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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3146-3152
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Respiratory Medicine, University of Leicester
School of Medicine, Glenfield Hospital, Leicester, UK.
Selective eosinophil accumulation is a hallmark of diseases such as
asthma. In a model of chronic eosinophilic inflammation, we have
previously shown that the tethering step in eosinophil adhesion is
mediated by PSGL-1 binding to P-selectin. The Th2-associated cytokine
IL-13 is of potential importance in allergic disease. We have therefore
investigated whether IL-13 can mediate eosinophil binding to human
umbilical vein endothelial cells (HUVEC) through P-selectin. IL-13
caused dose- and time-dependent increases of P-selectin expression, as
assessed by flow and laser scanning cytometry. A similar degree of
expression was observed with IL-4. There was no effect on E-selectin or
ICAM-1 expression. Tumor necrosis factor-
Eosinophils are important effector cells in a number of
allergic conditions, including asthma, allergic rhinitis, and eczema, and in related diseases such as nasal polyposis.1 A feature of these diseases is the selective accumulation of eosinophils, which
represent only a small proportion of the peripheral white blood cell
pool, without concomitant neutrophil influx. Understanding the
mechanisms of preferential eosinophil migration to tissue may provide
new targets for more selective therapeutic intervention for diseases
such as asthma without the risk of immunosuppression.2 Possible mechanisms for the selective eosinophil recruitment that have
been investigated are the adhesion pathways used by eosinophils but not
by neutrophils. The pathway that has received most attention in this
regard is VLA-4 binding to endothelial VCAM-1.3,4 VLA-4 is
not expressed by human neutrophils under physiological conditions, and
VCAM-1 expression is induced by the Th2 cytokines IL-4 and IL-13,
though expression is marginal unless tumor necrosis factor (TNF)- In resting human umbilical vein endothelial cells (HUVEC), P-selectin
is localized to intracellular Weibel-Palade bodies and is only
transiently expressed on the cell surface after stimulation with
secretagogues such as thrombin and histamine.13 This led to
the hypothesis that its principal role was to capture neutrophils during the immediate phase of cell recruitment.14 This was
supported by the observation that cytokines such as TNF- In addition to using flow cytometry to detect P-selectin expression by
HUVEC, we used laser-scanning cytometry (LSC) to overcome the problem
of having to trypsinize the cells. LSC represents a novel hybrid tool
for the cytometric analysis of cells that are found on a microscope
slide.22 It uses a 488-nm argon laser beam
directed through the optics of an Olympus microscope
(Melville, NY) and scans cells that are passed through this laser
spotlight by a high-precision stepper motor. Fluorescence-labeled cells are excited within the wavelength of the laser, and the emitted light
is transmitted back by the same optical path to photomultiplier tubes.
Because this allows for the scanning of even densely packed cells, we
postulated that this instrument would be ideally suited for the
analysis of adhesion receptor expression by confluent human endothelial
monolayers grown in chamber slides.
We therefore investigated using flow cytometry and a novel application
of LSC whether IL-13 could induce granular, luminal, and mRNA
P-selectin expression and whether the amount of surface expression
induced by IL-13 was sufficient to support eosinophil adhesion. We
found that IL-13, as well as IL-4, selectively induced P-selectin
expression at levels that were able to support eosinophil but not
neutrophil adhesion. VLA-4/VCAM-1 played a contributory role in
supporting adhesion by these cytokines. Thus, PSGL-1/P-selectin mediates an important pathway of selective eosinophil recruitment in
Th2-type inflammatory processes.
Reagents and antibodies
Cell culture
FACS analysis Confluent first-passage HUVEC were incubated for variable time periods in the presence of IL-4 (0, 0.2, 2, and 20 ng/mL), IL-13 (0, 0.5, 5, 5, and 50 ng/mL) or medium alone. Cells were harvested using trypsin (0.4%) and EDTA (0.02%; Life Technologies) and transferred to FACS analysis tubes for antibody labeling on ice. Cells were labeled for 10 minutes at optimized concentrations with primary and secondary antibodies, respectively, and washed with phosphate-buffered saline and 0.1% bovine serum albumin. Using a FACScan (Becton Dickinson) flow cytometer, HUVEC were gated using forward- and side-scatter settings that were optimized using EN4 anti-endothelial-specific fluorescence. Only viable cells (typically more than 95%), as determined by propidium iodide exclusion, were analyzed. Specific median fluorescence was determined after the subtraction of fluorescence for isotype-matched control antibody.Immunostaining for laser scanning cytometry After incubation for 48 hours in the presence of cytokines or medium alone and after stimulation with histamine 10 5 mol/L
(Sigma Aldrich) for 10 minutes, the monolayers were fixed with
paraformaldehyde 4% and washed twice. Cells were incubated for 60 minutes with first mAb ( -P-selectin or isotype-matched control
mAb). After 2 further washes, cells were incubated for 30 minutes with
Oregon green-conjugated rabbit--mouse second mAb (Molecular Probes
Europe BV, Leiden, The Netherlands), propidium iodide 20 µg/mL (Sigma
Aldrich), and pancreatic RNase A (200 ng/mL). To allow propidium iodide
to enter the cells, saponin 0.1% (Sigma Aldrich) was included during
the second antibody incubation step. For experiments in which granular
expression was assessed, saponin 0.1% was also included during the
first antibody incubation step. By including immunostaining against von
Willebrand factor (vWF) as a control von Willebrand factor is only
expressed within Weibel-Palade bodies of endothelial cells and has no
significant surface expression24it was assured that no
inadvertent intracellular staining of P-selectin was detected. Only
when saponin 0.1% was included with the first Ab staining buffer was
significant vWF staining seen. The microscope slide was finally
detached from the culture vessel, and a coverslip was applied with
glycerol. Immunostaining was performed on ice, and slides were kept in
the dark until analysis by LSC.
