Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 600-609
P-Selectin Expression by Endothelial Cells Is Decreased in Neonatal
Rats and Human Premature Infants
By
Diane E. Lorant,
Wenhua Li,
Niloufar Tabatabaei,
Michael K. Garver, and
Kurt H. Albertine
From The Nora Eccles Harrison Cardiovascular Research and Training
Institute, Department of Pediatrics, University of Utah, Salt Lake
City, UT.
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ABSTRACT |
Decreased adhesion of neutrophils to endothelial cells and delayed
transendothelial cell migration of neutrophils have been consistently
reported in neonatal animals and humans and contribute to their
susceptibility to infection. The delayed transmigration of neutrophils
is especially prevalent in premature neonates. To define the nature of
this defect, we used an in vivo animal model of inflammation and found
that radiolabeled leukocytes from adult rats transmigrated into the
peritoneum of other adult rats 5 times more efficiently than they did
in neonatal rats (P = .05). This indicated that defects in
neonatal neutrophils could not completely account for the delayed
transmigration. Delayed transmigration in the neonatal rats correlated
with a defect in the expression of P-selectin on the surface of their
endothelial cells. We found a similar P-selectin deficiency in
endothelial cells lining mesenteric venules and umbilical veins of
human premature infants when compared with term human infants. The
decreased P-selectin in premature infants was associated with decreased
numbers of P-selectin storage granules and decreased P-selectin
transcription. Decreased P-selectin expression on the surface of
endothelial cells in preterm infants may contribute to delayed
neutrophil transmigration and increased susceptibility to infection.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE PREVALENCE OF SEPSIS in newborn
infants is 1 per 1,000 live births, with a mortality rate of 20% to
75%.1 In premature infants, the prevalence is estimated to
be as high as 1 in 230 infants, with an even greater
mortality.2 Because there are in excess of 75,000 infants
less than 32 weeks gestation born prematurely each year in the United
States,3 the morbidity and mortality from sepsis in
premature infants continues to be a major health problem.
One reason for the increased susceptibility to infection in neonates is
a defect in the neonatal inflammatory response that results in delayed
accumulation of neutrophils at sites of microbial invasion. This defect
is related to the decreased adhesion of neutrophils to endothelial
cells and delayed transendothelial cell migration, both of which are
among the most consistently reported defects in the neonatal immune
system.1,4-6 Delayed transendothelial cell migration of
neutrophils has been demonstrated in vivo in neonatal animals by
intraperitoneal inoculation with various inflammatory mediators.
Between 4 and 6 hours after the inoculation, neonatal animals have a
markedly diminished peritoneal neutrophil count compared with
adults.7-9 Because neutrophils are the first cells to
arrive at the site of microbial invasion,10 delayed
neutrophil transmigration allows the invading microorganisms to
multiply unchallenged and ultimately predisposes newborns to an
increased bacterial load and overwhelming sepsis.11,12
The recruitment of neutrophils to sites of microbial invasion is
regulated by the sequential interaction of different classes of
adhesion molecules, resulting in an adhesion and activation cascade.13 The first step in adhesion is rolling of
neutrophils along the vessel wall. This is mediated by a class of
adhesion molecules termed the selectins. Three selectins have been
identified: L-, P-, and E-selectin. Selectins are critical in
neutrophil transmigration by virtue of their ability to tether
leukocytes in a reversible fashion under conditions of shear and flow,
a process that mediates their rolling on the endothelium of inflamed
vessels.14 Although all three selectins have been
implicated in mediating neutrophil rolling, P-selectin is most
important during the initial induction of neutrophil rolling after
endothelial cell stimulation.15 It is constitutively
synthesized by endothelial cells and stored in Weibel-Palade bodies
along with von Willebrand factor (vWF).16 Within minutes of
activation with certain agonists (phorbol myristate acetate, histamine,
thrombin, c5a, or oxidants), P-selectin is translocated to the plasma
membrane, where it supports rapid neutrophil adhesion.14
Investigations of neutrophil adhesion and transmigration in neonates
have focused on neonatal neutrophils as the defective cell type
responsible for delayed neutrophil adhesion to endothelial cells. The
deficient adhesion of neonatal neutrophils to endothelial cells has
been attributed to decreased expression of integrins,4 diminished expression of L-selectin,5 and a rigid
cytoskeleton that prevents redistribution of adhesion sites and normal
deformability and movement of neutrophils.17 However, the
premise that neonatal neutrophils are deficient in their expression of
cell adhesion molecules has recently been challenged.18-22
To date, studies have not examined endothelial cells despite the fact
that adhesion molecules expressed by endothelial cells are required for
neutrophil adhesion and transmigration.13 We hypothesized
that decreased or delayed expression of cell adhesion molecules on the
endothelial cell surface in newborns is a crucial factor in the delayed
transmigration of neutrophils. To test this hypothesis, we used an in
vivo model of peritoneal inflammation in newborn and adult rats. We
found that neutrophil transmigration in neonatal rats was delayed. This delay was associated with a defect in P-selectin expression on the
surface of neonatal endothelial cells. These studies were extended to
humans. We found that the expression of P-selectin on endothelial cells
is less in preterm neonatal infants compared with full-term neonates.
