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Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 283-290
Complement Fragment-Induced Release of Neutrophils From Bone
Marrow and Sequestration Within Pulmonary Capillaries in Rabbits
By
Hiroshi Kubo,
Lori Graham,
Nicholas A. Doyle,
William M. Quinlan,
James C. Hogg, and
Claire M. Doerschuk
From the Physiology Program, the Department of Environmental Health,
Harvard School of Public Health, Boston, MA; the Herman B Wells Center
for Pediatric Research and the Section of Pulmonology and Intensive
Care, the Department of Pediatrics, Indiana University, Indianapolis,
IN; and the Pulmonary Research Laboratory, University of British
Columbia, Vancouver, BC, Canada.
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ABSTRACT |
Infusion of complement fragments induces rapid sequestration of
neutrophils within the pulmonary capillaries. This study examined the
contributions of the bone marrow (BM) and the liver to the accumulation
of neutrophils within the lungs. Complement fragments induced the
release of neutrophils from the BM within 7 minutes of infusion, and
these neutrophils sequestered in the lungs immediately upon reaching
the pulmonary capillaries. Neutrophils expressing high levels of
L-selectin were preferentially retained within the pulmonary
microvasculature. By 30 minutes after the infusion was stopped, the
circulating neutrophil counts had increased, primarily because of
release from the BM. The number of neutrophils sequestered in the lung
had decreased by only 27%, and the number of neutrophils in the liver
increased by 223%. These studies indicate that complement fragments
induce the release of neutrophils from the BM far more rapidly than
previously described. These newly released neutrophils immediately
sequester within the lung, increasing the number of neutrophils
available to injure the lung many fold beyond the number that were
circulating before infusion. The preferential retention of
L-selectin-expressing neutrophils likely reflects the requirement for
L-selectin-mediated adhesion in maintaining sequestered neutrophils
within the pulmonary microvasculature. The number of circulating
neutrophils reflects a balance between pulmonary sequestration, rapid
release from the BM, and uptake by the liver and other organs.
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INTRODUCTION |
INFLAMMATORY MEDIATORS produced at sites
of infection or trauma often enter the bloodstream. Neutrophils and
other leukocytes express receptors for many mediators and become
activated when mediators bind to these receptors. Activated neutrophils
are thought to sequester within the microvasculature of the lungs and
other organs, contributing to tissue damage that results in vascular permeability changes and dysfunction.1-4 A complement
fragment, particularly C5a, is one such mediator that induces
neutrophil sequestration and neutropenia.1-5 Complement
fragments including C5a aid clearance of organisms from the blood
during sepsis, because both complement fragments and neutrophils are
required for intravascular Pseudomonas to sequester within the
lungs.6 Within the pulmonary microvasculature, the
sequestered neutrophils are located primarily within the capillaries,
and the majority are single rather than aggregated.5,7,8
After the complement fragments are cleared from the blood, the
circulating neutrophil counts recover, but the source of these
neutrophils and the fate of neutrophils sequestered in the lungs has
not been determined.
Previous studies have shown that the number of neutrophils sequestered
within the pulmonary capillaries at the end of a 15-minute infusion of
complement fragments is severalfold greater than the total number of
neutrophils circulating in the blood before the infusion.5,7,8 These data suggest that complement fragments rapidly induce the release of neutrophils from a mobilizeable pool. While C5a, formyl-methionyl-leucyl-phenylalanine
(fMLP), LTB4, granulocyte-macrophage colony-stimulating factor
(GM-CSF), and other neutrophil activators can induce release of
neutrophils from the bone marrow (BM),9,10 our studies
suggested that large numbers of neutrophils could be released more
rapidly than previously suspected.
Previous studies showed that the mechanism through which neutrophils
sequester in the pulmonary capillaries involves at least two sequential
steps.7,11,12 First, inflammatory mediators induce changes
in the neutrophil's cytoskeleton that result in decreased ability to
deform and pass through the narrow capillary bed, resulting in rapid
sequestration and neutropenia. Second, engagement of adhesion
molecules, both L-selectin and CD11/CD18, are required to maintain the
sequestered neutrophils within the microvasculature and, presumably, to
injure the endothelial cells. Van Eeden et al13 showed that
young neutrophils newly released from the BM express higher levels of
L-selectin than older circulating neutrophils. These observations led
to the hypothesis that newly released neutrophils expressing high
levels of L-selectin are preferentially retained within the pulmonary
microvasculature.
