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 Previous Article | Table of Contents
Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 1062-1069
Release of Polymorphonuclear Leukocytes From the Bone Marrow by
Interleukin-8
By
Takeshi Terashima,
Dean English,
James C. Hogg, and
Stephan F. vanEeden
From the Pulmonary Research Laboratory, University of British
Columbia, St. Paul's Hospital, Vancouver, Canada.
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ABSTRACT |
Several studies have shown that interleukin-8 (IL-8) causes a rapid
granulocytosis with the release of polymorphonuclear leukocytes (PMN)
from the bone marrow (BM) partially responsible for the granulocytosis.
This study was designed to quantitate the release of PMN from the BM by
IL-8 and measure the transit time of PMN through the marrow after IL-8
administration. The thymidine analogue, 5'-bromo-2'-deoxyuridine
(BrdU), was used to label dividing PMN in the marrow and follow their
release into the circulation after intravenous IL-8. This allowed us to
calculate the transit time of PMN through the mitotic and postmitotic
pools of BM. BrdU was infused intravenously into rabbits 24 hours
before IL-8 (2.5 µg/kg). IL-8 caused a rapid, transient
granulocytopenia (5.9 ± 0.4 at baseline v 0.2 ± 0.06 × 10/9L at 5 minutes, P < .05) followed
by granulocytosis (8.4 ± 0.1 at 30 minutes, P < .05)
associated with an increased number (0.3 ± 0.1 at baseline v
1.2 ± 0.6 × 109/L at 30 minutes, P < .05) and
percentage of band cells (P < .05), as well as a rapid
increase in the number of BrdU-labeled PMN (PMNBrdU) in the
circulation (0.09 ± 0.05 at baseline to 1.5 ± 0.6 × 109/L at 60 minutes, P < .05). The transit time
of PMN through both the mitotic and postmitotic pools of BM was not
affected by IL-8. To determine the marrow compartment from which the
PMN were mobilized by IL-8, we quantitated PMN movement from the
hematopoietic and sinusoidal compartments into the circulation. The
fraction of PMNBrdU in both compartments was higher than in
the circulating blood (P < .05) and the fraction and number
of PMNBrdU in the sinusoids decreased with IL-8 treatment
(P < .05). We conclude that the pool of PMN residing in the
BM venous sinusoids are rapidly released into the circulation after
administration of IL-8.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE REGULATION of circulating
neutrophil levels is an important feature of both the local and the
systemic response to inflammatory stimuli.1,2 Circulating
neutrophils can increase without increasing the total number of
neutrophils in the vascular system by mobilizing the cells marginated
along vessel walls.3 With increasing stress, neutrophils
are mobilized from the bone marrow (BM), which increases their number
in both the circulating and the marginated pools.4,5 During
their maturation process in the BM, the polymorphonuclear leukocytes
(PMN) increase their mobility, deformability, and chemotactic
responsiveness.6,7 Because immature PMN are larger and less
deformable than their mature counterparts,7,8 they
preferentially sequester in lung microvessels where they may play a
pivotal role in mediating inappropriate lung injury associated with
infection and sepsis.4,9
Interleukin-8 (IL-8) is produced by a wide variety of cell
types and functions as a potent and selective neutrophil
chemoattractant and activator inducing directional migration, release
of storage enzymes, and production of toxic metabolites in
PMN.10,11 Recent studies have shown that during both
experimentally induced inflammation and human clinical disease, IL-8
can be detected in plasma at concentrations of 1 to 8 ng/mL.12-14 In endotoxemia and after IL-1 administration, the increase of circulating IL-8 levels coincides with
the resolution of early granulocytopenia.12 Several
investigators have shown that intravenous (IV) administration of IL-8
induces a rapid granulocytopenia followed by
granulocytosis.15-18 Demargination of PMN from within the
vascular space may contribute to this granulocytosis, but these studies
suggest that the release of PMN from the BM contributes to the
transient granulocytosis induced by IL-8.15,17
The present study was designed to determine the effect of IV
IL-8 administration on the BM by calculating the transit time of PMN
through the mitotic and postmitotic marrow pools and by measuring the
release of new PMN into the circulation.5 Changes in the BM
hematopoietic tissue and venous sinusoids after IL-8 stimulation were
also assessed using morphometric analysis.
