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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2730-2737
By
From the Pulmonary Research Laboratory, University of British
Columbia, St Paul's Hospital, Vancouver, British Columbia, Canada; and
the Department of Neonatology, Carmel Medical Center, Faculty of
Medicine, Technion, Harif, Israel.
When active bone marrow release is induced by inflammatory stimuli,
it is associated with an increase in L-selectin expression on
circulating polymorphonuclear leukocyte (PMN). This contrasts sharply
with glucocorticoid-induced granulocytosis that is associated with
decreased L-selectin expression on PMN. The present study was designed
to determine if the reduced L-selectin expression observed after
glucocorticoid treatment is the result of suppression of L-selectin
synthesis in the bone marrow. New Zealand white rabbits treated with
dexamethasone (2.0 mg/kg, a single dose intravenously) were shown to
have decreased L-selectin expression on circulating PMN 12 to
24 hours after treatment (P < .01) with a return to baseline
levels by 48 hours. When dexamethasone was administered 48 hours after
the bone marrow PMN were pulse labeled with the thymidine analogue,
5'-bromo-2'-deoxyuridine (BrdU), L-selectin expression on
BrdU-labeled PMN released from the bone marrow was decreased (P < .01). Dexamethasone decreased L-selectin expression on
segmented PMN in the bone marrow (P < .05) but not on PMN
already in the circulation. We conclude that glucocorticoids decrease L-selectin expression on circulating PMN by downregulating L-selectin expression in the maturation pool of bone marrow and speculate that
this is an important glucocorticoid effect that influences the
recruitment of PMN into inflammatory sites.
L-SELECTIN HAS A crucial role in the
initial attachment of circulating polymorphonuclear leukocyte (PMN) to
vascular endothelium during the initiation of PMN recruitment into a
systemic inflammatory site.1-3 L-selectin expression is low
in the mitotic pool, increases as they mature in the postmitotic pool
of bone marrow,4,5 and is constitutively expressed on
circulating PMN.6 PMN released from the bone marrow by
inflammatory stimuli such as complement fragments, pneumonia, and
endotoxin express higher levels of L-selectin than their circulating
counterpart,7-9 and they progressively lose this L-selectin
as they age in the circulation.10
Glucocorticoids also induce the release of PMN from the bone
marrow,11-13 but recent studies have established that, in
contrast to inflammatory stimuli, this release is associated with a
decrease in L-selectin expression on bovine, rat, and human
PMN.14-17 The L-selectin expression on leukocytes is
regulated by proteolytic cleavage from the cell surface3
that may occur in the presence18,19 or absence of cell
activation in vitro.20-22 Rapid shedding of L-selectin
occurs in vivo after stimuli such as endotoxin and C5a.9,23
Glucocorticoid treatment, on the other hand, results in a slow decrease
in the L-selectin expression on circulating PMN that only becomes
detectable at 8 hours after administration in intact animals or
human14,16 and cannot be demonstrated in
vitro.20,24,25
The present study is based on the hypothesis that glucocorticoids
decrease the L-selectin expression on circulating PMN by releasing PMN
that express low levels of L-selectin from the bone marrow. To test
this hypothesis, we studied the effects of dexamethasone on the
L-selectin expression on PMN in the bone marrow, on PMN newly released
from the bone marrow into the circulation by glucocorticoids, and on
PMN already in the circulation.
Animals
Study Design
Effect of dexamethasone on L-selectin expression on all PMN in the
circulation.
Rabbits received 2.0 mg/kg of dexamethasone in 3 mL of saline (n = 7)
or 3 mL of saline (n = 6) IV. Blood samples were obtained from the
central ear artery at 24 hours and just before dexamethasone or saline
injection and at 2, 4, 6, 12, 24, 48, 72, 96, 120, 144, and 168 hours
after dexamethasone or saline injection.
Effect of dexamethasone on L-selectin expression on PMN already in
the circulation.