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MATERIALS AND METHODS |
In vivo transmigration studies.
Adult and 1-day-old Sprague-Dawley rats (Charles River Laboratories,
Wilmington, MA) received an intraperitoneal injection of either 0.01 mL/g body weight thioglycollate (Becton Dickinson, Cockeysville, MD) or
endotoxin tested-sterile phosphate-buffered saline (PBS; Sigma Chemical
Co, St Louis, MO). Thioglycollate was prepared per the manufacturer's
instructions, autoclaved, and kept at room temperature for 1 to 3 weeks
before use. At various times after the injection, the rats were killed
by cervical dislocation and the peritoneum was lavaged. The lavage was
performed 3 times with 0.1 mL/g 37°C PBS, 0.1% bovine serum
albumin, and 10 U/mL heparin. The total number of leukocytes in the
peritoneal fluid was measured with a Coulter Counter (Coulter
Electronics, Inc, Hialeah, FL), and the percentage of neutrophils was
determined by differential Wright stain. Neutrophil accumulation in the
lavage fluid was also estimated with a myeloperoxidase assay using the tetramethylbenzidine technique.23 We compared the
neutrophil count in the peritoneum between rats of the same age that
received PBS with those receiving thioglycollate. The percentage
increase in peritoneal neutrophils was calculated using the following
equation: % increase = (count in thioglycollate lavage-count in PBS
lavage)/(count in PBS lavage) × 100.
For the studies measuring transmigration of homologous leukocytes, we
collected blood from adult rats, isolated the leukocytes by hypotonic
lysis, and labeled them with 111Indium (111In),
as previously described.24 Total leukocytes were used in these studies, rather than isolated neutrophils, to avoid the possibility of neutrophil activation during additional isolation procedures. The leukocytes were resting as demonstrated by
quantification of surface L-selectin compared with leukocytes in whole
blood and by leukocyte morphology. They were round with no aggregation or rosetting with platelets. Labeled leukocytes (6.5 × 105 per gram body weight) were injected into newborn and
adult rats by an intracardiac puncture. Peripheral blood samples were
then collected from the rats every hour for a white blood cell count and measurement of radioactivity. There was approximately a 50% increase in the white blood cell count of both the neonatal and adult
rats. The amount of radioactivity in the blood was stable up to 2 hours
after the injection and the increase in the white blood cell count
remained elevated by 50% in both sets of animals. The rats then
received intraperitoneal PBS or thioglycollate, as described above.
Four hours after the intraperitoneal injection, the rats were killed,
the peritoneum was lavaged, and the radiolabeled adult leukocytes that
had transmigrated into the peritoneum and retrieved in the lavage fluid
were counted (Beckman Gamma 5500; Beckman Instruments,
Inc, Irvine, CA).
Immunohistochemistry.
Rat mesentery was collected at various times after intraperitoneal
injection and fixed in 4% paraformaldehyde. Human mesentery was
obtained at autopsy and fixed in Histochoice tissue fixative (Amresco,
Solon, OH). Tissue from individuals who died from an infectious process
or had an ongoing infection at the time of death was excluded from the
study (Table 1). Mesentery from rats and
humans was dehydrated in a graded series of acetone at 4°C and
embedded (Immunobed; Polysciences Inc, Warrington, PA) at 4°C.
Four-micrometer-thick sections were cut using glass knives and
transferred to coated slides (Vectabond; Vector Laboratories, Burlingham, CA). P-selectin was detected on rat mesenteric endothelial cells using a 1:200 dilution of the monoclonal antibody (MoAb) PB1.3
(Cytel Corp, San Diego, CA) that has been used extensively for
immunohistochemical analysis in other animal models of inflammation and
recognizes P-selectin expressed on the endothelial cell
surface.25,26 The MoAb S12 (16 µg/mL) was used to detect
the expression of P-selectin in human mesenteric endothelial cells
(provided by Rodger McEver, University of Oklahoma,
Oklahoma City, OK). S12 has been well characterized and used
extensively for immunohistochemical localization of P-selectin in
humans.16 Unlike MoAb PB1.3, MoAb S12 recognizes P-selectin
stored intracellularly as well as expressed on the endothelial cell
surface. A rabbit antibovine antiserum to PECAM-1 was used at a 1:1,000
dilution to detect PECAM-1 expression on rat endothelial cells
(provided by Steven Albelda, University of Pennsylvania Medical Center,
Philadelphia, PA). An MoAb was used to detect human PECAM-1 (1:200
dilution; Genosys Biotechnologies Inc, Cambridge, UK). Tissue sections
were incubated with the primary antibody overnight at room temperature.