This study tested the hypothesis that either complement fragments or
fMLP induce a rapid and massive release of neutrophils from the BM that
immediately sequester in the pulmonary microvasculature. The
sequestration of neutrophils expressing low compared with high amounts
of L-selectin was also determined. The release of neutrophils from the
BM, as well as their expression of L-selectin, was quantitated by
comparing neutrophils in simultaneously collected arterial and venous
blood samples. Finally, these studies examined the recovery of
circulating neutrophil counts after clearance of complement fragments
and the contributions of the BM, lung, and liver to this recovery.
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MATERIALS AND METHODS |
Preparation of complement-activated plasma.
Zymosan-activated plasma was used as a source of complement fragments
and was prepared as previously described.5,7,8 In brief,
zymosan A yeast (Sigma Z-4250; Sigma, St Louis, MO) was boiled for 15 minutes and centrifuged twice. Heparinized blood obtained from donor
rabbits was centrifuged. The plasma was incubated with the boiled
zymosan yeast (5 mg/mL plasma) for 30 minutes at 37°C. The activated
plasma was centrifuged to remove the yeast and was always used within 1 hour of preparation.
Protocol 1: Release of neutrophils from the BM.
Female New Zealand white rabbits (2.5 to 2.9 kg, n = 10) were
anesthetized using ketamine hydrochloride (70 to 100 mg/kg
intramuscular [im]) and acepromazine maleate (8 to 10 mg/kg im) with additional injections as needed to maintain anesthesia.
A butterfly catheter was placed in a marginal ear vein, and the animals
received heparin (100 U/kg intravenous). In 10 rabbits, an arterial
catheter was placed in the aorta through the carotid artery to sample
blood exiting the lungs. To sample blood entering the lungs, a venous catheter was passed through the right external jugular into the thoracic vena cava near the entrance to the right atrium, as estimated by the catheter length. In a second group of five rabbits, a second venous catheter was placed through the left external jugular vein into
the inferior vena cava and positioned at the origin of this vessel just
above the union of the common iliac veins and below the entrances of
the renal, hepatic, splenic, pancreatic, and mesenteric veins. Catheter
locations were confirmed at autopsy in all rabbits.
Rabbits with one central venous catheter near the right atrium received
an infusion of complement fragments (0.3 mL/kg/min, n = 5) or the
same volume of saline (n = 5) through the ear vein for 15 minutes.
Blood samples were drawn simultaneously from the aortic and vena caval
catheters before and at 0.5, 1, 2, 4, 7, 11, 15, 20, 25, 30, 35, 45,
60, 90, and 120 minutes after the start of the infusion for measurement
of circulating leukocyte and platelet counts. The heart was stopped by
injection of saturated potassium chloride at 120 minutes. The
circulating neutrophil, mononuclear, and platelet counts in the
arterial and venous blood samples were corrected for changes in
hematocrit and were compared at each time point.
Rabbits (n = 5) with two central venous catheters, one at the right
atrium and one at the origin of the inferior vena cava, received a
similar infusion of complement fragments and blood was simultaneously
sampled from the one arterial and two venous sites before and at 1, 4, 5.5, 7, 11, and 15 minutes of infusion.
A third group of rabbits (n = 5) was similarly anesthetized and
prepared. Catheters were placed in the aorta and in the thoracic vena
cava near the right atrium. These animals received an infusion of fMLP
(Sigma; 0.01 mg/mL infused at a rate of 0.3 mL/kg/min) for 15 minutes.
Arterial and venous blood samples were obtained before and at 0.5, 1, 2, 4, 7, 11, 15, 20, 25, 30, 35, 45, 60, 90, and 120 minutes after the
start of the infusion for measurement of circulating neutrophil counts
as described above.
Protocol 2: Expression of L-selectin on sequestering neutrophils.