To determine if dexamethasone had any effect on the L-selectin
expression on PMN already in the circulation, the PMN of donor rabbits
(n = 5) were labeled with the thymidine analogue,
5'-bromo-2'-deoxyuridine (BrdU; Sigma Chemical, St Louis,
MO), at 25 mg/kg/d for 7 days and the BrdU-labeled PMN harvested on day
8 were transferred to recipient rabbits as 15 mL of whole blood. This
method of labeling results in approximately 80% BrdU-labeling of PMN
and was previously described in more detail.26 The
recipients received 2.0 mg/kg of dexamethasone (Boucherville, Quebec,
Canada) in 3 mL of saline (n = 5) or 3 mL of saline (n = 4) IV 1 hour after the blood transfusion. This time point was selected
to administer dexamethasone because previous studies from our
laboratory using a similar method showed that labeled PMN infused into
rabbits reached stable counts in the circulation 1 hour after a blood
transfusion.26 Blood samples were obtained from the central
ear artery just before dexamethasone or saline injection and at 2, 4, 6, 12, and 24 hours after dexamethasone or saline injection. We have
selected these time points because the effects of dexamethasone on
demargination of PMN were shown to reach a peak at 6 hours after
treatment and to disappear 12 hours after treatment in Nakagawa et
al.27
Effect of dexamethasone on L-selectin expression on PMN released
from the bone marrow.
The PMN in the bone marrow of 12 rabbits were pulse labeled with IV
administration of 100 mg/kg of BrdU. The BrdU was infused at a
concentration of 10 mg/mL in normal saline over 15 minutes. Forty-eight
hours later, either 2.0 mg/kg of dexamethasone in 3 mL of saline (n = 6) or 3 mL of saline (n = 6) was administered intravenously. This time
point was selected because previous studies have shown a rapid release
of BrdU-labeled PMN from the bone marrow27,28 48 hours
after labeling. Blood samples were obtained from the central ear artery
just before dexamethasone or saline injection and at 12 and 24 hours
after dexamethasone or saline injection.
Effect of dexamethasone on L-selectin expression on PMN in the bone
marrow.
A 0.2-mL sample of bone marrow was aspirated from the left iliac bone
just before the intravenous injection of dexamethasone (2.0 mg/kg, n = 4) and from the right iliac bone 6 hours after dexamethasone injection
using an 18-G spinal needle. This time point was selected because
L-selectin on circulating PMN was still normal with little release of
BrdU-labeled PMN from the marrow by dexamethasone.27 Smears
were made immediately and allowed to air dry.
Flow Cytometry
Determination of BrdU-Labeled PMN in Circulation Sample preparation. One milliliter of blood collected in tubes containing acid-citrate-dextrose (ACD) was used to obtain leukocyte-rich plasma. Erythrocytes in the ACD blood sample were allowed to sediment for 25 to 30 minutes after the addition of an equal volume of 4% dextran (average molecular weight, 162,000; Sigma) in PMN buffer (138 mmol/L NaCl, 27 mmol/L KCl, 8.1 mmol/L Na2HPO47H20, 1.5 mmol/L KH2PO4, and 5.5 mmol/L glucose, pH 7.4). The resulting leukocyte-rich plasma was cytospun onto 3-aminopropryl-tri-ethoxysilane-coated slides by cytocentrifugation at 180g with a Cytospin 2 (Shandon Lab Products, Chestire, UK) for 4 minutes. The cytospin specimens were air-dried and stained using the alkaline phosphatase and anti-alkaline phosphatase (APAAP) method30 to determine the fraction of the BrdU-labeled PMN in each specimen. Immunoenzymatic staining of PMN for BrdU. Cytospin prepared from peripheral blood were stained for the presence of nuclear BrdU by the APAAP technique as previously descried.26 Briefly, the slides were fixed in methanol for 10 minutes and then digested at 37°C for 15 minutes in a 0.04% pepsin solution acidified to pH 2.5. Incubating slides in 2 N HCl at 37°C for 60 minutes denatured DNA in the samples. The 2 N HCl was neutralized by washing the slides three times with 0.1 mol/L borate buffer, pH 8.5, each for 10 minutes. The slides were incubated with 5% rabbit serum for 15 minutes before application of 2 µg/mL mouse MoAb against BrdU (DAKO Laboratories, Copenhagen, Denmark) for 60 minutes in a humidity chamber at room temperature. Nonimmune mouse IgG (5 µg/mL) and omission of the primary antibody were used as negative controls. A 1:20 dilution of rabbit antimouse IgG (DAKO) was applied for 30 minutes, followed by the antimouse IgG alkaline phosphatase-conjugated complex (DAKO) in a 1:50 dilution for 30 minutes. All antibodies were prepared in 50 mmol/L Tris hydrochloride and 150 mmol/L NaCl, pH 7.6 (TBS), with 1% bovine serum albumin, and slides were washed in 0.1% Tween 20 (Fisher Scientific, Fair Lawn, NJ) in TBS twice for 10 minutes between each antibody application. The alkaline phosphatase was developed for 20 minutes in 50 mL TBS at pH 8.7 after the addition of 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 naphtol AS-BI phosphate (Sigma) dissolved in 0.3 mL N,N-dimethylformamide. Endogenous alkaline phosphatase was blocked by the addition of 17.5 mg levamisole (Sigma) to the color reaction. The preparations were counterstained with Mayer's hematoxylin for 5 seconds, dehydrated through graded alcohol from 70% to 100% and xylene, and then mounted and evaluated on a Zeiss Universal Research light microscope (Model 2R; Zeiss, Oberkochen, Germany) at 400× magnification. Number of BrdU-labeled PMN in circulation. The total white blood cell (WBC) counts were determined on a model SS80 Coulter Counter (Coulter Electronics). Differential counts of PMN were obtained by counting 100 leukocytes in a randomly selected field of view on Wright's-stained blood smears. Using these values, the number of BrdU-labeled PMN was calculated. The number of BrdU-labeled PMN in the circulation of each recipient was expressed as a percentage of the total number of labeled PMN originally infused and corrected for the calculated blood volume31 of the recipient in the following manner: %PMNBrdUcirc = (PMNcirc × BV × fractionPMNBrdUrecipient) × 100/PMNBrdUinfused (1), where %PMNBrdUcirc represents the number of BrdU-labeled PMN in the circulation as a fraction of the total number of BrdU-labeled PMN infused; PMNcirc, the calculated number of PMN (per milliliter) in the circulation (WBC count × fraction of leukocyte that are PMN); BV, calculated blood volume (in milliliters); fractionPMNBrdUrecipient, the fraction of BrdU-labeled PMN on a cytospin of peripheral blood in the recipient; and PMNBrdUinfused, the number (per milliliter) of BrdU-labeled PMN infused (PMN count/mL × mL of blood infused × fractionPMNBrdU). Determination of L-Selectin Expression on BrdU-Labeled PMN in Circulation Double immunoenzymatic staining of PMN for L-selectin and BrdU. Cells on cytospins were stained for the presence of both cell surface L-selectin (red) and nuclear BrdU (blue) using the APAAP and a modification of a previously described method.9 Briefly, surface L-selectin was stained for in the first part of the double staining procedure followed by nuclear BrdU staining. Cells were fixed in acetone for 10 minutes and incubated with 5% rabbit serum for 15 minutes before application of 5 µg/mL of DREG-200 for 60 minutes in a humidity chamber at room temperature. Rabbit antimouse IgG, followed by the antimouse IgG alkaline phosphatase-conjugated complex, was applied as described earlier. Slides were washed in TBS twice for 10 minutes between each antibody application. The alkaline phosphatase was developed and then fixed with 1% paraformaldehyde for 20 minutes. After a 10-minute wash, slides were incubated in methanol for 10 minutes at room temperature and then at 37°C for 15 minutes in a 0.08% pepsin solution acidified to pH 2.5. Denaturing in HCl and neutralizing of HCl was performed as described earlier. These steps were followed by the second APAAP procedure, in which mouse MoAb against BrdU was used as the primary antibody and 0.1% Tween 20 in TBS, pH 7.6, was used to wash slides. The alkaline phosphatase was developed with a commercially available kit, HistoMark Blue (Kirkegaard and Perry, Gaitherburg, MD), for 10 minutes in the dark. Slides were washed with tap water, mounted in an aqueous medium (Gelvatol), and analyzed on a light microscope. Evaluation of L-selectin expression on BrdU-labeled PMN. BrdU-labeled PMN was divided into three groups according to the intensity of surface staining of L-selectin using an arbitrarily designated grading system. Strong represents cells with more than 75% of their surface stained deep red, weak represents cells with positive and/or less than 75% of their surface stained deep red, and negative represents cells with no (background) red stain. The slides were coded and examined without knowledge of the group or the sampled time. Fields were selected in a systematic randomized fashion and 100 cells were evaluated per specimen. All cells of interest in a selected field were evaluated, except if the cell was broken or overlapping with other cells. This grading system was evaluated for both interobserver and intraobserver variability. Determination of L-Selectin Expression on PMN in Bone Marrow Immunoenzymatic staining of PMN for L-selectin. Bone marrow smears were fixed in acetone for 10 minutes before immunocytochemical staining using the APAAP technique and DREG-200 to label L-selectin as described earlier. The preparations were counterstained with Mayer's hematoxylin for 20 seconds, dehydrated through graded alcohol from 70% to 100% and xylene, mounted, and evaluated on a light microscope. One hundred cells in randomly selected fields were evaluated and graded according to the intensity of staining for L-selectin as strong, weak, or negative. Just segmented and band PMN were evaluated. Statistical Analysis All values are expressed as means ± SEM. Temporal changes in mean fluorescence intensity and BrdU-labeled PMN counts in the circulation were analyzed using an analysis of variance for repeated measurements. The interobserver and intraobserver variation of the grading the intensity of staining for L-selectin were evaluated by calculating the Pearson coefficient of mean-square contingency (R2) for each grade and expressing R2 as a fraction of the maximum possible value, R2max, which represent the value of the Pearson 2 coefficient if the there is 100% agreement in
the grading score between or within observers; thus, R2max = ±0.82.32 Results in immunocytochemical
staining were analyzed using paired or two-sample t-test, and
Bonferroni corrections were made for multiple comparisons. Statistical
significance was defined as a P value of less than .05.