A biotinylated IgG was used as the secondary antibody (Vector
Laboratories) at a 1:200 dilution for 1 hour at room temperature. The
avidin-biotin immunoperoxidase technique (Vectastain ABC Reagent;
Vector Laboratories) was used to detect biotinylated secondary
antibody. Immunostaining negative controls included omission of the
primary antibody or secondary antibody.
Procurement of human umbilical vein endothelial cells.
Thirty umbilical cords were collected from preterm deliveries,
excluding those suspected of having chorioamnionitis. Infants were
delivered preterm because of pregnancy induced hypertension (5),
placental abruption (6), an incompetent cervix (4), multiple gestation
(7), placental insufficiency (3), a motor vehicle accident (1),
precipitous delivery in the emergency room (2), maternal cholecystectomy (1), and posterior urethral valves with oligohydramnios (1). Of the preterm deliveries, 16 were vaginal and 14 were by cesarean
section. Term deliveries were all of healthy infants, none of whom were
admitted to an intensive care nursery. Mothers of preterm infants were
exposed to various medications such as betamethasone, tocolytics,
antihypertensives, and antibiotics. The only medication consistently
administered to mothers delivering preterm was betamethasone.
Analysis of P-selectin, vWF, and ICAM-1 by enzyme-linked
immunosorbent assay (ELISA).
Endothelial cells were isolated from the umbilical vein with
collagenase and counted using a hemocytometer. An equal number of
endothelial cells were lysed in a detergent lysis buffer (Tris-buffered saline, pH 7.4, 1% Triton-x, 1% 0.1 mol/L phenylmethylsulfonyl fluoride). The cell lysates were centrifuged, protein assays (Bio-Rad Laboratories, Hercules, CA) were performed on the supernatant, and
equal concentrations of protein were used for sandwich ELISAs. The
ELISAs for P-selectin and vWF were performed as previously described.27 The antihuman P-selectin MoAbs W-40 and S-12
and purified membranous P-selectin were used for the P-selectin ELISA (all provided by Rodger McEver). Rabbit antihuman vWF (Dako Corp, Carpinteria, CA), goat antihuman factor VIII-related antigen (Atlantic Antibodies, Scarborough, ME), and purified von Willebrand factor (provided by Jerry Roth, University of Washington, Seattle, WA) were
used for the vWF ELISAs. The ICAM-1 ELISAs were performed with the
PREDICA kit (Genzyme, Cambridge, MA).
Transmission electron microscopy.
Fresh umbilical cords from preterm and term infants were cut in
cross-section, fixed with 2.5% glutaraldehyde and 1% paraformaldehyde in cacodylate buffer, postfixed with 1% osmium tetroxide, and embedded
in epoxy resin. Thin sections were cut across the umbilical vein's
endothelial cells, counterstained with uranyl acetate and lead citrate,
and observed with an Hitachi H-7100 transmission electron microscope
(Hitachi Instruments Inc, Mountainview, CA). For each
cord, 10 nonoverlapping, calibrated fields were evaluated for the
number of Weibel-Palade bodies per cubic micrometer of nonnuclear
cytoplasm (numerical density), as previously described.28 Briefly, the numerical density (Nv) of Weibel-Palade bodies
was determined by the formula Nv=n
A/A(D + T),29 where nA
is the number of Weibel-Palade bodies counted in an area (A) of a
section (2.5 µm2), D is the mean diameter of
Weibel-Palade bodies, and T is the section thickness (75 nm). The
dimensions (long and short axis) of Weibel-Palade bodies were
determined for 10 Weibel-Palade bodies per cord. The Weibel-Palade body
dimensions and shape were the same between groups (preterm and term).
The average dimensions were 116 ± 19 × 148 ± 30 nm (mean ± SD), meaning that the bodies are elliptical.
RNAse protection assay.