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MATERIALS AND METHODS |
Experimental Animals
Adult female New Zealand White rabbits (n = 26; weight, 1.8 to 2.4 kg)
were used in this study. All of the experiments were approved by the
Animal Experimentation Committee of the University of British Columbia.
Experimental Protocols
Release of PMN from the BM.
Following a protocol that has previously been described in detail,
5 -bromo-2-deoxyuridine (BrdU) (Sigma Chemical Co, St Louis, MO) was infused through the marginal ear vein at a concentration of 10 mg/mL in normal sterile saline solution over a period of 5 minutes to deliver a pulse dose of 100 mg/kg.5 The animals were then allowed to recover for 24 hours before they were anesthetized with ketamine hydrochloride (80 to 100 mg/kg intramuscular
[IM]) and xylazine (10 to 15 mg/kg IM). Polyethylene
tubing catheters of appropriate size were placed in the marginal ear
vein, the right carotid artery, and the right jugular vein. Each
catheter was connected through a three-way stopcock to a syringe
containing heparinized saline. After a baseline sample was drawn,
saline (n = 4) or rabbit recombinant IL-8 (a kind gift from Genentech Inc, San Francisco, CA) (n = 4) was administered IV as a rapid bolus
(2.5 µg/kg) into the marginal ear vein. Blood samples were obtained
simultaneously from the carotid artery and the jugular vein 3, 5, 15, 30, 60, 90, and 180 minutes after the IV IL-8 administration. After
each blood sample, the catheter was flushed with an equal volume of
heparinized saline.
Leukocyte Counts
Blood samples were analyzed as follows: 1 mL was collected in standard
Vacutainer tubes containing potassium ethylene diamine tetra-acetic
acid (Becton Dickinson, Rutherford, NJ) for blood cell counts, which
were determined on a model SS80 Coulter Counter (Coulter Electronics,
Hialeah, FL) and differential white blood cell (WBC) and band cell
counts were done on Wright's stained blood smears; 1 mL samples were
collected in acid-citrate-dextrose (ACD) for the preparation of
leukocyte-rich plasma (LRP). Erythrocytes in the ACD blood samples 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
Na2HPO4 7H2O, 1.5 mmol/L
KH2PO4, and 5.5 mmol/L glucose, pH 7.4). The
resulting LRP was cytospun at 1,600 rpm for 4 minutes to obtain a
monolayer of cells on slides coated with
3-aminopropryl-tri-ethoxysilane, which was used to determine the number
of BrdU-labeled PMN (PMNBrdU) per 100 PMN counted in random
fields of view.
Immunocytochemical Detection of PMNBrdU
A mouse monoclonal antibody against BrdU and the alkaline phosphatase
antialkaline phosphatase (APAAP) method was used to stain for the
presence of BrdU incorporated into the DNA of PMN in cytospins made of
LRP as previously described.4,5 All slides were evaluated
on a Zeiss Universal Research light microscope (Model IIR;
Oberkochen, Germany) at 400× magnification.
Evaluation of PMNBrdU and Calculation of BM Transit
Times
PMN with any nuclear stain were counted as BrdU-labeled.
PMNBrdU were divided into three groups according to the
intensity of nuclear staining using an arbitrarily designated grading
system: weakly positive (staining of less than 5% of the nucleus: G1); moderate positive (staining of 5% to 80% of the nucleus: G2); and
highly positive (staining of more than 80% of the nucleus: G3). This
grading system was designed to evaluate the transit time of the myeloid
cells that were in their last division in the mitotic pool when exposed
to BrdU (highly positive or G3), those that were in the middle
(moderately positive or G2), and those that were in their first
division (weakly positive or G1).
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. This method of evaluating PMNBrdU has a small
intraobserver and interobserver variability and has previously been
described in more detail.5
This method to calculate the transit time of PMN through the different
pools in the marrow was derived from the work of Maloney and
Patt19 who used tritiated thymidine to label dividing cells in the BM of dogs. It is based on the assumption that most of the
mitotic cells in the BM incorporate BrdU into nuclear DNA over a short
period and that the recirculation and reuse of BrdU is
minimal.20 The assumption that the highly labeled PMN are myelocytes that incorporate BrdU during their last division and did not
dilute it by subsequent divisions allowed us to calculate the transit
time of this single generation of cells through the postmitotic or
maturation pool. Similarly, by assuming that the weakest stained PMN
represents myeloid cells where the BrdU was incorporated during their
early division stages and diluted with subsequent divisions allowed the
transit time of these cells through both the mitotic and postmitotic
pools of the BM to be calculated. All of these transit times were
corrected for the normal removal of PMN from the
circulation.21
Transit time of PMN through the BM.