Effect of Dexamethasone on L-Selectin Expression on all PMN in the Circulation Dexamethasone decreased the mean fluorescence intensity of L-selectin on circulating PMN. The maximum effect was seen at 12 hours after dexamethasone treatment and L-selectin levels were back to pretreatment levels at 48 hours (Fig 1). Figure 2 shows a typical flow cytometry result of L-selectin measurement from one of the dexamethasone-treated rabbits. The curve was shifted to the left by dexamethasone at 12 hours after treatment. Although the mean fluorescence intensity was still low at 24 hours after treatment, there was a distinct double peak in the curve at 24 hours. A new population on PMN with normal baseline levels of L-selectin (right) appears, with still a significant population expressing low levels of L-selectin (left). CD18 was also measured as a marker of cell activation, but it did not change with dexamethasone treatment (data not shown).
Effect of Dexamethasone on L-Selectin Expression on PMN Already in the Circulation Figure 3 shows the clearance of BrdU-labeled PMN from the circulation of recipients. After transfusion of the BrdU-labeled PMN, 1 hour was allowed to achieve a stable count of BrdU-labeled PMN in the circulation.26 The fractions of the transfused BrdU-labeled PMN at baseline (1 hour after blood transfusion) were similar in both the dexamethasone-treated rabbits (40% ± 2.8%) and control rabbits (38% ± 9.1%). In control rabbits, BrdU-labeled PMN present in the circulation gradually decreased and almost disappeared by 24 hours after treatment. BrdU-labeled PMN increased after dexamethasone treatment (P < .05 at 2 to 6 hours compared with control), then decreased and almost disappeared by 24 hours after treatment. Figure 4 shows the PMN stained for both L-selectin (red) and BrdU (blue). Interobserver and intraobserver variation in the immunocytochemical evaluation of L-selectin expression was small, with the R2/R2max values greater than 0.75.7 The cells graded as either strong or negative gave the most reproducible results and were used to compare differences. The influence of the double labeling procedure on the presence of surface L-selectin and nuclear BrdU expression was evaluated by comparing the number of positive PMN for each antigen with paired slides stained for a single antigen using APAAP method as described above in 10 randomly selected slides. No significant difference was found (data not shown).
Effect of Dexamethasone on L-Selectin Expression on Younger PMN
Recently Released From the Bone Marrow
Effect of Dexamethasone on L-Selectin Expression on PMN in the
Bone Marrow
This study confirms the previous reports showing that glucocorticoids
decrease the L-selectin expression on circulating PMN. It extends these
observations by demonstrating that this decrease was due to a decrease
in L-selectin expression on PMN in the bone marrow and not in the
circulation. This glucocorticoid-induced decrease in L-selectin
expression on PMN in the maturation pool of the bone marrow suggests
that modulation of L-selectin expression on PMN in the bone marrow may
be an important mechanism of glucocorticoid-induced reduction in
neutrophilic inflammation.
The authors thank Beth Whalen, Corinne Rocchini, Daniela Zamfir, and
Jennifer Hards for technical supports; Stuart Green for photography;
and Yulia D'yachkova for statistical analysis.
Submitted July 30, 1998; accepted December 14, 1998.
Supported by Grant No. 4219 from the Medical Research Council of Canada.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Stephan F. van Eeden, MD, PhD, Pulmonary
Research Laboratory, University of British Columbia, St Paul's
Hospital, 1081 Burrard St, Vancouver, BC, Canada V6Z1Y6; e-mail:
svaneeden{at}prl.pulmonary.ubc.ca.
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ABSTRACT
INTRODUCTION
P < .05 compared with baseline;
P < .01 compared with control; Dex, dexamethasone.
B activity.
gene and protein synthesis that would inhibit
the translocation of NF-