Endothelial cells were isolated from freshly collected human umbilical
cords by incubation with collagenase and then lysed in TRIzol reagent
(GIBCO BRL, Grand Island, NY). Total RNA was isolated in TRIzol
according to the manufacturer's instructions. The RNA probe was
generated by in vitro transcription using T7 polymerase and a template
containing a P-selectin cDNA insert extending from bp 316 to 647. This
region encodes the entire lectin-like domain of P-selectin necessary
for binding.30 A full-length human P-selectin cDNA in
pGEM-9Zf(
) vector was cut by the restriction enzyme Sac
I, which leaves a 647-bp cDNA insert in the vector. The resulting
plasmid was linearized with BamHI. An RNAse protection assay
was performed with the RPAII kit (Ambion Inc, Austin, TX) to detect and
quantitate P-selectin mRNA per the manufacturer's instructions.
Statistics.
The percentage increase in peritoneal neutrophils in newborn and adult
rats was compared using an unpaired t-test. Unpaired t-tests also were used to compare the means of the ELISAs. A
Mann-Whitney rank sum test was performed on the mean numerical density
of Weibel-Palade bodies. Significance was defined at the P < .05 level.
The animal studies were approved by the institutional animal care and
use committee. The institutional review board approved all studies
using human tissue and endothelial cells.
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RESULTS |
Delayed accumulation of neutrophils in the inflamed peritoneum of
neonatal rats is not accounted for by a neutrophil defect.
Four hours after intraperitoneal injection of thioglycollate, there
were significantly fewer neutrophils in the fluid obtained by
peritoneal lavage of neonatal rats compared with fluid retrieved from
adults. Both the absolute neutrophil number and the percentage increase
in neutrophil count were greater in the adult rats. In 9 experiments,
there was a fivefold greater increase in peritoneal neutrophils in
adults compared with newborns (P = .01). Additionally, the
percentage increase in myeloperoxidase activity in lavage fluid from
adults was 23 times greater than that in samples from the newborn rats.
These results are similar to those in previous studies7-9
and confirmed that there is impaired accumulation of neutrophils at
extravascular sites in response to inflammatory stimuli in neonatal
animals. The defect in accumulation of neutrophils in the peritoneum of
neonatal rats in our experiments was not due to lower circulating
neutrophil numbers or impaired release of neutrophils from the bone
marrow. In the 9 experiments, there was initially no significant
difference in blood neutrophil number in samples from adults vs.
neonates, and 4 hours after intraperitoneal injection of
thioglycollate, the number of blood neutrophils was actually about
twofold higher in samples from neonates compared with adults (P = .03).
To determine whether leukocytes from an adult animal can accumulate
normally in the peritoneum of a neonatal animal challenged with
thioglycollate, leukocytes from adult rats were isolated, pooled,
radiolabeled with 111In, and injected into allogenic adult
and newborn rats. Rats then received an intraperitoneal injection of
thioglycollate or PBS, as described above. The percentage increase in
radiolabeled leukocytes in the peritoneal fluid from adults was
fivefold greater than that in peritoneal fluid from newborns
(Fig 1) and was almost exclusively
neutrophils. This experiment indicated that the neonatal defect could
not be overcome by transfusion of adult leukocytes that were competent
to target and transmigrate and that the phenotype of the neonatal
leukocyte alone does not account for the impaired accumulation.
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| Fig 1.
Transfused leukocytes from adult rats do not transmigrate
efficiently in neonatal rats. The ordinate indicates the percentage
increase (see Materials and Methods) in cpm of radiolabeled adult
leukocytes that transmigrated into the peritoneum of neonatal and adult
rats. The bars represent the mean ± SEM of values from 4 rats in each
group. The difference in the percentage increase in peritoneal
leukocytes between the newborn and the adult is statistically
significant (P = .05).
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In the original experiments measuring the transmigration of autologous
neutrophils, the number of neutrophils in peritoneal fluid of neonatal
rats increased with time after injection with thioglycollate and by 24 hours equaled the number in samples from adult rats. This early defect
in accumulation of neutrophils with preserved ability to accumulate at
later time points was similar to that reported in previous studies of
neonatal animals and also to that in adult mice genetically deficient
in P-selectin that were challenged with intraperitoneal
thioglycollate.31
P-selectin is not expressed on the surface of endothelial cells from
newborn rat mesentery.
The expression of P-selectin was examined in situ in endothelium in
neonatal and adult rat mesentery at various times after the
intraperitoneal injection of PBS or thioglycollate. By 15 minutes after
the injection, P-selectin was detectable on the surface of endothelial
cells in adult rats injected with thioglycollate but not in adult rats
injected with PBS. The maximum expression of P-selectin in the adults
was 30 minutes after the injection (Fig
2A). The expression was sustained, but weaker, from 60 minutes to 4 hours after the injection. In contrast to the tissue from adult rats,
the endothelial cells lining the mesenteric vessels of newborn rats
injected with thioglycollate did not stain for P-selectin early after
challenge (Fig 2B). Endothelial cells in mesentery from 1 newborn rat
of the 11 studied stained weakly positive for P-selectin 120 minutes
after thioglycollate injection. As in the adult rats, no newborn rats
injected with PBS stained positively for P-selectin. Therefore, the
delay in the intraperitoneal accumulation of neutrophils in neonatal
rats coincided with decreased expression of P-selectin on endothelial
cells from neonatal rats.