Laser scanning procedure Differentially treated areas on the microscope slide were scanned individually by using the × 20 objective and 5-mW laser output. The LSC captures each cell by software contouring around areas of fluorescence if they are sufficiently distinct from the surrounding background. Using nuclear staining with PI as the threshold parameter, single endothelial cells were identified by their nuclear fluorescence as detected by the photomultiplier tube in the red channel. Green surface fluorescence of each endothelial cell was sampled by defining a perimeter around the nucleus where green fluorescence was quantified. The dimensions of this perimeter were set by adding a defined number of pixels to the threshold contour so that overlap of adjacent cells was avoided. Specific mean fluorescence was determined within this perimeter after the subtraction of fluorescence for the isotype-matched control, and it was corrected for mean perimeter size. Using this correction, the specific mean fluorescence value per pixel (µm2) of cell surface area was established.Confocal microscopy Using a Leica (Heidelberg, Germany) confocal microscope, fluorescent images of the same monolayers were obtained.Duplex reverse transcription-polymerase chain reaction Semiquantitative analysis of P-selectin mRNA was performed using the primer dropping method as previously described.25 HUVEC total RNA was isolated by TRIzol reagent (Life Technologies). Total RNA (2 mg) was used for first-strand cDNA synthesis. Poly A+ RNA was selected using oligo-dT primers (Life Technologies) at 70°C for 10 minutes. Reverse transcription (RT) was performed at 37°C for 1 hour in 20 mL containing 0.2 mmol/L deoxynucleotide triphosphate, 10 mmol/L dithiothreitol, ribonuclease inhibitor RNasin (Promega, Southampton, UK), 1 × RT buffer, and Superscript RT (Life Technologies) overlaid with mineral oil, followed by 5 minutes at 95°C. Polymerase chain reaction (PCR) was performed using primers (Cruachem, Glasgow, UK) specific for P-selectin (5' primer: TGC TCA GAA CTA CAT GT; 3' primer: AGG ACT CGG GTC AAA TG) and GAPDH (5' primer: GGG AAG CTC ACT GGC ATG GCC TTC C; 3' primer: CAT GTG GGC CAT GAG GTC CAC CAC). The P-selectin primers were designed to coamplify both the soluble (260 bp) and the membrane-bound (380 bp) alternately spliced isoforms. Fifty-milliliter PCR reactions contained 25 pmol each primer, 0.2 mmol/L dNTP, 1 × PCR buffer, 1 U BioTaq DNA polymerase (Bioline, London, UK), template cDNA, and 1.5 mmol/L MgCl added as a "hot start" during the denaturation (94°C, 2 minutes). In duplex reactions, secondary primers were added at the appropriate cycle during the denaturation step as determined for each primer set in preliminary experiments. PCR reactions were resolved on an agarose gel, and scanned images of the bands were quantified using National Institutes of Health Image software for IBM-PC (Scion, Frederick, MD).Eosinophil and neutrophil purification Granulocytes were isolated from 100 mL blood from healthy donors by dextran sedimentation, slow centrifugation (200g) to remove platelets, and centrifugation on Histopaque 1083 (Sigma Aldrich) followed by negative immunomagnetic selection using CD16 Microbeads and the MACS system (Miltenyi Biotec, Bisley, UK) to purify eosinophils. Eosinophils and neutrophils had more than 98% purity as assessed by Kimura staining and more than 95% viability on trypan blue exclusion testing.Parallel flow chamber assay First-passage HUVEC were grown to confluence in 2-well chamber slides (Nunc) coated with 0.1% plasma fibronectin (Sigma Aldrich) and incubated in the presence of IL-4 (20 ng/mL) or IL-13 (5 ng/mL) for 48 hours. Using a parallel flow chamber (provided by M. Lawrence, University of Virginia), Perthése 0.5-mm silicone gasket (Osteotec, Christchurch, UK) and tubing-purified eosinophils or neutrophils were drawn over the monolayer by a Harvard apparatus 22-syringe pump (Edenbridge, UK). Flow rates of 1.5 mL/min (1.5 dyn/cm2) were used. Using inverted videomicroscopy (Zeiss Axiovert25, Jena, Germany), total cell accumulation was quantified by counting neutrophils and eosinophils in 15 to 20 video frames after a 2-minute interaction for each condition. For blocking experiments, eosinophils or HUVEC were preincubated for 10 minutes with specific mAb. Flow experiments were performed at room temperature unless otherwise stated.