Rabbits (n = 15) were similarly prepared as described in protocol 1. An arterial catheter was placed in the aorta through the carotid
artery. A venous catheter was inserted through the external jugular
vein into the vena cava near the right atrium. Three groups of animals
were studied: Group 1: infusion of complement fragments for 15 minutes
and studied immediately (n = 5); group 2: infusion of complement
fragments for 15 minutes and studied at 45 minutes (ie, 30 minutes
after the infusion was stopped) (n = 5); and group 3: infusion of
unactivated plasma for 15 minutes and studied at 45 minutes (n = 5).
Blood was simultaneously sampled from the arterial and venous catheters
before and 11 and 15 minutes after in all three groups and also at 30 and 45 minutes after the infusion of complement fragments was begun in
groups 2 and 3. The circulating leukocyte, neutrophil, and lymphocyte
counts were determined in each sample. At each time point,
leukocyte-rich plasma was prepared by incubating 0.5 mL blood with 0.4 mL 4% dextran (100 to 200 kD; final concentration, 1.9%) to sediment
the red blood cells (RBCs).5 Cytospins were immediately
prepared and stored at  20°C for immunocytochemical studies.
L-selectin expression was evaluated in cytospins obtained from the
arterial and venous blood samples using immunocytochemical techniques
as previously described.14 In brief, after the cytospins were warmed to room temperature, they were fixed with acetone:methanol 1:1 for 90 seconds and transferred to 0.05 mol/L Tris-buffered saline
(pH 7.6). The alkaline phosphatase-anti-alkaline phosphatase technique
was used to localize the primary antibodies.14 In brief,
the sections were incubated with either the anti-L-selectin antibody
DREG 200, kindly provided by Dr Eugene C. Butcher (Stanford University), or nonspecific mouse IgG (Sigma) for 30 minutes. After washing with Tris-buffered saline, anti-mouse Ig
(Dakopatts Z259; Dako Corp, Carpinteria, CA) was applied for 30 minutes, followed by the alkaline phosphatase-anti-alkaline
phosphatase complex (D651; Dako Corp) for 30 minutes. After repeating
the incubations with the secondary antibody and the complex, the
alkaline phosphatase substrate containing new fuchsin was added for 20 minutes. After washing, the slides were counterstained with hematoxylin and mounted in aqueous mounting media.
Randomly sampled fields at 400× magnification in two cytospins from
each experiment were examined by two observers (L.G. and C.M.D.). All
the neutrophils in each field were evaluated and graded 0-3. Fields
were selected until 100 to 150 neutrophils/cytospin were evaluated. In
this grading system, a grade of 0 was assigned to neutrophils showing
no staining, grade 1 corresponded to very sparse granular staining,
grade 2 corresponded to moderate granular staining, and grade 3 corresponded to dense staining. The fraction of neutrophils showing
each grade of staining were determined. The absolute number of
neutrophils at each grade was calculated by multiplying the total
number of neutrophils in the blood sample by the percent of neutrophils
that showed that grade of staining. The number of neutrophils showing
grades 0 and 1 (low expression) and grades 2 and 3 (high expression)
were combined. The numbers of neutrophils expressing low and high
amounts of L-selectin were compared in the arterial and venous blood at
each time point. Intra- and inter-observer variability was less than
10% when all four grades were evaluated and was virtually zero when
only low and high expression were compared.
Protocol 3: Changes in the accumulation of neutrophils in lungs,
liver, and blood during and at 30 minutes after infusion of complement
fragments.
The effect of infusion of complement fragments on the number of
neutrophils sequestered in the lungs and liver was determined in the
same animals as studied in protocol 2. At the end of the experiment,
the animals were administered an intra-arterial injection of saturated
potassium chloride, the chest was rapidly opened, the base of the heart
ligated, and the lungs were fixed by instillation of 6% glutaraldehyde
in phosphate buffer at 25 cm H2O with the vessels clamped
for morphometric determination of neutrophil numbers. The vasculature
of one lobe of liver was clamped and the lobe was fixed in formalin.