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| Fig 2.
Newborn rats have a defect in expression of P-selectin on
endothelial cells after an inflammatory stimulus. The photomicrographs
in this figure are of samples collected 30 minutes after an
intraperitoneal injection of thioglycollate and are representative of
the 11 rats studied in each group. Postcapillary mesenteric venules
were identified by their morphology. The sections were viewed with
Nomarski differential interference contrast optics. All four panels
were magnified 500×. (A and B) P-selectin expression. P-selectin
expression was detected with the MoAb PB1.3. (A) is representative of
mesenteric venules in the adult rat. The arrows demonstrate positive
brown immunostaining of endothelial cells lining the vessels. (B) is
representative of the newborn rat. The arrow points to endothelial
cells lining newborn mesenteric venules that did not stain. (C and D)
PECAM-1 expression. (C) is a section of adult mesentery and (D) is a
section of newborn mesentery. Positive brown immunostaining for PECAM-1
is demonstrated covering the full height of the cytoplasm of
endothelial cells (arrows).
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Platelet endothelial cell adhesion molecule-1 (PECAM-1) was used as a
control adhesion molecule that is expressed by endothelial cells,
because it is expressed early in development.32 Endothelial cells from both newborn and adult rats constitutively expressed PECAM-1
on their surface and the intensity of the staining was equivalent in
tissues from rats of the two age groups (Fig 2C and D). In contrast,
neither the newborn nor the adult rats expressed intercellular adhesion
molecule-1 (ICAM-1) on the surface of endothelial cells lining
mesenteric venules 4 hours after the intraperitoneal injection of
thioglycollate. However, adult rats did exhibit surface expression of
ICAM-1 when endothelial cells in skin were examined 24 hours after
local injection of lipopolysaccharide as a positive control (not shown).
P-selectin expression is decreased on the surface of endothelial
cells from human newborn mesentery.
To determine whether endothelial cells from human neonates have the
same deficiency in P-selectin expression as observed in neonatal rats,
mesenteric tissue was obtained at autopsy from patients of various ages
for immunohistochemistry. Eleven samples from individuals ranging in
age from 18 weeks gestation to 10 years of age were studied. There was
intense staining for P-selectin in endothelial cells from older
children ranging in age from 3 to 10 years, and there was consistent
staining of the endothelial cells from term human neonates
(Fig 3A). Faint staining for P-selectin was
seen in endothelial cells at 27 weeks of gestation. Endothelial cells
from fetuses ranging in age from 18 to 22 weeks of gestation did not
stain positively for P-selectin (Fig 3B). As in the animal model, the
age of the subject did not affect the intensity of staining for PECAM-1
on endothelial cells lining the mesenteric venules (Fig 3C and D). By
immunohistochemical analysis of human tissues, P-selectin was not
expressed early in development and the expression of P-selectin
increased with advanced gestational age.
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| Fig 3.
Expression of P-selectin is developmentally regulated in
humans. Mesentery was collected during the autopsy of individuals of
varying age and processed as in Fig 2. The sections were viewed with
Nomarski differential interference contrast optics. All four panels are
magnified 165×. (A and B) P-selectin expression. P-selectin
expression was detected with the MoAb S12. (A) is from a term infant
who died because of a congenital heart defect and (B) from a 22-week
gestation infant delivered prematurely because of an incompetent
cervix. There is immunostaining for P-selectin on mesenteric
endothelial cells of the term infant (arrows), but no immunostaining
was detected on the endothelial cells from the 22-week gestation
infant. The arrow in (B) points to endothelial cells lining newborn
mesenteric vessels that did not stain. (C and D) PECAM-1 expression:
These sections are from the same tissue blocks as in (A) and (B). (C)
is a section of mesentery from the term infant and (D) is from the
22-week gestation infant. The arrows in both figures indicate
endothelial cells immunostained for PECAM-1.
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Endothelial cell stores of P-selectin and von Willebrand factor
increase with gestational age in humans.
Endothelial cells from umbilical veins of preterm infants contained
approximately half the amount of P-selectin as those from term infants
(Fig 4A). As with P-selectin, the
endothelial cell stores of vWF were diminished in the human umbilical
vein endothelial cells (HUVECs) from preterm infants (Fig
4B). In contrast, ICAM-1 levels were not lower when measured in the
same cellular lysates (142.6 ng/mg protein in cells from preterm
infants v 78.6 ng/mg protein in endothelial cells from term
infants).
