By
From the * Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Interleukin (IL)-8, a C-X-C chemokine, activates integrin-mediated adhesion of neutrophils.
Presentation of IL-8 on the endothelial cell surface may promote leukocyte extravasation. We
found that cultured human microvascular endothelial cells from the intestine (HIMEC) and
from nasal polyps (PMEC), but not human umbilical vein endothelial cells (HUVEC), contained IL-8 in intracellular granules that coexpressed von Willebrand factor (vWf ). This observation was corroborated by the immunohistochemical observation of double-positive granules
(IL-8+vWf+) in vessels of small and large intestine, nasal mucosa, and skin, whereas umbilical
cords revealed no endothelial IL-8. After treatment of HIMEC or PMEC with histamine or
thrombin, a dramatic increase in supernatant IL-8 concentration was observed within 3 min,
whereas no increase in IL-8 was detected in supernatants of identically treated HUVEC cultures. Histamine or thrombin treatment also caused IL-8-containing granules to rapidly disappear from HIMEC. In HUVEC, IL-8-containing granules were inducible by treatment with
recombinant human IL-1 Adhesion of neutrophils to endothelial cells (EC) is an
early, requisite event in the acute inflammatory response. Such binding of neutrophils can be rapid and transient, occurring within minutes under some conditions, or
developing over hours depending on the factors that incite
inflammation or tissue damage. According to the current
paradigm, these interactions involve a stepwise engagement of juxtaposed neutrophil- and EC-adhesion molecules, first
selectins and their counterreceptors, which mediate neutrophil tethering and rolling, then In fact, the entire process of inflammatory neutrophil recruitment can be emulated in vivo by injection of IL-8 into
the tissues (19). Moreover, neutrophil influx is severely
impaired after intraperitoneal injection of thioglycollate in
mice deficient in the IL-8 receptor homologue (23). However, it has been noted that soluble chemoattractant gradients cannot persist on the blood-EC interface; they are
likely to be washed away by the blood flow (24).
Therefore, the observation of in situ binding of IL-8 to EC
in human skin (27), and the enhanced ability of immobilized chemokines to attract and activate leukocytes in vitro
(28), led to the theory that chemokines bound to the EC
membrane may effectively promote leukocyte-EC adhesion in vivo. Furthermore, tissue-derived IL-8 can be transcytosed and presented on the surface of EC (29), but such
translocation of IL-8 requires its release or production in
the tissue and its subsequent transport to the EC surface.
Here we describe that IL-8 can also be released within
minutes from Weibel-Palade bodies in resting (i.e., not
previously exposed to proinflammatory reagents in vitro)
cultured human intestinal microvascular EC (HIMEC) and
nasal polyp-derived microvascular EC (PMEC) after stimulation with histamine or thrombin. Such storage of a
chemokine may be a novel principle in the biology of leukocyte adhesion that would serve to rapidly increase local
chemokine concentrations at the EC surface and enable a
higher level of specificity than that provided by classical
chemoattractants such as PAF.
Reagents
Cytokines and Cell Culture Reagents.
Surgical Department B, University of Oslo, The National Hospital,
Rikshospitalet, N-0027 Oslo, Norway
Abstract
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
for 24 h; additional histamine treatment doubled IL-8 secretion
from HUVEC in the same rapid manner observed for mucosal EC. These data suggested that
IL-8 prestored in microvascular endothelial cells may provide a rapid pathway for specific activation of neutrophil adhesion at sites of acute inflammation.
Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
2-integrins and immunoglobulin-like intercellular adhesion molecules, which mediate
firm neutrophil adhesion (1). The most rapid recruitment
of neutrophils may be initiated by P-selectin, a transmembrane glycoprotein constitutively present in the Weibel-Palade bodies of EC (4, 5); this selectin is translocated within
minutes to the cell surface membrane after stimulation with a
number of secretagogues, including thrombin or histamine
(6). On the EC surface, P-selectin supports the initial rolling
of neutrophils both in vitro (7) and in vivo (8, 9). However,
this rolling is of a transient nature, and firm adhesion through
integrin binding requires neutrophil activation through G
i-linked receptors (7, 10, 11). Such activation can be accomplished by broadly acting leukocyte activators (3, 12) such as
platelet-activating factor (PAF [13, 14]) or by activators that
are more specific for neutrophils, such as the chemotactic
cytokine (chemokine) IL-8 (15).
Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and rh basic fibroblast growth factor were obtained
from R&D Systems Ltd. (Abingdon, UK). MCDB 131, cycloheximide, dibutyryl cAMP, histamine, thrombin, epidermal growth factor, and hydrocortisone were from Sigma Chemical
Co. (St. Louis, MO), and Endothelial-SFM, FCS, and gentamicin from Life Technologies Ltd. (Paisley, UK).
Primary Antibodies and Secondary Conjugates.
mAbs to human IL-8 (clone NAP-1, IgG1; and clone LS04/2A2, IgG1) were gifts from Dr. C. Svanborg (University of Lund, Lund, Sweden) and Dr. C. Mackay (LeukoSite, Inc., Cambridge, MA), respectively; rabbit anti-human von Willebrand factor (vWf, IgG fraction) and FITC- labeled swine anti-rabbit IgG conjugate were purchased from Dakopatts A/S (Glostrup, Denmark); biotinylated horse anti-mouse IgG was from Vector Laboratories, Inc. (Burlingame, CA), and streptavidin-Texas Red conjugate from GIBCO BRL (Gaithersburg, MD).Cell Culture
HIMEC and PMEC were isolated from segments of small intestine or from nasal polyps and cultured as described (30, 31). In brief, cells were dispersed from minced tissue by collagenase/dispase treatment, plated, and grown to confluence in Endothelial- SFM containing 2.5% FCS, 1 µg/ml hydrocortisone, 0.5 mM dibutyryl cAMP, 50 µg/ml gentamicin, and 0.25 µg/ml amphotericin-B. EC were selected from primary cultures by using paramagnetic beads armed either with mAb to CD31 (positive selection) or with mAb to CD44 (negative selection [31]). HIMEC and PMEC were subsequently maintained in MCDB 131 as described for HUVEC (see below).
HUVEC were isolated as described by Jaffe et al. (32) and cultured in MCDB 131 containing 7.5% FCS, 10 ng/ml epidermal growth factor, 1 µg/ml hydrocortisone, 50 µg/ml gentamicin, and 0.25 µg/ml amphotericin-B. All cultures were used at passage levels 1-8, were uniformly positive for vWf, and contained <1% contaminating cell types determined as described elsewhere (30, 31).
ELISA Experiments
HIMEC, PMEC, and HUVEC were grown to confluence in
96-well trays (Becton Dickinson, San Jose, CA). At the start of
the experiment, resting or IL-1-activated (100 U/ml, 24 h) EC
monolayers were washed twice. Cells were subsequently incubated at 37°C in culture medium with or without histamine (0.1 mM) or thrombin (3 U/ml). Cell culture supernatant fluids were
analyzed for IL-8 with an ELISA kit according to the recommendations of the manufacturer (R&D Systems Ltd.). In our hands,
this assay provided an analytical sensitivity of 51 pg/ml.
Immunocyto/histochemistry
Confluent monolayers of EC grown on LabTek chamber slides
(Nunc, Inc., Roskilde, Denmark) were fixed (10 min, pH = 7.4)
in 0.5% periodate-lysine-paraformaldehyde (33) or in 4% paraformaldehyde (PFA). Tissue specimens from histologically normal
small (n = 2) and large (n = 2) intestine, skin (n = 2), nasal mucosa (n = 3), and umbilical cord (n = 1) were snap frozen and
stored at 70°C. Cryosections were cut at 4 µm and fixed in 4%
PFA for 5 min at 23°C. All subsequent incubation steps except
the last washing step were performed with 0.1% saponin for permeabilization. Cell monolayers or tissue sections were first incubated with mAb to IL-8 (10 µg/ml) for 1 h or overnight, respectively; then with rabbit IgG anti-human vWf (1:1,400) combined
with biotinylated horse anti-mouse IgG (1:250) for 1 h; and finally with streptavidin-Texas red conjugate (1:200) combined
with FITC-labeled swine anti-rabbit IgG (1:50) for 0.5 h. Negative controls were incubated with irrelevant isotype- and concentration-matched primary antibodies. All fixations and incubations
took place at room temperature.
