IL-6), and the -chemokines are
thought to be important mediators for the production and mobilization of
monocytes from the bone marrow
(22,
25). Lung cells such as
alveolar macrophages and epithelial cells are a major source for these
mediators when they process microorganisms and inhaled ambient particles
(2,
9,
16,
32,
35).
This study was designed to develop a nonisotopic method to quantify the release of monocytes from the bone marrow, measure their half-life in the circulation, and calculate their transit through the bone marrow in rabbits. The thymidine analog 5'-bromo-2'-deoxyuridine (BrdU) was used to label dividing monocytes in the bone marrow and follow their release into the circulation. The monocytes that were labeled with BrdU in the marrow were specifically identified as monocytes in the peripheral blood using the monoclonal antibody RbM2 that is specific for rabbit monocyte lysosomal antigen (23, 26, 44). The kinetics of these cells was examined both in the normal control state and in a well-established model of pneumococcal pneumonia in rabbits (33).
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MATERIALS AND METHODS |
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Female New Zealand White rabbits (n = 28; weight, 2.4 ± 0.3 kg) were used in this study, and the protocol was approved by the Animal Experimentation Committee of the University of British Columbia.
Experimental Design
Release of BrdU-labeled monocytes from bone marrow. The time from BrdU incorporation into dividing monocyte precursors in the marrow until their appearance in the circulation was determined by infusing BrdU (100 mg/kg; Sigma Chemical, St. Louis, MO) at a concentration of 10 mg/ml in sterile saline into the marginal ear vein over a period of 15 min. Blood samples were obtained from the central ear artery just before (baseline) and at intervals from 2 to 72 h after BrdU infusion was used to measure total leukocyte counts and determine the number of BrdU-labeled monocytes (MOBrdU). Sedation [Fentanyl (20 µg/kg) and droperidol (1 mg/kg)] was administered by subcutaneous injection to facilitate blood collection.
Clearance of MOBrdU from the circulation. To determine
the clearance of monocytes from the circulation, whole blood containing
labeled monocytes was transfused from donor rabbits to serum compatible
recipients. In a preliminary study, donor rabbits (n = 3) were given
BrdU at a dose of 100 mg/kg every 2 h for a total of 5 injections, and blood
was obtained from the central ear artery of these rabbits before and at 4, 8,
18, 21, 24, 27, 30, and 48 h after the first BrdU injection to determine the
time of maximum number of monocytes were labeled with BrdU. On the basis of
this result, the BrdU-labeled monocytes were harvested from another three
donor rabbits and transfused to six recipients as 15 ml/kg of whole blood by a
method previously described in detail
(3,
20). The donor-recipient pair
was chosen by their serum compatibility that was confirmed by microscopic
evidence of hemolysis and agglutination after the recipient serum and donor
red cells were combined. Leukocyte-rich plasma (LRP) made from the donor
rabbit blood was incubated with recipient serum in microwells where trypan
blue uptake was used to determine cytotoxicity using recipient LRP and
recipient serum as a control
(29). Counts of 20% or more
dead cells over controls were considered incompatible. The clearance of
MOBrdU from the circulating blood was measured from blood samples
obtained from the central ear artery of the recipient rabbits before and at 30
min, and 1, 2, 4, 6, 12, 24, 36, and 48 h after the blood transfusion. The
number of MOBrdU in the circulation of each recipient was expressed
as a fraction of the total number of labeled monocytes originally infused and
corrected for the calculated blood volume
(27) of the recipient in the
following manner
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Effect of pneumonia on monocyte transit time through the marrow. Rabbits (n = 6) were anesthetized with ketamine hydrochloride (80 to 100 mg/kg, intramuscularly) and xylazine (10 to 15 mg/kg, intramuscularly) and challenged with intrabronchial instillation of either Streptococcus pneumoniae (1.5 x 109 organisms mixed with 1 ml of saline and colloidal carbon) or vehicle (1 ml of saline mixed with colloidal carbon to mark the area of instillation for histological sampling) as a control 4 h after treatment with BrdU. This time point was selected for instillation because first MOBrdU appeared in the circulation 4 h after BrdU administration. Briefly, streptococci or vehicle was instilled into the lower lobe of the lung by inserting a pediatric feeding tube through the tracheal rings and positioned via fluoroscopy as previously described in detail (17, 33). The animal was then allowed to recover, and the peripheral blood samples were taken just before (baseline) and at intervals from 4 to 168 h after BrdU injection and analyzed for the presence of MOBrdU in the circulation as described below.