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| Fig 4.
Endothelial cell stores of P-selectin and von Willebrand
factor increase with gestational age in human neonates. ELISAs were
performed on HUVEC lysates and normalized to total cellular protein.
These graphs summarize the data from 12 term infants (>38 weeks) and
15 preterm infants (18 to 31 weeks). (A) P-selectin. The bars represent
the mean ± SD. The amount of P-selectin was significantly less in
preterm HUVECs compared with term HUVECs (P = .003). When
individual samples were examined, the amount of P-selectin generally
correlated with gestational age, although there was variation from
sample to sample. (B) von Willebrand factor. The bars represent the
mean ± SD. The amount of vWf was significantly less in preterm HUVECs
compared with term HUVECs (P = .005).
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Maternal betamethasone was administered to hasten lung maturity in 77%
of the preterm infants whose HUVECs were used in this study. To
determine the effect of betamethasone on endothelial cell stores of
P-selectin, HUVECs from umbilical cords of term infants were grown in
the presence of either 0.02 µg/mL of betamethasone (the concentration
predicted to be present in cord blood after the mother receives 12 mg
intramuscular betamethasone33), 10 µg/mL betamethasone,
or 100 ng/mL oncostatin M. Oncostatin M is an endothelial cell agonist
that has been shown to increase P-selectin mRNA and protein in human
endothelial cells.34 Both concentrations of betamethasone
decreased the amount of ICAM-1 in HUVECs (53% and 49% decrease in
cells treated with 0.02 or 10 µg/mL betamethasone, respectively) and
increased P-selectin in HUVECs (not shown). The increase when the
HUVECs were exposed to 0.02 µg/mL betamethasone (76% increase) was
comparable to that upon exposure to oncostatin M (79% increase).
Decreased P-selectin in endothelial cells from premature human
infants is associated with fewer Weibel-Palade bodies.
HUVECs were collected from 6 preterm infants ranging from 23 to 26 weeks of gestation and from 6 term infants greater than 38 weeks of
gestation. Ultrastructural observation was performed by an observer
blinded to the gestational age of the infants from whom the endothelial
cells were collected and showed that intact endothelial cells lined all
of the umbilical cords. Weibel-Palade bodies were morphologically
identified35,36 as small, membrane-bound, rod-shaped
organelles of moderate density that were located in the cytoplasm of
the umbilical cord endothelial cells. There were few Weibel-Palade
bodies in endothelial cells of very premature infants and the number
increased with increasing gestational age (Figs 5 and 6).
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| Fig 5.
Preterm umbilical vein endothelial cells have fewer
Weibel-Palade bodies than term cells. HUVECs were viewed by
transmission electron microscopy for quantitative morphology. The mean
numerical density (NV) represents the number of
Weibel-Palade bodies per cubic micrometer of nonnuclear cytoplasm. The
difference between term and preterm endothelial cells is significant
(P = .01).
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| Fig 6.
The number of Weibel-Palade bodies per HUVEC is less in
endothelial cells from preterm infants. This figure is representative
of the data in Fig 5. (A) is from a 23-week gestation infant and (B) is
from a term infant. Here there are 4 to 5 times as many Weibel-Palade
bodies (arrows) in the cytoplasm of the term infant compared with the
preterm infant.
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mRNA for P-selectin is decreased in endothelial cells from premature
neonates.
Umbilical cords were collected from 6 preterm infants ranging in age
from 22 to 26 weeks and were paired with umbilical cords from 6 term
infants born at approximately the same time, and P-selectin mRNA levels
were measured. Endothelial cells were isolated from the veins and
lysed, and the amount of P-selectin mRNA in each sample was quantitated
with an RNAse protection assay. In each pair of samples, there was more
mRNA for P-selectin in the HUVECs from term infants compared with the
HUVECs from preterm neonates (Fig 7). There
was considerable variation in mRNA in these samples that may have been
due to the variable period of time between delivery of the placenta and
processing of the samples.
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| Fig 7.
P-selectin mRNA is diminished in HUVECs from preterm
cords. P-selectin mRNA was quantitated in each sample with an RNAse
protection assay (see Materials and Methods) and normalized to GAPDH
mRNA in the same sample. The ratio of the relative intensities of
P-selectin mRNA compared with GAPDH mRNA is shown in the bar graph with
the corresponding lane below. The combined relative densitometric units
in the term infants was 4.6× greater than in the preterm infants
(P = .016).
