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Release of Polymorphonuclear Leukocytes From the Bone Marrow
by Interleukin-8
By Takeshi Terashima, Dean English, James C. Hogg, and Stephan F. van Eeden
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 mg/kg). IL-8 caused a rapid, transient granulocytopenia (5.9 6 0.4 at baseline v 0.2 6 0.06 3 10/9L at 5 minutes,
P F .05) followed by granulocytosis (8.4 6 0.1 at 30 minutes,
P F .05) associated with an increased number (0.3 6 0.1 at
baseline v 1.2 6 0.6 3 109/L at 30 minutes, P F .05) and
percentage of band cells (P F .05), as well as a rapid increase
in the number of BrdU-labeled PMN (PMNBrdU) in the circulation (0.09 6 0.05 at baseline to 1.5 6 0.6 3 109/L at 60
minutes, P F .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 F .05) and
the fraction and number of PMNBrdU in the sinusoids decreased with IL-8 treatment (P F .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.
r 1998 by The American Society of Hematology.
T
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.
HE 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-1a administration, the
increase of circulating IL-8 levels coincides with the resolution
From the Pulmonary Research Laboratory, University of British
Columbia, St. Paul’s Hospital, Vancouver, Canada.
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: [email protected].
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9203-0010$3.00/0
1062
MATERIALS AND METHODS
Experimental Animals
Adult female New Zealand White rabbits (n 5 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, 58-bromo-28-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 5 4) or
rabbit recombinant IL-8 (a kind gift from Genentech Inc, San Francisco,
CA) (n 5 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.
Blood, Vol 92, No 3 (August 1), 1998: pp 1062-1069
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BONE MARROW RELEASE OF NEUTROPHILS BY IL-8
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 4003 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
1063
sample was drawn from the central ear artery, and either saline (n 5 4)
or rabbit recombinant IL-8 (n 5 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:
DN(Dt) 5 Ntj 2 Nti exp 2 (kDt)
where: DN 5 number of labeled cells released from the marrow in the
time interval Dt; ti, tj 5 the initial and successive time intervals; Dt 5
tj 2 ti; and k 5 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 5 6) or saline (n 5 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.
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1064
TERASHIMA ET AL
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 8003 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 5 BW 3 K 3 0.045 3 Volume fraction of venous sinusoid of
marrow tissue where: BW 5 body weight and K 5 correction factor to
change weight into volume.
These values were used to calculate the total number of PMNBrdU in
the venous sinusoids:
Total number of PMNBrdU in sinusoids 5
VS 3 Volume fraction of PMNBrdU in venous sinusoid/143 fL
where 143 fL is the volume of rabbit PMN fixed in paraformaldehyde.25
Statistical Analysis
All values are expressed as mean 6 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.
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 6 0.4 to 0.2 6 0.08 3 109/L at 3 minutes and 0.2 6 0.06 3
109/L at 5 minutes (P , .05 v baseline). Circulating PMN
numbers returned to baseline levels by 15 minutes, exceeded
Fig 1. The effect of IV IL-8 (2.5 mg/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 F .05 v baseline). Circulating
PMN numbers returned to pretreatment levels by 15 minutes, exceeded these levels by 30 minutes (*P F .05 v baseline), remained
elevated up to 90 minutes (*P F .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 F .05 v baseline). Values are
mean 6 SEM (n 5 4).
these levels by 30 minutes (8.4 6 0.1 3 109/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 6 0.1 3
109/L at baseline to 0.8 6 0.2 3 109/L at 15 minutes and peaked
at 1.2 6 0.6 3 109/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).
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
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BONE MARROW RELEASE OF NEUTROPHILS BY IL-8
1065
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.
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
Fig 2. The release of PMNBrdU into the circulation after IV IL-8 (2.5
mg/kg) (n 5 4) or saline (n 5 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 F .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 6
SEM.
baseline at 180 minutes. The control group showed no change
during the 180-minute period.
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 6 0.06 v 1.0 6 0.2 3 109/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% 6 1.9% v 100.3% 6 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 6 0.02 3 109/L v 0.3 6 0.01 3
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% 6 64.2% to 44.7% 6 15.2% at 3 minutes and
17.4% 6 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 6 0.002 v 0.27 6 0.1 3 109/L, P , .05). Figure
3C shows that the arterio-venous ratio of PMNBrdU across the
lung decreased from a baseline value of 100.8% 6 37.1% to
28.9% 6 8.4% at 3 minutes and 19.2% 6 2.4% at 5 minutes.