Tissue blocks were prepared from the lungs and the liver (n = 2 of
each from each of five animals). The tissue was embedded in
methacrylate and 1- to 2-µm sections were cut. In the lungs, the
number of neutrophils and RBCs within the capillaries were counted in
20 400× fields of peripheral lung tissue for each rabbit, and the
accumulation of neutrophils was expressed as both the number of
neutrophils/1,000 RBCs and neutrophils/20 fields. In the liver, the
number of neutrophils within the sinusoids was counted in 20 400×
fields and expressed as neutrophils/20 fields.7 In the
blood samples, the number of neutrophils was expressed as neutrophils
per milliliter of circulating blood. The number of neutrophils in each
location was compared after the 15-minute infusion of complement
fragments and at 30 minutes after cessation of infusion of complement
fragments or plasma.
To determine the absolute number of neutrophils sequestered within the
liver, the volume of liver was determined by ligating all vessels to
maintain the hepatic blood volume and measuring the volume of
displacement when the excised liver was placed in a partially
fluid-filled graduated cylinder. The volume fraction of the liver
tissue occupied by neutrophils was calculated by multiplying the number
of neutrophils/field of liver tissue (determined above) by the surface
area of a rabbit neutrophil (32.16 µm2)15
and dividing by the surface area of the microscopic field at 400×
magnification (112,401 µm2). The total number of
neutrophils in the liver was determined by multiplying the volume
fraction occupied by neutrophils times the volume of the liver and
dividing by the volume of a rabbit neutrophil
(1.37 × 10 10 mL).15
Statistics.
Analyses of variance were used to compare the neutrophil counts over
time, the accumulation of neutrophils in the lungs and the liver, and
L-selectin expression.16 The modified Bonferroni correction
was used to correct for multiple comparisons when significant overall
differences were identified.17 A probability of less than
0.05 for the null hypothesis was accepted as indicating a statistically
significant difference. Data are expressed as mean ± SEM unless
otherwise noted.
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RESULTS |
Protocol 1: Release of neutrophils from the BM.
The neutrophil counts in the arterial and venous blood samples between
0 and 20 minutes from beginning the infusion of complements are shown
in Fig 1A and for the entire 2-hour
duration of the experiment in Fig 1B. The counts in the arterial and
venous blood were similar before infusion of complement fragments (Fig
1A). By 30 seconds after the start of the infusion, the neutrophil counts in the arterial blood samples, blood exiting the lungs, decreased rapidly and almost no neutrophils were present in these samples by 1 minute (Fig 1A). In contrast, the neutrophil counts in the
venous blood entering the lungs did not decrease until 1 minute, and 2 minutes of infusion was required for neutropenia to occur (Fig 1A).
This arterio-venous difference indicated that the majority of the
neutrophils were sequestering within the lungs.
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| Fig 1.
Circulating neutrophil counts in the arterial and venous
blood during and after infusion of complement fragments. The counts between 0 and 20 minutes are shown in (A), and (B) shows the counts for
the entire 2-hour duration of the experiment. The venous counts ( )
decreased more slowly than the arterial counts ( ) in the first 2 minutes of the infusion. Both arterial and venous counts were decreased
at 2 to 4 minutes. The neutrophil counts in the arterial blood samples
remained low throughout the entire infusion. However, the counts in the
venous samples began to increase by 7 minutes, and continued to rise
throughout the infusion. After the infusion was stopped, neutrophil
counts in both arterial and venous blood increased similarly, reaching
peak values that were twofold to threefold above baseline values by 45 minutes (30 minutes after the infusion was stopped). The counts
returned to baseline values by 90 minutes. *Significantly greater than
the neutrophil counts in the arterial blood, P < .05.
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Both the venous and the arterial neutrophil counts remained decreased
until 7 minutes when the neutrophil counts in the venous but not the
arterial samples began to increase (Fig 1A). This arterio-venous
difference continued until after the 15-minute infusion of complement
fragments was discontinued. The increase in the venous counts together
with the arterio-venous difference indicated that neutrophils were
being released into the venous circulation and immediately sequestering
within the pulmonary microvasculature.
By 20 minutes, when the infusion had been discontinued for 5 minutes,
both the arterial and venous neutrophil counts increased similarly (Fig
1A). At 45 minutes there was a significant rebound in the numbers of
neutrophils in both samples that was greater than the baseline values
(Fig 1B). Finally by 90 minutes this rebound recovered, and values
similar to those obtained at baseline were observed. No change in
neutrophil counts and no arterio-venous differences were observed at
any time point when rabbits received infusion of saline instead of
complement fragments.