Leukocyte Counts
One milliliter of blood was collected in standard Vacutainer tubes containing EDTA (Becton Dikinson, Rutherford, NJ), and WBC counts were determined on a model SS80 Coulter counter (Coulter Electronics, Hilaeh, FL). Differential WBC counts were done by counting 200 leukocytes in randomly selected fields of view on Wright-Giemsa stained blood smears.
Immunohistochemical Detection of BrdU-labeled Leukocytes in the Circulation
Sample preparation. Blood collected in acid-citrate-dextrose (ACD) was used to obtain LRP. Erythrocytes in the ACD blood sample were sedimented for 2530 min by the addition of an equal volume of 4% dextran (average molecular weight, 162,000; Sigma) in polymorphonuclear leukocytes (PMN) buffer (in mmol/l: 138 NaCl, 27 KCl, 8.1 Na2HPO4 7H2O, 1.5 KH2PO4, and 5.5 glucose, pH 7.4). The resulting LRP was cytospun onto slides precoated with 3-aminopropyl-tri-ethoxysilane by cytocentrifugation at 1,200 revolutions/min with a Cytospin 2 (Shandon Scientific, Runcorn, UK) for 4 min. The cytospun specimens were air-dried and fixed in acetone for 10 min.
Double immunoenzymatic staining of monocytes for RbM2 and BrdU.
Monocytes were identified using RbM2 (ICN Biomedicals, Aurora, OH), a
monoclonal antibody specific for rabbit monocyte lysosomal antigen
(23,
26,
44). Cells on cytospins were
stained for the presence of both monocyte cytoplasm RbM2 antigen (red) and
nuclear BrdU (blue) using the alkaline phosphatase and anti-alkaline
phosphatase (APAAP) method (5)
and a modified double immunolabeling technique previously described
(19,
34). Briefly, cells on the
cytospin were incubated with 6 mg/ml of N-octyl
-D-glucopyranoside (Sigma) as a permeabilizer for 7 min and
then with 5% rabbit serum for 15 min before application of 5 µg/ml of RbM2
for 90 min in a humidity chamber at room temperature. As a negative control,
nonspecific mouse immunoglobulin G1 (IgG1) at 5 µg/ml was used. A 1:20
dilution of rabbit anti-mouse IgG (DAKO Laboratories, Copenhagen, Denmark) was
applied and followed by the anti-mouse IgG alkaline phosphatase-conjugated
complex (DAKO) in a 1:50 dilution for 20 min, respectively. This procedure was
repeated in the same concentration for 15 min. All antibodies were prepared in
50 mmol/l Tris · HCl and 150 mmol/l NaCl, pH 7.6 (TBS), with 1% bovine
serum albumin, and slides were washed in TBS twice for 10 min between each
antibody application. The alkaline phosphatase was developed for 20 min in 50
ml of TBS at pH 8.7 after the addition of a mixture of 0.5 ml of 4% sodium
nitrite, 0.2 ml of 5% fuschin (Merck, Rahway, NJ) in 2 mol/l HCl, and 50 mg
naphthol AS-B1 phosphate (Sigma) dissolved in 0.3 ml of
N,N-dimethylformamide. Endogenous alkaline phosphatase was blocked by
the addition of 17.5 mg of levamisole (Sigma). Specimens were then fixed with
1% paraformaldehyde for 15 min before being stained for the presence of
nuclear BrdU using a second APAAP procedure as described in detail previously
(3,
19,
34). The alkaline phosphatase
was developed with a commercially available kit, HistoMark Blue (Kirkegaard
and Perry, Gaithersburg, MD), for 10 min in the dark. Slides were washed with
tap water, mounted in an aqueous medium (Gelvatol), and evaluated on a Zeiss
Universal Research light microscope (Model 2R; Zeiss, Oberkochen, Germany) at
x400 magnification.