IL-4 and IL-13 cause dose- and time-dependent increases of P-selectin expression on human endothelial cells that persist for at least 48 hours The expression of P-selectin on unstimulated HUVEC was either absent or barely detectable by flow cytometry. In contrast, both IL-4 and IL-13 induced a consistent, if modest, increase in expression of P-selectin on the surface of the endothelial cells (Figures 1A and 1D). This increase was readily apparent by 24 hours and was maintained up to at least 48 hours in culture (Figure 1B). The increased P-selectin expression in response to IL-13 was selectively inhibited by anti-IL-13 antibodies (data not shown). The increase was dose dependent (Figure 1C). The increase in P-selectin expression was similar in degree to the increase in VCAM-1 expression. No differences were observed between the effects of IL-4 and those of IL-13 (Figure 1D) on either P-selectin or VCAM-1 expression. Expression of E-selectin was not detectable on IL-4, IL-13, or unstimulated HUVEC. ICAM-1 was constitutively expressed by unstimulated HUVEC, and expression was unaffected by either IL-4 or IL-13. As previously described, TNF- induced the expression of
E-selectin, VCAM-1, and ICAM-1 but did not induce P-selectin
expression. TNF- or IL-1 , in combination with IL-4 or IL-13, had
no effect on P-selectin beyond that seen with IL-4 and IL-13 alone (all
data not shown).
Expression of mRNA for both soluble and surface P-selectin increases
in response to allergic cytokine stimulation
Eosinophils but not neutrophils adhere to IL-4- and
IL-13-stimulated human endothelial cells in flow predominantly
through P-selectin/PSGL-1
One of the novel findings of this study is that the Th2
cytokine IL-13 selectively induced chronic expression of P-selectin on
cultured endothelial cells, providing further evidence that this
receptor is important in mediating Th2-type inflammatory responses and
in particular eosinophil trafficking. Until recently, most attention
regarding selective pathways of eosinophil adhesion has concentrated on
the role of VLA-4 binding to VCAM-1 because this receptor is not
expressed by neutrophils. Furthermore, VCAM-1 is selectively induced by
IL-426 and IL-13.7 However, in the absence of
costimulation with other cytokines such as TNF- We wish to thank R. McEver (University of Oklahoma, Oklahoma City), R. Lobb (Biogen Inc, Cambridge, MA), F. Sanchez-Madrid (Servicio de
Immunologica, Madrid, Spain) and H. K. Nieuwenhuis (University Hospital
Utrecht, Utrecht, Netherlands) for their generous antibody gifts. We
are grateful to D. Ireland for her invaluable assistance with the
primer design. We also wish to thank the staff of the Department of
Obstetrics and Gynaecology of Leicester Royal Infirmary Hospital for
their assistance with the collection of umbilical cords.
Submitted June 3, 1999; accepted December 22, 1999.
Supported by the British Lung Foundation and the National
Asthma Campaign UK.
Reprints: Andrew J. Wardlaw, Department of Respiratory
Medicine, Glenfield Hospital, Groby Road, Leicester LE3 9QP, UK;
e-mail: aw24{at}le.ac.uk.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction
induced the
expression of VCAM-1, E-selectin, and ICAM-1 but had no effect on
P-selectin expression. IL-13 increased the production of mRNA for
surface and soluble variants of P-selectin. Under flow conditions,
eosinophils, but not neutrophils, showed enhanced binding to
IL-13 and to IL-4-stimulated HUVEC compared to
medium-cultured cells. Eosinophil adhesion was completely inhibited by
a blocking monoclonal antibody against PSGL-1 and P-selectin. Anti-VLA-4 and anti-VCAM-1 antibodies inhibited binding to a lesser extent. Thus, at physiologic levels of expression induced by Th2 cytokines, P-selectin/PSGL-1 supported eosinophil but not neutrophil adhesion. This mechanism is likely to be a key event leading to the
selective accumulation of eosinophils in allergic inflammation.
(Blood. 2000;95:3146-3152)
and lipopolysaccharide, which induce the expression of
other cell adhesion molecules by the up-regulation of rapid-response
genes, have no effect on P-selectin expression in humans, though
importantly these cytokines do increase P-selectin expression in
mice.
B transcription site,