The transit time of PMN through the mitotic and postmitotic pools of
the BM were measured using a technique that is fully described
elsewhere.5 Briefly, BrdU was infused at a pulse dose of
100 mg/kg. Twenty-four hours later, a baseline sample was drawn from
the central ear artery, and either saline (n = 4) or rabbit recombinant
IL-8 (n = 5) was administered IV as a rapid bolus (2.5 µg/kg) into
the marginal ear vein. Blood samples were obtained from the central ear
artery at intervals from 6 to 168 hours after IL-8 administration,
cytospins prepared from LRP, and used to calculate the transit times
through the BM, as previously described.5
The transit time of PMN from the BM to the circulation was corrected
for the disappearance (half-life, T1/2) of
PMNBrdU in the circulation. In previous studies, we have
reported that the T1/2 of PMNBrdU in rabbits is
270 minutes or 4.5 hours using a whole blood transfusion method.21 Therefore, this rate of exponential loss of
PMNBrdU from the circulation was used to calculate the
number of PMNBrdU released from the BM and the transit time
through the different pools in the marrow in the following manner:
where:
 N = number of labeled cells released from the marrow in the time
interval t; ti, tj = the initial and successive time intervals; t = tj  ti; and k = In2/T1/2.
These calculations were made for each 6-hour interval and a histogram
was drawn showing the distribution of PMNBrdU released from
the BM during each 6-hour interval. The mean transit times for all
PMNBrdU and the different populations of labeled PMN (G1,
G2, and G3) were calculated individually for each animal and were
compared among the groups.
Changes in BM compartments.
BrdU was infused at a pulse dose of 100 mg/kg 24 hours before IL-8 (n = 6) or saline (n = 3) administration as described above. The animals
were anesthetized with ketamine hydrochloride (80 to 100 mg/kg IM) and
xylazine (10 to 15 mg/kg IM), and polyethylene catheters (16G; Jelco
I.V. catheters, Tampa, FL) were placed in the abdominal aorta and
inferior vena cava. Both catheters were directed distally and clamped
proximally to perfuse the lower limbs selectively. The animals were
killed with an overdose of sodium pentobarbitone, after which the lower
limbs of the animals were perfused through the arterial line with Krebs
buffer (6.9 g/L NaCl, 0.35 g/L KCl, 0.29 g/L
MgSO4 7H2O, 0.16 g/L KH2PO4, 2.1 g/L NaHCO3, 2.0 g/L glucose, 0.28 g/L
CaCl2) from a reservoir with a 30 cm H2O
perfusion pressure and drained from the venous line. Perfusion was
continued until no more blood was visible in the solution draining from
the venous catheter. The perfusate was replaced by 10% formalin to
perfusion fix the BM (100 mL). Both femurs were removed and emerged in
B5 fixative (60 g/L HgCl2, 12.5 g/L CH3COONa,
4% formalin) overnight. BM was harvested from the femurs by carefully
removing the cortical bone of the femur and the BM tissue was embedded
in glycolmethacrylate for histological analysis (see below).
BM samples were processed for histology by embedding them in
glycolmethacrylate (GMA) using a modification of a method previously described for immunohistochemical analysis.22 The BM tissue was cut in 1- to 2-mm thick slices, washed well with phosphate-buffered saline (PBS), then dehydrated through graded alcohol from 30% to
100%. The specimens were then infiltrated overnight with GMA resin
monomer (JB4; PolyScience, Ltd, Warrington, PA) containing 0.9% benzoyl peroxidase-solution A. The infiltrated tissue was placed
in embedding molds and GMA chemically polymerized by a mixture of 200 mL of JB4 solution B to 5 mL of solution A (JB4; PolyScience, Ltd) for
1 hour. Polymerized resin blocks were brought to room temperature and
2-mm sections cut with a Sorval JB4 microtome fitted with a glass knife
(made with an LKB Knife maker Type 7801, Stockholm, Sweden). Sections
were floated on a room temperature water bath, transferred to glass
slides, air dried overnight at 37°C, and stained with hematoxylin
and eosin (H&E). These sections were used for the evaluation of the
fraction of metamyelocytes, band cells, and segmented PMN in the BM.