In an additional group of rabbits, blood was drawn simultaneously from
catheters in the distal vena cava near its origin from the common iliac
veins, the proximal vena cava near the right atrium above the entrance
of renal and hepatic veins, and in the thoracic aorta as the blood
leaves the lungs. The circulating neutrophil counts were similar at
these three sites before infusion of complement fragments (Fig 2). By 4 minutes of infusion the counts at all three sites were significantly
decreased. By 15 minutes the counts in the proximal venous catheter had
increased, confirming the results described in Fig 1. Importantly, the
circulating neutrophil counts in the distal venous blood had also
increased by the same amount as those in the proximal venous blood (Fig 2). These results indicate that neutrophils
were not released from organs that directly enter the vena cava between
the distal and proximal venous catheters, including the liver and
kidneys, or from organs including the spleen, pancreas, and small
intestines that drain into the portal vein, through the liver, and then
into the inferior vena cava.
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| Fig 2.
Circulating neutrophil counts in the arterial blood
exiting the lungs sampled from the thoracic aorta ( ), the proximal
venous blood entering the lungs sampled near the right atrium (),
and the distal venous blood sampled from the origin of the inferior vena cava near the union of the common iliac veins (). The
neutrophil counts increased by a similar degree in the proximal and
distal venous blood by 7 minutes of infusion, and this increase
persisted throughout the infusion. *Significantly greater than the
neutrophil counts in the arterial blood, P < .05.
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Circulating platelet counts decreased more slowly than the neutrophil
counts (Figs 3A and B). The arterial counts
began to decrease by 2 minutes of infusion and reached 30% to 35% of
their baseline value by 7 minutes (Fig 3A). The arterial counts
declined more quickly than the venous counts, indicating that platelets were sequestering within the lungs. No arterio-venous difference was
observed after sequestration was complete (Fig 3A). The circulating platelet counts recovered by 45 minutes and no rebound was observed (Fig 3B). Circulating mononuclear cell counts decreased less, falling
to 60% to 70% of their baseline value within 4 minutes of infusion,
and there was no arterio-venous difference between 7 and 15 minutes
(data not shown). Recovery occurred within 10 to 15 minutes after the
infusion was discontinued, and no rebound was observed.
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| Fig 3.
Circulating platelet counts in the arterial and venous
blood during and after infusion of complement fragments. The counts between 0 and 20 minutes are shown in (A), and (B) shows the counts for
the entire 2-hour duration of the experiment. The venous counts ()
decreased more slowly than the arterial counts () in the first 4 minutes of the infusion. The platelet counts in both the arterial and
the venous blood samples remained low throughout the entire infusion.
After the infusion was stopped, platelet counts in both arterial and
venous blood increased similarly to baseline values by 45 minutes (30 minutes after the infusion was stopped). *Significantly greater than
platelet counts in the arterial blood, P < .05.
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Infusion of fMLP induced neutropenia as rapidly and completely as
complement fragments (Fig 4A and B). An
initial arterio-venous difference was observed at 0.5 and 1 minute of
infusion (Fig 4A). Arterial counts remained low throughout the infusion
while venous counts increased by 7 minutes, and this arterio-venous
difference persisted until the infusion was stopped (Fig 4A). Both
arterial and venous counts recovered by 30 minutes after infusion (Fig 4B). In contrast to complement fragments, the neutrophil counts continued to increase rather than returning to baseline values by 90 minutes.
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| Fig 4.
Circulating neutrophil counts in the arterial and venous
blood during and after infusion of fMLP. The counts between 0 and 20 minutes are shown in (A), and (B) shows the counts for the entire
2-hour duration of the experiment. The venous counts () decreased
more slowly than the arterial counts () in the first 2 minutes of
the infusion. Both arterial and venous counts were decreased at 2 to 4 minutes. The neutrophil counts in the arterial blood samples remained
low throughout the entire infusion. However, the counts in the venous
samples began to increase by 7 minutes, and continued to rise
throughout the infusion. After the infusion was stopped, neutrophil
counts in both arterial and venous blood increased similarly, reaching
peak values more than threefold baseline values by 120 minutes (105 minutes after the infusion was stopped). *Significantly greater than
the neutrophil counts in the arterial blood, P < .05.