Evaluation of MOBrdU. Monocytes with both cytoplasmic stain (red), and nuclear stain (blue) were counted as BrdU-labeled (MOBrdU). Cytospin slides were coded and evaluated by investigators without knowledge of their origin. MOBrdU were evaluated on the light microscope in random field of view by counting 200 monocytes per slide. Results are expressed as the percentage of monocytes or the number per milliliter of blood that is BrdU-labeled.
Calculation of the Half-Life of Monocytes
The exponential decrease of the number of MOBrdU in the
circulation is described by the equation
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Because the half-life (T1/2) can be estimated as the time at which Nt is NTmax x 1/2, the late-decay equation for T1/2 becomes T1/2 = ln 2/k. The constant k was calculated with the random-effects regression method (7). The confidence interval (4) was obtained by deriving the lower and upper bounds of the 95% confidence interval for the slope, k.
Transit Time of MOBrdU through the Bone Marrow
The transit time on monocytes through the bone marrow was calculated from
the appearance of BrdU-labeled cells in the circulation over time and
corrected for the disappearance (T1/2) of the
cells in the circulation (33).
The T1/2 of MOBrdU was applied to
calculate the transit time of monocytes through the marrow in the following
manner
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These calculations were made for 2 h intervals for the first 24 h and 6 h intervals for the later time course, and a histogram was drawn showing the distribution of MOBrdU released from the bone marrow during each interval. The mean transit time for MOBrdU was calculated for each animal.
Statistical Analysis
All values are expressed as means ± SE. Data were analyzed using a repeated measure analysis of variance (ANOVA) over time, and the effect of multiple comparisons was corrected using the Bonferroni method. Transit time was compared by one-way ANOVA, followed by Fisher's protected least significant difference test as the post hoc test among the groups. Comparisons of transit time or half life between monocytes and PMN were made by Student's t-test. A corrected P < 0.05 was considered significant throughout the study.
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RESULTS |
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Figure 1 shows double immunolabeling of monocytes for both cytoplasm RbM2 (red) and nuclear BrdU (blue). All RbM2-positive cells (monocytes) were classified as either BrdU positive or negative. The influence of the double-labeling procedure on the presence of cytoplasm RbM2 and nuclear BrdU expression was evaluated by comparing the number of positive monocytes of all nucleated cells for each antigen with paired slides stained for a single antigen in 10 randomly selected sides. There was no difference in the number between single- or double-stained cells.
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Clearance of MOBrdU from the Circulation
Figure 2 shows the
appearance of labeled monocytes in the circulation of donors after BrdU
injection at a dose of 100 mg/kg every 2 h for 5 injections. The number
increased gradually, and 80% of the circulating monocytes were labeled 24
h after the initial injection of BrdU. After the transfusion of donor whole
blood containing 83 ± 2.0% of labeled monocytes, the number of
MOBrdU in the circulation of the recipients fluctuated to a peak at
1 h, rapidly decreased over the next 6 h, and then relatively slowly declined
over the next 48 h (Figure 3).
The time required to achieve the maximal number of MOBrdU in the
circulating blood of the recipients (Tmax) was 1 h and was
applied to the rate decay equation to calculate the
T1/2 of monocytes in the circulation. The
calculated T1/2 of MOBrdU in the
circulation using this method was 12.7 h with a 95% confidence interval of
10.715.6 h, which is significantly longer the 4.5 h with 95% confidence
interval of 4.14.9 h reported for PMN in rabbits using the same
technique (3).
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Transit Time of Monocytes through the Bone Marrow
Figure 4 shows the appearance of MOBrdU in the circulation after pulse labeling with 100 mg/kg BrdU. The first MOBrdU appears in the circulation 4 h after labeling, followed by a rapid increase in the percentage of MOBrdU to reach a peak at 18 h (34.3 ± 5.8% of circulating monocytes labeled), and then a slow decline over the next 72 h (at 24 h: 21.0 ± 2.3%, at 48 h: 12.7 ± 2.7%, at 72 h: 8.7 ± 2.0%). The absolute number of circulating monocytes was stable during the study period. The calculated mean transit time of MOBrdU through the bone marrow in control rabbits was 38.1 ± 3.1 h, which was much shorter than the values for PMN (95.6 ± 3.6 h) (P < 0.001) previously reported from our laboratory using a similar technique (33).