Immunohistochemical Detection of PMNBrdU Cells
Immunohistochemical staining of plastic embedded sections of BM for the
presence of BrdU was performed by the APAAP method. DNA in tissue was
denatured in 1 N HCl at 60°C for 20 minutes, followed by washing
with water and air drying. Slides were then immersed in xylene for 10 minutes, dried, and washed with water. BM tissue was then digested with
0.25 mg/mL protease type XIV at 37°C for 90 minutes, which was
followed by neutralization in two washes of Tris buffered saline
(TBS). After nonspecific binding sites were blocked by
incubation with 5% normal rabbit serum for 15 minutes, the specimens
were incubated with 10 µg/mL mouse anti-BrdU antibody (Dako,
Glostrup, Denmark) prepared with 1% bovine serum albumin
in TBS at 37°C in a humidified chamber for 1 hour. Nonspecific mouse IgG1 at 10 µg/mL was used as a negative control. Incubation in
a 1:20 dilution of rabbit antimouse IgG (Dako) for 30 minutes was
followed by 30 minutes in a 1:50 dilution of a mouse monoclonal APAAP
complex (Dako). Slides were washed in 0.1% Tween 20 in TBS for 10 minutes after each antibody application. The alkaline phosphatase was
developed as described above. The preparations were counterstained with
Toluidine Blue O, dried, and mounted in an aqueous medium.
Evaluation of PMNBrdU in the BM
PMNBrdU in the BM were counted at 800× magnification
using a Nikon Microphot-fx light microscope (Nikon, Tokyo, Japan) with
Bioview, an image processing system (Infrascan Inc, Richmond, British
Columbia, Canada). A minimum of 20 randomly selected fields were
evaluated per slide and a minimum of 100 sinusoids per animal. In these fields of view, the total number and the percentage of
PMNBrdU were counted both in the sinusoids and
hematopoietic tissue. The volume fraction of venous sinusoids in the
marrow was determined using a point counting technique23
where random fields of view generated by a computer program and a
200-point counting grid superimposed on the microscope image allowed a
calculation of total points falling on the different BM tissue.
Assuming that the BM represents 4.5% of total body
weight,24 the volume of venous sinusoids (Vs)
was calculated as follows:
VS = BW × K × 0.045 × Volume fraction of
venous sinusoid of marrow tissue where: BW = body weight and K = correction factor to change weight into volume.
These values were used to calculate the total number of
PMNBrdU in the venous sinusoids:
where
143 fL is the volume of rabbit PMN fixed in
paraformaldehyde.25
Statistical Analysis
All values are expressed as mean ± standard error of mean (SEM).
Analysis of variance (ANOVA) was used for continuous data and to
compare IL-8 and control groups. Bonferonni corrections were done for
multiple comparisons. Student's paired t-test was used to
compare the data between arterial and venous samples and P < .05 was accepted as statistically significant.
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RESULTS |
Release of PMN From the BM
Leukocyte counts.
A bolus of IL-8 induced a rapid decline in circulating PMN numbers
(Fig 1A) from the baseline value of 5.9 ± 0.4 to 0.2 ± 0.08 × 109/L at 3 minutes and
0.2 ± 0.06 × 109/L at 5 minutes (P < .05 v baseline). Circulating PMN numbers returned to baseline
levels by 15 minutes, exceeded these levels by 30 minutes (8.4 ± 0.1 × 109/L, P < .05 v baseline),
remained elevated up to 90 minutes (P < .05), and returned to
baseline by 180 minutes. The increase in circulating PMN counts was
accompanied by an increase in band cells (Fig 1B). The number of band
cells increased from 0.3 ± 0.1 × 109/L at
baseline to 0.8 ± 0.2 × 109/L at 15 minutes and
peaked at 1.2 ± 0.6 × 109/L at 30 minutes
(P < .05). The percentage of band cells change in a similar
pattern (P < .05). The band cell counts did not change in the
control group (data not shown).
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| Fig 1.