Fig 3. The arterio-venous difference in PMN (A), band cell (B), and
PMNBrdU counts (C) after IV IL-8 (2.5 mg/kg) (n 5 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 F .05 v baseline. Values are mean 6 SEM.
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1066
TERASHIMA ET AL
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.
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 6 2.5 hours in
the IL-8 group and 98.1 6 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 6 1.4 hours and through
the entire pool was 125.5 6 1.9 hours in the control group. The
difference between these two values (G1-G3, 64.8 6 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
Fig 4. The appearance and disappearance of PMNBrdU in the
circulation after IV IL-8 (2.5 mg/kg) (n 5 5) or saline (n 5 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 6 SEM.
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 5 not
significant). The G3 cells (representing the transit time through
the postmitotic pool of the BM) peaked at 36 hours (Fig 4B) and
IL-8 did not change the differential leukocyte counts in the
postmitotic pool in the BM (metamyelocyte; 31.7% 6 4.2% v
27.5% 6 1.8%, band cells; 37.3% 6 2.6% v 43.2% 6 1.8%, and
segmented PMN; 31% 6 1.7% v 28.8% 6 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 BrdUlabeled 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).
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 3 106 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 6 19.4 3 106 compared with 13.2. 6 0.9 3 106 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 3 106, which is
Table 1. PMN Transit Time Through the BM
Group
No.
All PMN (h)
G3 (h)
G1 (h)
G1-G3 (h)
Control
IL-8
4
5
98.1 6 1.2
97.2 6 2.5
60.7 6 1.4
60.3 6 2.6
125.5 6 1.9
122.7 6 1.3
64.8 6 2.9
62.4 6 2.7
Values are mean 6 SEM. All PMN represents the total transit time
through the BM. G1 represents the transit time through the entire
pool. G3 represents the transit time through the postmitotic pool in
the BM. G1-G3 represents the transit time through the mitotic pool in
the BM (see text for explanation).
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BONE MARROW RELEASE OF NEUTROPHILS BY IL-8
Fig 5. The percentage of PMNBrdU in the circulation, sinusoids, and
hematopoietic tissue in the BM 60 minutes after IL-8 (n 5 6) or saline
(n 5 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 F .05) and a decrease in the PMNBrdU venous
sinusoids (P F .05). Compared with the hematopoietic tissue, the
percentage of PMNBrdU in the sinusoids was lower in the IL-8 group
(P F .05), but not in the controls. Values are mean 6 SEM. *P F .05
control versus IL-8 group, #P F .05 hematopoietic tissue versus
sinusoids in the IL-8 group.
<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.
Fig 6. The total number of PMN and the PMNBrdU in the venous
sinusoids 60 minutes after IL-8 (n 5 6) or saline (n 5 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 F .05). Values are mean 6 SEM. *P F .05 control versus IL-8
groups.
1067
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
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1068
TERASHIMA ET AL
Table 2. Number of PMN and BRDU-Labeled PMN in the Circulation, BM Venous Sinusoids and Hematopoietic Tissue
Circulation
Venous Sinusoids
Hematopoietic Tissue
Group
PMN
PMNBrdU
PMN
PMNBrdU
PMN
PMNBrdU
Control
IL-8
631 6 99
1,041 6 158*
13 6 0.9
80 6 19*
226 6 24
132 6 10*
94 6 7
36 6 10*
9,079 6 427
9,447 6 345
3,250 6 365
3,785 6 245
Values (3106/kg) represents the total number of either PMN or PMNBrdU and are expressed as the mean 6 SEM. These calculations assume the
BM is 4.5% of body weight.24
*P , .05 between control (n 5 3) and IL-8 (n 5 6) groups.
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-1b, TNF-a, 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-a 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 (6 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.
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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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1998 92: 1062-1069
Release of Polymorphonuclear Leukocytes From the Bone Marrow by
Interleukin-8
Takeshi Terashima, Dean English, James C. Hogg and Stephan F. vanEeden
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