The immunostained slides were examined and analyzed with a confocal laser scanning microscope (MRC 600; Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) or a fluorescence microscope (model E800; Nikon, Tokyo, Japan) equipped with a cooled CCD camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan).
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Results |
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In contrast to HUVEC, unstimulated HIMEC (Fig. 1, a, c, and e) and PMEC (data not shown) contained IL-8 in intracellular vesicle-like granules as revealed by staining with mAbs LS04/2A2 and NAP-1. Staining of nonpermeabilized monolayers revealed no reactivity (data not shown). The observed subcellular localization of IL-8 was similar to that described for vWf (34) and P-selectin (4, 5), which both are stored in Weibel-Palade bodies. Therefore, we performed paired immunostaining with an antibody to vWf (Fig. 1, b, d, and e) and found coexpression of the two proteins in most granules. In addition, mAb NAP-1 revealed perinuclear IL-8 reactivity compatible with its presence in the Golgi apparatus (data not shown), as also reported for other cytokines (35), but this staining pattern was not observed with mAb LS04/2A2.
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We observed a higher constitutive secretion of IL-8 into the culture supernatant in unstimulated HIMEC and PMEC than in HUVEC (Fig. 2, and data not shown). However, treatment of HIMEC (Fig. 2) and PMEC (data not shown) monolayers with histamine (0.1 mM) or thrombin (3 U/ml, data not shown) more than doubled the level of IL-8 over unstimulated cultures within 5 min (Fig. 2). Consistent with the lack of detectable granule-associated IL-8 in HUVEC, these agents had no effect on IL-8 levels (Fig. 2). To exclude enhanced protein synthesis as a possible explanation for the increased levels of IL-8 in the supernatant, HIMEC and HUVEC were incubated with the protein synthesis inhibitor cycloheximide (1 mg/ml) for 24 h before histamine stimulation. Despite further reduction of the low level of constitutive IL-8 secretion observed in both cell types, cycloheximide had no effect on the amount of IL-8 released in response to histamine (Fig. 3).
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Immunocytochemical staining of resting and histamine-stimulated HIMEC (Fig. 4) revealed that the observed increase of secreted IL-8 was accompanied by disappearance of IL-8+ granules, most apparent after 15 min (Fig. 4 b). Double staining also revealed a reduction of vWf+ granules after histamine stimulation (data not shown).
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This disparity between small vessel-derived EC and HUVEC observed in vitro was substantiated by immunohistochemical detection of IL-8 in small vessels of small intestine (Fig. 5, a and c) but not in EC of the umbilical vein (data not shown). The IL-8 staining of these vessels was observed in granules that costained for vWf (Fig. 5, b and c). Small vessels of the normal skin, nasal mucosa, and large intestine also contained IL-8+ granules (data not shown).
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After activation with rhIL-1 (100 U/ml) for 24 h, IL-8+ granules coexpressing vWf appeared in HUVEC (data not shown),
and strongly elevated IL-8 secretion levels were observed
for both HIMEC and HUVEC after such treatment (Fig.
6). In HIMEC, IL-1 treatment enhanced the staining intensity of IL-8+ granules (data not shown). We next tested
if histamine would effect the release of IL-8 from cytokine-activated EC and found almost doubling of the level of released IL-8 in both cell types within 15 min (Fig. 6).
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Discussion |
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This study reports that the chemokine IL-8 is stored in
resting microvascular EC and can be rapidly released to the
luminal EC surface after activation by secretagogues such as
histamine and thrombin. This availability may play a crucial
role in providing rapid recruitment of neutrophils because
prestored P-selectin, which is also made available at the endothelial surface after such activation (6), merely permits
transient rolling of neutrophils (7). Subsequent firm adhesion appears to require the activation of 2-integrins,
which can be achieved by stimulation of leukocytes with
broadly acting classical chemoattractants or with leukocyte type-specific chemokines affording a higher level of specificity (7, 10, 11). The classical chemoattractant PAF is the
only known adhesion-activating factor that can be made
available at the endothelial surface within minutes of EC
stimulation (18). However, PAF activates several types of
leukocytes that can both roll on P-selectin and adhere
firmly to integrin counterreceptors on EC (3, 12, 14). On
the other hand, the ability of IL-8 to activate integrin-
mediated adhesion appears restricted to neutrophils, and its
rapid secretion from previously resting microvascular EC as
described here may provide a higher level of specificity in
the earliest phases of neutrophil recruitment.