The effect of IV IL-8 (2.5 µg/kg) on the circulating
PMN and band cell counts. IL-8 induced a rapid decline in circulating PMN (A) that was significant at 3 to 5 minutes (*P < .05 v baseline). Circulating PMN numbers returned to pretreatment
levels by 15 minutes, exceeded these levels by 30 minutes (*P < .05 v baseline), remained elevated up to 90 minutes
(*P < .05), and returned to baseline by 180 minutes. The
increase in circulating PMN counts was accompanied by a similar
increase in band cells (B). The number of band cells increased at 15 minutes, peaked at 30 minutes, and returned to baseline at 180 minutes
after IL-8 (*P < .05 v baseline). Values are mean ± SEM (n = 4).
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Release of PMNBrdU into the circulation.
Figure 2A shows the percentage of
PMNBrdU in the circulation after IL-8 administration. At
time 0 (24 hours after BrdU labeling), the percentage of
PMNBrdU in the circulation was less than 2% in both
groups. A bolus of IV IL-8 induced a rapid increase in the percentage
of PMNBrdU in the circulation, which was observable at 3 minutes. This increase peaked at 5 minutes and remained higher than
baseline for up to 90 minutes (P < .05 v baseline).
Figure 2B shows the absolute number of PMNBrdU in the
circulation after IL-8 administration. This number increased and peaked
at 60 minutes, then gradually declined until the values were similar to
baseline at 180 minutes. The control group showed no change during the
180-minute period.
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| Fig 2.
The release of PMNBrdU into the circulation
after IV IL-8 (2.5 µg/kg) (n = 4) or saline (n = 4). BrdU was
infused 24 hours before the IL-8 was administered. At time 0, the
percentage of PMNBrdU in the circulation was less than 2%
(A). IL-8 induced a rapid increase in the percentage of
PMNBrdU in the circulation, which remained higher than the
controls for 90 minutes (*P < .05 v control). (B)
Shows the absolute number of PMNBrdU in the circulation.
After IV IL-8, the number of circulating PMNBrdU increased
and peaked at 60 minutes, then gradually declined to baseline levels at
180 minutes. The control group showed no change in either the
percentage or the number of PMNBrdU during the 180-minute
study period. Values are mean ± SEM.
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Sequestration of PMN in the lung after IL-8.
The number of PMN was lower in the arterial blood than in the venous
blood 5 minutes (0.2 ± 0.06 v 1.0 ± 0.2 × 109/L, P < .05) after IL-8.
Figure 3A shows that the arterio-venous ratio of PMN, expressed as a percentage (arterial values divided by
venous values times 100) was lower at 5 minutes than at baseline after
IL-8 (22.5% ± 1.9% v 100.3% ± 12.0%, P < .05). The number of band cells was also lower in the arterial blood
than in the venous blood at 5 minutes (0.1 ± 0.02 × 109/L v 0.3 ± 0.01 × 109/L,
P < .05). Figure 3B shows the arterio-venous ratio of the band cells across the lung with a decreased ratio from a baseline value
of 154.9% ± 64.2% to 44.7% ± 15.2% at 3 minutes and 17.4% ± 3.3% at 5 minutes (P < .05). The number of
PMNBrdU was lower in the arterial blood than in the venous
blood at 5 minutes (0.019 ± 0.002 v 0.27 ± 0.1 × 109/L, P < .05). Figure 3C shows that the
arterio-venous ratio of PMNBrdU across the lung decreased
from a baseline value of 100.8% ± 37.1% to 28.9% ± 8.4% at
3 minutes and 19.2% ± 2.4% at 5 minutes. These results show a
transient arterio-venous gradient of PMN, band cells, and
PMNBrdU across the lung with the granulocytopenia induced
by IL-8. These arterio-venous (A-V) gradients returned to
baseline values within 15 minutes and remained unchanged during the
granulocytosis.
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| Fig 3.
The arterio-venous difference in PMN (A), band cell (B),
and PMNBrdU counts (C) after IV IL-8 (2.5 µg/kg) (n = 4). Values are the ratio of arterio-venous counts and are expressed as
a percentage (see text for formula). These ratios were lower than
baseline values at 3 minutes for PMN and 3 and 5 minutes for band cell and PMNBrdU after IL-8 administration. *P < .05 v baseline. Values are mean ± SEM.
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Transit Time of PMNBrdU Through the BM
The circulating PMN or band cell counts did not change significantly
from baseline in either the IL-8 or control groups (data not shown).