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| |
DISCUSSION |
The etiology of delayed neutrophil transmigration in neonates has been
extensively studied. Although there are many studies that have focused
on defects in neonatal neutrophils, there are no studies in the
literature that address the possibility of defects in neonatal
endothelial cells. Using an animal model, we show that leukocytes from
adult rats do not transmigrate as efficiently in neonatal rats when
compared with adults (Fig 1). This experiment is the first to
demonstrate a defect in accumulation of adult leukocytes at an inflamed
site in a neonatal host. Our study demonstrates that P-selectin, a
rapidly expressed endothelial cell adhesion molecule shown to be
essential for both neutrophil rolling early after endothelial cell
stimulation15 and neutrophil transmigration in
vivo,31 has diminished expression in the newborn rat. Given the complexity of whole animal studies, factors other than decreased expression of endothelial cell adhesion molecules also may be contributing to the delay in transmigration in the neonatal rats in our
experiments. For example, newborns have been reported to have decreased
levels of interleukin-8 (IL-8),37 granulocyte-macrophage colony stimulating factor,38 and tumor necrosis
factor.39,40 Thus, impaired expression of signaling
molecules may also contribute to delayed transmigration in neonates in vivo.
We found that P-selectin expression on endothelial cells in humans also
increased with maturation. However, there was not a complete absence of
P-selectin expression in term infants as in 1-day-old rats. These
findings are in accordance with previous reports of decreased
P-selectin expression on activated neonatal platelets compared with
adult platelets,41 with a particularly low P-selectin
expression on the surface of activated platelets from very low birth
weight infants.42 In the autopsy specimens used in this
study, P-selectin expression on mesenteric endothelial cells was first
detected by immunohistochemistry at approximately 27 weeks of
gestation. We were not able to determine if the amount of P-selectin
was decreased in term infants compared with adults. We attempted to
answer this question with endothelial cells obtained from newborn human
foreskin compared with human adult dermal endothelial cells. However,
these studies were not informative. We found that P-selectin expression
was highly dependent on the degree of confluence of the cells. There
was as much as a fourfold difference in the amount of P-selectin per
milligram of protein in endothelial cells, depending on the degree of
confluence (not shown). This made experiments using endothelial cells
grown in culture very difficult to interpret with regard to the amount
of P-selectin or adhesion and transmigration dependent on P-selectin.
However, although cell adhesion and transmigration assays are
frequently performed using HUVECs, neutrophils transmigrate across
endothelial cells lining postcapillary venules in the systemic circulation.43 Therefore, the immunohistochemical results
demonstrating decreased expression of P-selectin on endothelial cells
lining postcapillary venules in premature infants (Fig 3) may be more predictive of decreased adhesion and transmigration in vivo.
Our experiments also demonstrate that endothelial cell stores of
P-selectin are significantly diminished in preterm human neonates
compared with term neonates. This was demonstrated by ELISA (Fig 4A)
and by an RNAse protection assay (Fig 7) using HUVECs. A variable that
may have influenced the amount of P-selectin in these studies was the
administration of glucocorticoids to the mother. Although
glucocorticoids are reported to decrease the synthesis of E-selectin
and ICAM-1,44,45 they do not decrease the constitutive or
induced expression of P-selectin in human endothelial
cells.46 We found that exposure to betamethasone actually
increased endothelial cell P-selectin. These data indicate that the
levels of P-selectin measured in endothelial cells from preterm
neonates (Fig 4A) were not due to suppression by exogenous steroids and
may have been even lower if they had not been exposed to
glucocorticoids in utero.
Subsequent experiments were designed to determine the etiology of
decreased P-selectin expression in preterm neonates. Because P-selectin
is stored in Weibel-Palade bodies along with vWF, the amount of vWF
stored in preterm endothelial cells was measured and found to also be
diminished compared with term cells (Fig 4B). Therefore, we postulated
that there were fewer Weibel-Palade bodies in endothelial cells from
preterm infants. The decreased numbers of Weibel-Palade bodies in
endothelial cells was demonstrated by transmission electron microscopy
(Figs 5 and 6). These results are similar to those in a previous report
in which Kagawa and Fujimoto47 collected HUVECs from
artificially aborted fetuses of 12, 19, and 33 weeks of pregnancy and
from full-term deliveries. They found that the number and size of
Weibel-Palade bodies per HUVEC was decreased from 12 to 19 weeks of
gestation and then rapidly increased from 33 weeks to full term.