Our data convincingly show that human microvascular
EC in intestinal and airway mucosa, as well as in skin, contain IL-8 in intracellular granules that also contain vWf,
clearly indicating that these granules are Weibel-Palade
bodies (34). Indeed, immunoelectron microscopy of IL-1-
activated HUVEC revealed IL-8 in the Weibel-Palade bodies (36), thus supporting our conclusion. Moreover,
histamine or thrombin induced a rapid release of IL-8 from
HIMEC and PMEC, in a time course virtually identical to
that described for translocation of P-selectin to the EC surface (6), whereas resting HUVEC failed to release IL-8
above background levels. The observed level of IL-8 released to the culture supernatant probably underestimated the true concentration obtainable in the microenvironment
between the surface membranes of EC and adhering leukocytes. First, EC in vivo possess binding sites for IL-8 (24)
that are grossly lost upon culture of HIMEC (our unpublished data) or EC from other tissue sources (37, 38).
Second, the recently described presentation of transcytosed
IL-8 on EC microvilli (29), paralleling that of 4
7 and
L-selectin on leukocytes (39, 40), raises the interesting
question as to whether prestored endothelial IL-8 may be
presented in the same manner, thereby enhancing the
availability of IL-8 to its leukocyte receptors. On the other
hand, in vitro studies have suggested that as few as 300 molecules of IL-8 per µm2 are sufficient for activation of
adhesion to occur (Campbell, J.J., personal communication).
The constitutive secretion of IL-8 measured at various
time points also indicated a difference in the resting state
between microvascular EC and HUVEC, possibly pointing
to a fundamental biological difference between small and
large vessels. However, after activation with rhIL-1, both
HUVEC and HIMEC secreted elevated levels of IL-8; in
this case, IL-8+ vesicles appeared in HUVEC, as also described by Wolff et al. (36). The biological significance of
the latter finding is uncertain, but it may reflect a common
sorting signal in the IL-8 sequence, whereas the absence of
IL-8 inside unstimulated HUVEC might merely reflect the
low constitutive level of IL-8 synthesis. To this end, it is
interesting to note that the microvascular EC described in
this study all derive from organs or tissues that border the external environment and may thus experience chronic inflammatory stimulation, possibly resulting in a higher level
of constitutive IL-8 expression. The molecular mechanisms
that allow such constitutive differences to persist over
weeks in vitro remain to be elucidated.
In conclusion, we describe a novel mechanism by which IL-8 can be made available at the EC surface within minutes of activation. Such presentation may provide a rapid pathway for specific activation of neutrophil adhesion at sites of acute inflammation.
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Footnotes |
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Address correspondence to Guttorm Haraldsen at his current address, Veterans Administration Medical Center (154B), Bldg. 101, Rm. C4-101, 3801 Miranda Ave., Palo Alto, CA 94304. Phone: 650-493-5000 ext. 63133; Fax: 650-858-3986; E-mail: guttormh{at}cmgm.stanford.edu
Received for publication 23 March 1998 and in revised form 22 July 1998.
We thank Drs. Eugene C. Butcher, James J. Campbell, and Finn-Eirik Johansen for helpful discussions, Drs. C. Svanborg and C. Mackay for providing mAbs, and Dr. Henrik Huitfeldt for assistance with the confocal microscope. Drs. Edward P. Bowman and Michael Delay kindly commented on the manuscript, and Ms. Gunn Jamne, Ms. Liv Mangschau, and Ms. Inger Johanne Ryen are gratefully acknowledged for their expert technical assistance. We are also grateful to the staffs at the Maternity Unit, Department of Gynecology, and at Surgical Department B, Rikshospitalet, for their kind assistance in obtaining tissue specimens.
This study was supported by the Norwegian Cancer Society, the Research Council of Norway, and Anders Jahre's Fund. G. Haraldsen and F.L. Jahnsen are Postdoctoral Fellows of the Norwegian Cancer Society and the Research Council of Norway, respectively.
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