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Behavior of MOBrdU through the Marrow into the Circulation during Pneumonia
Figure 5 shows the percentage of BrdU-labeled monocytes in the circulation in rabbits with and without a focal pneumococcal pneumonia. No change was seen in monocytes or PMN counts in the circulation during the study period (data not shown) with a significant increase in band cell counts at 16 h after instillation of pneumococci (1.34 ± 0.35 x109/l vs. 0.59 ± 0.16 x109/l, S. pneumoniae-challenged vs. control; P < 0.05). The fraction of MOBrdU rapidly increased after the instillation to peak at 10 h (S. pneumoniae-challenged) and 12 h (control) with a more rapid clearance of MOBrdU from the circulation in the S. pneumoniae-challenged group (2448 h; P < 0.05). The transit time of MOBrdU through the marrow was significantly shorter in the S. pneumoniae-challenged group compared with vehicle instillation (22.6 ± 0.6 vs. 27.3 ± 1.8 h; P < 0.05) or S. pneumoniae-challenged group compared with no instillation (22.6 ± 0.6 vs. 38.1 ± 3.1 h; P < 0.01) (Table 1).
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DISCUSSION |
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Monocyte turnover and behavior have been studied in human (14, 39), mouse (37, 38), and rat (41) using radiolabeled purified monocytes or pulse labeled of dividing cells with [3H]thymidine ([3H]TdR) intravenously for the calculation of their cell cycle time, transit through the bone marrow, circulation, and life span in the tissue. The purification of monocytes is difficult and time consuming, and labeling with radioisotopes also requires decontamination and disposal of radioactive waste. The ability to label the dividing cells in vivo and transfer them to recipient animals in whole blood avoids all of these difficulties. However, transfusing blood cells or blood products between outbred animals could potentially cause histocompatibility reactions. In this study, we performed serum cross-match between donors and recipients for red cell (agglutination microscopically) and leukocyte [cytotoxicity test (29)] compatibility. This method does not exclude all possible histocompatibility reactions, but several studies from our laboratory using this transfusion method have shown no measurable immune reaction either clinically (fever, diarrhea, lost of appetite, convulsion, or death) or in blood cell counts, marrow stimulation, and leukocyte activation (3, 19, 20). Although we cannot exclude mild activation of leukocytes, histocompatibility reactions are unlikely to be a significantly confounding factor using this method of transfusing labeled leukocytes.
Our preliminary experiments showed that the short transit time of monocytes
through the marrow required that the donors receive 5 injections of BrdU over
8 h to get 85% of the monocytes in the peripheral blood labeled at 24 h
(Fig. 2). Transfer of these
labeled cells in whole blood from the donor animals to serum-compatible
recipients showed that the calculated T1/2 of the
BrdU-labeled monocytes in the circulation was 12.7 h (95% confidence interval
of 10.7 and 15.6 h). This value is shorter than the previously reported values
of
24 h measured using 51Cr- or 111In-labeled
monocytes in rabbits and calculating T1/2 over
shorter periods in rabbits (6,
10,
21). It is also longer than
T1/2 of PMN in the circulation of rabbits
measured using the same technique
(3). After transfusion of
labeled whole blood, the percentage of BrdU-labeled monocytes remaining in the
circulating pool at steady state (1 h) was 41%
(Fig. 3), suggesting that the
remaining 59% of the monocytes had moved to the marginated intravascular pool.
These results are consistent with the studies in mice
(37,
40) and humans
(15) where the marginated pool
has been estimated to be
60 and
75%, respectively. In contrast,
using indium-labeled monocytes in rabbits, Ohgami and colleagues
(21) showed that
85% of
monocytes had shifted from the circulating to the marginated pool at 1 h in
rabbits and that the majority of the marginated cells were in the liver and
lung. Because of minimal cell manipulation with this whole blood method in
contrast to purification and isotope labeling, we suspect that the present
results more closely represent the behavior of monocytes in vivo.