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Protocol 2: Expression of L-selectin on sequestering neutrophils.
Before infusion of complement fragments, about 20% to 25% of the
neutrophils circulating in either the arterial or the venous blood
expressed low levels of L-selectin while 75% to 80% expressed high
levels, resulting in a ratio of high to low expressors of 3.8 (Table
1). At both 11 and 15 minutes of complement
fragments, the total number of neutrophils in either the venous or
arterial blood decreased compared with baseline values (Table 1).
However, the number of neutrophils in the 11- and 15-minute venous
samples was significantly greater than in the arterial samples, as was also found in protocol 1. This increase in the venous samples was
caused by an increase in the number of neutrophils that were expressing
high levels of L-selectin, and this increase is reflected in the
significantly higher ratio of high:low expressors in venous compared to
arterial blood (Table 1).
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Table 1.
Number of Neutrophils Expressing Low and High Levels of
L-Selectin in the Arterial and the Venous Blood Before, During, and After Infusion of Complement Fragments
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The total number of neutrophils in the arterial and venous blood at 30 and 45 minutes was increased and there was no arterio-venous difference. However, the ratio of high:low L-selectin expressors was
higher in both the venous and the arterial blood samples at 30 and 45 minutes than in blood obtained before infusion (Table 1).
Protocol 3: Changes in the accumulation of neutrophils in lungs,
liver, and blood during and at 30 minutes after infusion of complement
fragments.
Infusion of complement fragments for 15 minutes induced a significant
increase in the number of neutrophils within the pulmonary capillaries
and a decrease in the number of circulating neutrophils (Fig
5), as shown previously in Fig 1. No change
in the number of neutrophils was observed in the sinusoids of the liver
at this time. However, at 45 minutes (30 minutes after the infusion was stopped), the number of neutrophils in the liver had increased by
223%, and the number of neutrophils in the lungs had decreased by 27%
(Fig 5). The circulating counts had returned to baseline values.
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| Fig 5.
Accumulation of neutrophils in the blood, pulmonary
capillaries, and hepatic sinusoids. Complement fragments induced a
significant decrease in the circulating neutrophil counts at 15 minutes
and a significant increase at 30 minutes after the infusion was
stopped. The neutropenia was accompanied by a significant increase in
the number of neutrophils within the pulmonary capillaries. However, the number of sequestered neutrophils decreased only by 27% by 30 minutes after the infusion. In contrast, infusion of complement fragments did not induce an increase in the number of neutrophils within the hepatic sinusoids after 15 minutes, but caused a 2.3-fold increase within 30 minutes after the infusion was stopped.
*Significantly different from the same organ in animals that did not
receive complement fragments, P < .05. **Significantly
different from the same organ in animals studied immediately after the
15 minutes of complement fragments, P < .05.
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The volume of liver was 86 ± 3 mL. The total number of neutrophils in
the liver was 0.570 × 109 in the rabbits that did not
receive infusions of plasma, 0.468 × 109 at the end of
the 15-minute infusion of complement fragments, and 1.51 × 109 in rabbits given a 15-minute infusion of complement
fragments and studied 30 minutes later. These data indicate that 1.04 × 109 neutrophils accumulated in the liver during the 30 minutes after the infusion was stopped.
| |
DISCUSSION |
Release of neutrophils from the BM or other organs was hypothesized to
contribute to neutrophil accumulation in the lungs induced by
complement fragments because the increase in the number of neutrophils
accumulating within the pulmonary capillaries was much larger than the
number circulating within the total blood volume before
infusion.5,7,8 This new study shows that release of
neutrophils begins to occur within 7 minutes of infusion, more rapidly
than the previously reported 0.5 to 2.0 hours,9,10 where
this early release was likely masked by the coincident neutropenia. This release is most likely from the BM for two reasons. First, the
increase in the neutrophil counts was similar in blood sampled from the
distal vena cava near its origin at the union of the common iliac veins
and blood sampled from the proximal vena cava near the right atrium
(Fig 2). This observation that the neutrophil counts in
venous blood proximal to the entrance of venous drainage from
the liver, spleen, intestines, kidneys, pancreas, and other abdominal organs were not greater than the counts in the distal venous
blood indicates that there was no significant release of neutrophils by
these organs during the 15-minute infusion of complement fragments.
