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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 600-609
By
From The Nora Eccles Harrison Cardiovascular Research and Training
Institute, Department of Pediatrics, University of Utah, Salt Lake
City, UT.
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.
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.
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.
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( 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.
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).
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.
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.
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).
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).
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.
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.
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.
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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Pediat |
TOP
ABSTRACT
INTRODUCTION
) 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.
-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies.
J Clin Invest
84:92, 1989