Figure 4A shows the percentage of
PMNBrdU in the circulation for 7 days after IL-8
administration. A slow increase in the percentage of
PMNBrdU was seen between 0 and 12 hours followed by a rapid
increase to peak levels between 36 and 48 hours, then a slow decline
was seen over the next 5 days. This was not different from the control group (P = not significant). The G3 cells
(representing the transit time through the postmitotic pool of the BM)
peaked at 36 hours (Fig 4B) and G1 cells (represented the total transit
time ) peaked between 96 and 120 hours (Fig 4C). There was no
difference between the IL-8- and saline-treated animals.
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| Fig 4.
The appearance and disappearance of PMNBrdU
in the circulation after IV IL-8 (2.5 µg/kg) (n = 5) or saline (n
= 4). (A) Shows all the PMNBrdU. In both groups there was
a slow increase between 0 and 12 hours followed by a rapid increase to
peak levels between 36 and 48 hours and then a slow decline over the
following 5 days. G3 cells (representing the transit time through the
postmitotic pool) peaked at 36 hours (B) and G1 cells (representing the
transit time through both the mitotic and postmitotic pools) peaked
between 96 and 120 hours in both group (C). No differences were
apparent between groups. Values are mean ± SEM.
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Based on the assumptions outlined in Materials and Methods and fully
discussed elsewhere,16 the calculated mean transit time of
all PMNBrdU through the BM was 97.2 ± 2.5 hours in the
IL-8 group and 98.1 ± 1.2 hours in the control group.
Table 1 summarizes the data and shows that
the mean transit time through the postmitotic pool was 60.7 ± 1.4 hours and through the entire pool was 125.5 ± 1.9 hours in the
control group. The difference between these two values (G1-G3, 64.8 ± 2.9 hours) represents the transit time through the mitotic pool.
Therefore, IL-8 did not change the PMN transit time through either the
mitotic or the postmitotic pools of the BM.
Changes in BM Compartments
IL-8 did not change the differential leukocyte counts in the
postmitotic pool in the BM (metamyelocyte; 31.7% ± 4.2% v 27.5% ± 1.8%, band cells; 37.3% ± 2.6%
v 43.2% ± 1.8%, and segmented PMN; 31% ± 1.7%
v 28.8% ± 2.1%, control v IL-8 group). IL-8 also did not affect the percentage of BrdU-labeled segmented or band cells
in the hematopoietic tissue in the BM (data not shown). Figure 5 compares the percentage of
BrdU-labeled cells in the peripheral blood, marrow sinusoids, and
hematopoietic tissue. In the control group, there was no difference in
the percentage of PMNBrdU between the venous sinusoids and
the hematopoietic tissue (P < .5), but values in both of
these compartments were higher than in the circulating blood (P < .05). IL-8 increased the percentage of PMNBrdU in the
circulating blood (P < .05) and decreased the value in the
venous sinusoids (P < .05) with no change in the
hematopoietic compartment. This increased the gradient of
PMNBrdU between the hematopoietic tissue and venous
sinusoids (P < .05) in the IL-8 group.
Figure 6 shows that IL-8 also decreased the total number of PMN and the PMNBrdU in the marrow sinusoids
(P < .05).
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| Fig 5.
The percentage of PMNBrdU in the circulation,
sinusoids, and hematopoietic tissue in the BM 60 minutes after IL-8 (n
= 6) or saline (n = 3). The percentage of PMNBrdU was
higher in the hematopoietic tissue and venous sinusoids than in the
circulating blood in both the IL-8 and control groups. IL-8 caused an
increase in PMNBrdU in the circulating blood (P < .05) and a decrease in the PMNBrdU venous sinusoids
(P < .05). Compared with the hematopoietic tissue, the
percentage of PMNBrdU in the sinusoids was lower in the
IL-8 group (P < .05), but not in the controls.
Values are mean ± SEM. *P < .05 control versus IL-8 group,
#P < .05 hematopoietic tissue versus sinusoids in the IL-8
group.
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| Fig 6.
The total number of PMN and the PMNBrdU in
the venous sinusoids 60 minutes after IL-8 (n = 6) or saline (n = 3). Values are expressed as the number of PMN per sinusoid. IL-8
decreased total number of PMN and PMNBrdU in the sinusoids
compared with controls (P < .05). Values are mean ± SEM.
*P < .05 control versus IL-8 groups.