Second, neutrophil sequestration in muscle tissue, the major tissue
other than BM that drains into the blood sampled by the distal
catheter, is actually increased during infusion of complement
fragments5 and, therefore, is unlikely to be the site of
neutrophil release.
In fact, the number of neutrophils released into the distal venous
circulation completely accounts for the observed numbers accumulating
in the lungs, as described by the following calculations. The total
number of neutrophils circulating in the blood immediately before
infusion of complement fragments was calculated by multiplying the
number of neutrophils per milliliter of blood at this time by the blood
volume of the rabbit (60 mL/kg) and was equal to 6.1 × 108 neutrophils/rabbit. The number of neutrophils released
into the venous circulation that immediately sequestered within the
lungs was calculated by multiplying the difference in neutrophil counts per milliliter of blood between the venous and arterial blood samples
from 4 to 15 minutes of infusion by the cardiac output, previously
measured to be 496 mL/min,5 and was found to be 1.9 × 109 neutrophils/rabbit. Therefore, the predicted total
number of neutrophils available to accumulate within the pulmonary
capillaries was 6.1 × 108 + 1.9 × 109 = 2.5 × 109 neutrophils/rabbit. In control rabbits with no
injury, the number of marginated neutrophils within the pulmonary
capillaries was 2.1 × 109
neutrophils/rabbit,5 so that the number of accumulated
neutrophils after infusion of complement fragments was predicted to be
the circulating + newly released + normally marginated neutrophils = 6.1 × 108 + 1.9 × 109 + 2.1 × 109 = 4.6 × 109 neutrophils/rabbit. This
compares closely to the experimentally observed number of neutrophils
accumulated within the lungs of complement-treated rabbits of 4.9 × 109.5 Therefore, the complement
fragment-induced increase in the accumulation of neutrophils within the
pulmonary circulation can be completely explained by sequestration of
both previously circulating neutrophils and those newly released from
the BM.
Infusion of fMLP, another potent neutrophil activator, induced a
similar neutropenia due to sequestration within the lungs and a similar
release of neutrophils from the BM. These data suggest that this rapid
release of neutrophils is not induced solely by complement fragments
but may be a response to a number of neutrophil activators and
chemotactic factors.
This release from the BM was unique to neutrophils, as neither
mononuclear cells nor platelets showed a similar behavior. The
sequestration of platelets occurred after neutrophils had sequestered,
suggesting that the formation of neutrophil-platelet aggregates were
not the mechanism through which neutrophil sequestration occurred. In
fact, these data suggest that platelets may actually be adhering to
sequestered neutrophils.
The mechanism through which complement fragments and other inflammatory
mediators induce this release of neutrophils is not clear. It is
possible that neutrophils still in the BM bind to BM matrix by
L-selectin-mediated interactions, and newly released neutrophils are
known to express more L-selectin than the average circulating
neutrophil.13 Complement fragments could induce activation
of the protease that cleaves L-selectin on neutrophils within the BM,
resulting in their release. However, this possibility seems unlikely in
view of the observations that the number of neutrophils within the BM
and the circulating blood in L-selectin-deficient mice are not
altered.12,18,19 In addition, Jagels et al20 showed that neither L-selectin nor CD11/CD18 was required for neutrophils to emigrate from the BM into the blood using antibodies against these molecules in rabbits. In fact, no known adhesion molecule
appears to mediate this response, as mice deficient in P-selectin,
E-selectin, L-selectin, CD18, or ICAM-1, either singly or in
combination, have no apparent defect in the mobilization of neutrophils
from the BM.12,18-29 Alternatively, complement fragments
and other inflammatory mediators may stiffen neutrophils within the BM,
similar to the changes in biomechanical properties observed in
circulating neutrophils and in vitro.11,30-33 This stiffening of neutrophils that are adherent to the BM stroma may cause
them to loosen and de-attach, resulting in release. In addition, inflammatory mediators may cause retraction of the reticular cells that
line the adventitial surface of the venous sinusoids of the BM,
widening the opening through which neutrophils are
released.34-36
The pulmonary microvasculature preferentially retained neutrophils that