Without cell storage granules, the half life of P-selectin protein in
endothelial cells is likely to be short. Disdier et al48
found that, when a P-selectin cDNA was transfected into cells with
storage granules and capable of regulated secretion, it was
concentrated in storage granules, whereas the fate of P-selectin in
cells without storage granules is rapid degradation in
lysosomes.49 Green et al50 found that, when
P-selectin was transfected into neuroendocrine cells, the fraction of
P-selectin that was not targeted to secretory granules was rapidly
degraded in lysosomes. These studies indicate that decreased numbers of
Weibel-Palade bodies in endothelial cells in vessels of premature
infants may result in increased P-selectin degradation and consequent
diminished endothelial cell stores and surface expression of P-selectin
in response to inflammatory stimuli.
Many cell adhesion molecules, including E-selectin, ICAM-1, VCAM-1, and
PECAM-1, are synthesized in the early stages of
development.32,51,52 In contrast, the amount of P-selectin
message was diminished in preterm endothelial cells compared with cells
from term infants, identifying a second mechanism for decreased levels
of the protein. The decreased amount of P-selectin mRNA in preterm
infants could be the result of decreased transcription, because factors
reported to drive P-selectin transcription in humans, such as
IL-4,34 may be deficient in neonates.53 mRNA
stability has been reported to be altered in mononuclear leukocytes
from neonates.54 Therefore, the stability of mRNA for
P-selectin, which has a long half life in excess of 12 hours in term
HUVECs,34 may be altered in premature endothelial cells.
An alternative explanation for decreased detection of P-selectin in
preterm infants is that they may make an isoform of P-selectin not
detected by the MoAbs or RNAse protection assay described here.
Although the MoAbs we used to detect P-selectin have been reported to
recognize human P-selectin precursor proteins and all reported human
P-selectin isoforms,55,56 there remains the possibility of
an embryonic isoform of P-selectin synthesized by preterm neonates not
detected by these antibodies. However, the RNAse protection assay used
in these studies protects the entire lectin-like domain of P-selectin
that is necessary for adhesion.30 Therefore, an isoform of
P-selectin with an alteration in the transcript of this domain may not
be able to bind the P-selectin ligand.
Decreased P-selectin protein in endothelial cells from preterm infants
compared with term infants is associated with both decreased amount of
P-selectin mRNA and fewer P-selectin storage granules in endothelial
cells from preterm infants. Consistent with previous studies
demonstrating the importance of P-selectin in recruitment of
neutrophils into inflamed peritoneum,31,57 we demonstrate
that diminished expression of P-selectin in neonatal rats is associated
with delayed neutrophil transendothelial cell migration in vivo. The
relative deficiency of P-selectin in endothelium of preterm human
infants compared with term infants may contribute to the neutrophil
targeting defect and the increased susceptibility to infection with
increasing prematurity in humans. Increased susceptibility to systemic
infection with Streptococcus pneumoniae has been reported in
P-selectin-deficient mice.58 Additionally, P-selectin has
been reported to participate in various disease states, including some
that would affect premature infants, such as intestinal reperfusion
injury,26,59,60 a component of necrotizing enterocolitis,
and cerebral ischemia and reperfusion injury.61 Therapeutic
interventions are currently being designed to block adhesion to
P-selectin to prevent reperfusion injury mediated by
leukocytes.61-63 Given the relative deficiency of
P-selectin in neonatal animals and preterm neonatal humans, the
therapeutic targeting of P-selectin would unlikely be efficacious in
these patients.
| |
ACKNOWLEDGMENT |
The authors thank Donelle Benson, Nancy Chandler, and Deborah Dykstra
for excellent technical assistance; Diana Lim for preparation of the
figures; Ed Klatt and Cheryl Coffin for the autopsy tissue samples; and
Andy Weyrich, Guy Zimmerman, Tom McIntyre, and Steve Prescott for
thoughtful discussion. We also thank Rodger McEver for the contribution
of antibodies and P-selectin, Steven Albelda for the contribution of
antibodies to PECAM-1, and Jerry Roth for purified vWF.
| |
FOOTNOTES |
Submitted September 22, 1998; accepted March 12, 1999.
Supported by the Nora Eccles Harrison Foundation, a Special Center of
Research in Lung Injury Grant (P50HL50153) sponsored by the National
Institutes of Health, a Physician Scientist Award (HL-02726) from the
National Institutes of Health (D.E.L.), an Established Award (9640261N)
from the American Heart Association (D.E.L.), a Shared Instrumentation
Grant (K.H.A.) from the National Institutes of Health (S10-RR 10489),
and a Grant-In-Aid (K.H.A.) from the American Heart Association (96014370).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Diane E. Lorant, MD, Department of
Pediatrics, Indiana University, Methodist Hospital, 1701 N Senate Blvd,
Indianapolis, IN 46202.
| |
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TOP
ABSTRACT
INTRODUCTION