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The total number of PMN and PMNBrdU calculated in the
venous sinusoids was lower in the IL-8 group than in the control group
(Table 2, see Materials and Methods for
calculation). Assuming that the number of PMNBrdU in the
sinusoids in the control group represents a baseline value, it follows
that IL-8 released 57.5 × 106 PMNBrdU
from the venous sinusoids. Using a total blood volume of 56.4 mL/kg in
rabbits,24 the number of PMNBrdU in the
circulation 60 minutes after IL-8 administration was 80.8 ± 19.4 × 106 compared with 13.2. ± 0.9 × 106 in the control group. Assuming that 50% of the
PMNBrdU that are released into the general circulation
marginate along vessel walls,25 the calculated number of
PMNBrdU added to the intravascular pool by IL-8 treatment
was 135.2 × 106, which is  2 times the calculated
number released from the venous sinusoids.
| |
DISCUSSION |
The results of this study confirm that intravenous IL-8 causes a
transient neutropenia followed by a neutrophilia. The neutropenia was
associated with an arterio-venous difference of PMN across the lung,
which suggests sequestration of cells in the lung. During the
neutrophilia, there was no A-V difference of PMN across the lungs
consistent with release of PMN from marginated pool in the lung into
the circulation. The neutrophilia was associated with rapid release of both segmented and band cells from the BM. IL-8 released PMN from the BM without changing their transit time through either the mitotic or the postmitotic marrow pools. The quantitative histologic studies of the marrow showed that the majority of PMN that
are released from the marrow after IL-8 administration come from a pool
in the venous sinusoids of the BM.
Several investigators have reported that IV IL-8 induces a
granulocytopenia followed by a granulocytosis.15-18 Ley et
al18 suggested that this granulocytosis results from
demargination rather than BM release because there was no consistent
increase in circulating nonsegmented PMN (band cells) in rabbits
receiving human recombinant IL-8 (hrIL-8). Van Zee et al16
reported similar results in baboons receiving hrIL-8. Studies from
our26 and other laboratories27,28 have shown
that the lung is the major site of PMN margination in rabbits. However,
in the present study, we were unable to show an arterio-venous
difference consistent with the release from the pulmonary circulation
during the granulocytosis phase of the IL-8 response. This suggests
that the PMN leave the lung at the same rate as they enter it during
the granulocytosis phase.
Hechtman et al15 showed that IL-8 induced a granulocytosis
with an increase in band cells in rabbits, which was supported by
studies from Jagels and Hugli,17 suggesting a significant release of PMN from BM stores. The results of our study provide several
lines of evidence that IL-8 stimulates the BM to release PMN into the
circulation. There was a rapid increase in the number of PMN in the
circulation 30 minutes after IL-8 administration (Fig 1A). This
increase was accompanied by an increase in the number and percentage of
band cells, which provides definitive evidence for the BM release of
PMN (Fig 1B). There was also a rapid increase in the number and the
fraction of PMNBrdU in the circulation, which confirms that
these cells come from the BM (Fig 2A and B).
Interestingly, Laterveer et al29,30 have demonstrated a
rapid mobilization of hematopoietic progenitor cells into the
circulation of monkeys29 and mice30 after a
single dose of IV IL-8. They reported that the maximum circulating
level of progenitors was 30 minutes after the IL-8, which corresponds
to the peak release of granulocytes in our study. Our results show that
granulocyte release from the BM venous sinusoids accounts for
approximately 50% of the IL-8-induced granulocytosis. Furthermore,
IL-8 also did not change the transit time of PMN through the mitotic or postmitotic pools in the marrow (Fig 4). Taken together, these results
suggest the presence of a pool of hematopoietic progenitors in the BM
sinusoids available for immediate mobilization into the circulation
with IL-8.
The effect of IL-8 on the BM was transient in that circulating PMN,
band cell, and PMNBrdU counts returned to baseline levels
by 6 hours. Interestingly, there was no change in the calculated
transit time of PMN through either the mitotic or the postmitotic pools
in the BM (Fig 4A through C). However, IL-8 did decrease the number of
BrdU-labeled cells in the marrow sinusoids and increase the gradient in
BrdU-labeled cells from the hematopoietic tissue to the sinusoids.
These results suggest that IL-8 mobilizes PMN out of the BM venous
sinusoids, which could increase the traffic of PMN from the
hematopoietic tissue into the sinusoidal compartment.