were expressing high levels of L-selectin. This retention likely
reflects the requirement for L-selectin-mediated adhesion to keep
neutrophils within the capillaries and arterioles/venules once they are
sequestered.12,37 Low L-selectin-expressing neutrophils may still stiffen in response to complement fragments, reducing their
deformability and resulting in entrapment, but without L-selectin they
eventually pass through the capillary bed within 2 to 5 minutes.12,37 Alternatively, the high expression of
L-selectin may be an epiphenomenon, reflecting some other feature of
neutrophils newly released from the BM. For example, these newly
released neutrophils may be larger in volume or they may be less
deformable than older neutrophils.38
After the infusion of complement fragments was stopped, the number of
circulating neutrophil counts rapidly increased. By 30 minutes, the
number of neutrophils in the lungs was reduced by 27%. In contrast,
the number of neutrophils in the liver, which was not increased at the
end of the infusion, had increased by 223% within 30 minutes, an
increase of 1.04 × 109 neutrophils. These data support
the hypothesis that the increase in circulating neutrophils is most
likely caused by continued release from the BM without an inflammatory
stimulus to sequester, while neutrophils released from the lungs do not
circulate but are cleared by the liver, for the following reasons.
First, Van Eeden et al13 have shown that the expression of
L-selectin on neutrophils within the BM is higher than that on normally
circulating neutrophils, while we have shown that neutrophils within
the capillaries of rabbits given infusion of complement fragments for
15 minutes have an average reduction of 72% in the expression of
L-selectin due to complement fragment-induced shedding using
quantitative ultrastructural immunohistochemistry.39 The
observation in the present study that the ratio of high to low
L-selectin-expressing neutrophils was higher in blood samples obtained
after the infusion was stopped than in samples obtained immediately
before the infusion therefore support the hypothesis that the increase
in circulating counts was caused by release of neutrophils from the BM.
Second, a decrease of 27% in the number of sequestered neutrophils
within the lungs corresponds to the release of 1.32 × 109 neutrophils (27% × 4.9 × 109 total
sequestered neutrophils5). This value is very similar to
the 1.04 × 109 neutrophils that sequestered in the liver
within 30 minutes after cessation of complement fragments, suggesting
that the neutrophils released from the lungs may not circulate well and
are rapidly cleared by the liver. Taken together, these data suggest
that the increase in circulating neutrophil counts is caused by
continued release of neutrophils from the BM rather than from the lung. Because neutrophils sequestered in the lungs shed most of their L-selectin39 and L-selectin expression on most circulating
neutrophils is high, the neutrophils released from the lungs appear to
circulate poorly. Whether the neutrophils accumulating in the liver
injure hepatocytes and contribute to multiorgan failure1-4
remains to be determined.
In summary, infusion of complement fragments induces a rapid release of
neutrophils, most likely from the BM, that immediately sequester on the
first passage through the lungs. Neutrophils expressing high levels of
L-selectin are preferentially sequestered within the lungs. After the
infusion of complement fragments is stopped, the circulating neutrophil
counts recover, most likely due to continued release of neutrophils
from the BM. Neutrophils that were sequestered in the lungs appear to
be released from the pulmonary microvasculature too slowly to account
for the increase in the circulating neutrophils, to circulate poorly,
and to rapidly accumulate in the liver. These studies show the
complex nature of neutrophil kinetics during complement
fragment-induced sequestration and the need to consider the balance
between the many compartments that contribute to the distribution of
neutrophils within the body.
| |
FOOTNOTES |
Submitted April 30, 1997;
accepted February 14, 1998.
Supported by Public Health Service Grants No. HL48160 and
HL33009.
Address reprint requests to Claire M. Doerschuk, MD, Harvard School of
Public Health, I-305, 665 Huntington Ave, Boston, MA 02115; e-mail:
cdoersch{at}hsph.harvard.edu.
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 |
The authors thank Taffy Hooser and Bonnie Meek for preparation of
histologic sections.
| |
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Abstract
Introduction
Abstract