In the experiments where a single dose of BrdU was given to pulse label the dividing monocytes in the marrow, the first BrdU-labeled monocytes appeared in the circulation 4 h after the BrdU injection (Fig. 4). This represents the minimum time from DNA synthesis of the last division in the marrow until release of the monocytes into the circulation. This rapid release of monocytes after labeling suggests that monocytes translocate from the marrow to the peripheral blood soon after their last division and do not mature in the marrow. This contrasts with PMN that have a distinct maturation pool in the marrow where they reside before their release into the circulation (1, 33). The rapid release of monocytes from the marrow and their long T1/2 in the circulation suggest that they mature in the circulation before they migrate into the tissues.
Assuming that all mitotic cells in the bone marrow incorporated BrdU into
nuclear DNA over a short period and that rapid clearance of the BrdU minimized
recirculation and reuse of BrdU
(12,
24), we calculated that the
transit time of monocytes through the bone marrow in normal rabbits was 38.1
± 3.1 h. This is shorter than in humans (48.2 h) but longer than
in rats (
32 h) and mice (28.1 h)
(37,
39,
41). Comparison of the present
values for monocyte transit to the previously reported transit time of PMN
through the marrow measured using the same technique showed that PMN transit
is significantly longer (95.6 ± 3.6 h vs. 38.1 ± 3.1 h)
(33). The present results also
show that the introduction of a focal pneumonia significantly shortened the
transit time of monocytes through the marrow
(Table 1). Interestingly, even
the instillation of the vehicle into the lung also reduced the transit time of
monocytes through the marrow, suggesting that just a mild inflammatory insult
in the alveolar space (32)
results in a monocytic response from the marrow.
Studies from several laboratories including our own have shown that
alveolar macrophages are a potent source of inflammatory mediators
(2,
16,
32,
35), and we have shown that
several of these mediators have the ability to stimulate the bone marrow
(18,
28,
31,
36). The hematopoietic growth
factors, GM-CSF and M-CSF, IL-6, and the -chemokines are thought to be
important mediators for the production and mobilization of monocytes from the
bone marrow (22,
25). Terashima and colleagues
(30) showed an increase in
circulating IL-6 and G-CSF in subjects with community-acquired pneumonia,
suggesting that these two cytokines contribute to the bone marrow response to
bacterial pneumonia. Interleukin-6 is considered an important multifunctional
cytokine involved in the regulation of a variety of cellular responses,
including hematopoiesis. It is a permissive factor for monocytic colony
formation by human hematopoietic progenitor cells
(11). These workers showed
that antibody against IL-6 almost completely inhibited the growth of monocytic
colonies without decreasing the number of granulocytic colonies. The
importance of IL-6 in monocytopoiesis was further demonstrated in
serum-deprived bone marrow cultures where the addition of exogenous IL-6 to
cultures stimulated with GM-CSF resulted in increased numbers of monocytic
colonies. We suspect that IL-6 combined with the colony stimulating factors
released from the inflammatory site in the lung is critically important in the
production and release of monocytes from the marrow during acute lung
inflammation.
The recruitment of leukocyte into a site of acute lung inflammation is characterized by an early recruitment of PMN (12 h), followed by a delayed accumulation of monocytes (6, 8, 10, 42). Monocytes are recruited into lung tissues from the circulation most likely from the large marginating pool of monocytes (6, 17, 21) in the lung. Our data indicate a rapid release of monocytes from the marrow with pneumonia (Fig. 5) that occurs before the PMN are released (33). Because the migration of monocytes into the lung is delayed compared with PMN, we speculate that these new monocytes enter the marginating pool of cells where they mature before their migration into sites of inflammation.
In summary, we have developed a nonradioisotopic method to measure monocytes behavior in vivo. This method allows the release of monocytes from the marrow to be quantified and their T1/2 in the circulation pool to be estimated. This novel technique will allow the phenotypic and functional characterization of monocytes that are newly released from the marrow to be compared with fully mature monocytes as they participate in the inflammatory response.
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DISCLOSURES |
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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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