In the control group, the fraction of PMNBrdU in the
sinusoids was similar to the fraction in the hematopoietic tissues and
higher than those in the circulation (Fig 5), suggesting the presence of a pool of PMN in the venous sinusoids that does not circulate. We
postulate that these cells remain attached to the sinusoidal endothelium after migration from the hematopoietic compartment and that
IL-8 reduces this attachment to release them into the circulation. We
calculate that 50% of the PMNBrdU released into the
circulation by IL-8 come from a pool located in the marrow sinusoids
and the rest are transferred from the hematopoietic tissue into the
sinusoids and then into the peripheral blood. The mechanism(s)
responsible for this preferential release of PMN from the sinusoids by
IL-8 is not known. We have previously shown that the surface expression
of the adhesion molecule, L-selectin, on PMN decreases when these cells
translocate from the BM to the circulation22 and speculate
that L-selectin is involved in keeping PMN attached to sinusoidal
endothelial. Circulating stimuli, such as IL-8, could influence this
adhesive interaction by shedding L-selectin and release PMN into the
circulation.
Factors capable of stimulating the BM to release PMN include
granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, and IL-3.31,32
G-CSF is known to shorten the PMN transit time through the mitotic and postmitotic pools in the marrow.33 Van Zee et
al16 have shown that IV IL-8 infusions were not associated
with the appearance of other cytokines such as IL-1 , TNF-, or
IL-6 in the plasma,34 suggesting that IL-8 has a direct
effect on the BM rather than an indirect effect via other inflammatory
mediators. Furthermore, the relatively late appearance of IL-8 in the
circulation during endotoxemia and sepsis12 suggests that
IL-1 and TNF- may induce the biosynthesis of IL-8, which contributes
to the leukocytosis seen during sepsis.
The short-lived neutropenia induced by IL-8 was accompanied by a
transient arterio-venous gradient of PMN, band cells, and PMNBrdU across the lung, suggesting sequestration in the
pulmonary microvessels. PMN activation decreases cell deformability,
which has been shown to be the principal factor responsible for their
sequestration in lung microvessels.35 The short half-life
(± 8 minutes) of IL-8 in the circulation suggests that this effect
should be brief and reversible.16
The neutrophilia that followed the IL-8-induced neutropenia was not
associated with an arterio-venous gradient of newly released PMN (band
cells and PMNBrdU) across the lung (Fig 3B and C). Lichtman
and Weed7 have shown that PMN harvested from the
postmitotic pool in the marrow are larger and less deformable than
their counterparts in the circulation. These physical characteristics
make immature PMN more prone to sequester in the lung microvessels
because of the size discrepancy between PMN and pulmonary
capillaries.25 Studies from our laboratory have shown that
PMN released from the marrow during pneumococcal pneumonia4
and endotoxemia9 preferentially sequester in lung
microvessels. The absence of an A-V difference during the neutrophilic
period in this study suggests that the cells released from the marrow
by IL-8 behave like fully mature circulating PMN.
In summary, the data presented here show that IV IL-8 stimulates the BM
to release PMN without affecting PMN transit time through either the
mitotic or the postmitotic marrow pools. The data also demonstrate the
presence of a pool of PMN in BM sinusoids that are rapidly mobilized
into the circulation after intravenous IL-8 administration. We
speculate that PMN are attached to the sinusoidal endothelium after
migration from the hematopoietic tissues and that this PMN-endothelial
interaction is reduced when PMN are mobilized from the marrow sinusoids
by IL-8 into the peripheral blood. These observations extend the
previously defined functions of IL-8 and provide insight into how this
molecule acts to release PMN from the marrow.
| |
FOOTNOTES |
Submitted December 11, 1997;
accepted March 25, 1998.
Supported by Grant No.MRC 4219 from the Medical Research Council of
Canada (Ottawa, Ontario) and by the B.C. Lung Association (Vancouver,
B.C.).
Address reprint requests to Stephan F. van Eeden, MD, UBC
Pulmonary Research Laboratory, St. Paul's Hospital, 1081 Burrard St,
Vancouver, B.C. V6Z 1Y6; e-mail: svaneeden{at}prl.pulmonary.ubc.ca.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Genentech Inc for supplying the recombinant rabbit IL-8, Jenny
Hards for helping with the immunohistochemistry, Lorri Verburght for
doing the statistical analysis, Stuart Greene for photography, and
Heather Hogg for reviewing the manuscript.
| |
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Abstract
Introduction
Abstract
