1 Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3GE, United Kingdom; 2 Department of Environmental Health Sciences, School of Hygiene and Public Health and 3 Division of Pulmonary and Critical Care Medicine, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205; and 4 Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland
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ABSTRACT |
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In vivo,
eosinophils localize to airway cholinergic nerves in antigen-challenged
animals, and inhibition of this localization prevents antigen-induced
hyperreactivity. In this study, the mechanism of eosinophil
localization to nerves was investigated by examining adhesion molecule
expression by cholinergic nerves. Immunohistochemical and functional
studies demonstrated that primary cultures of parasympathetic nerves
express vascular cell adhesion molecule-1 (VCAM-1) and after cytokine
pretreatment with tumor necrosis factor- and interferon-
intercellular adhesion molecule-1 (ICAM-1). Eosinophils adhere to these
parasympathetic neurones after cytokine pretreatment via a
CD11/18-dependent pathway. Immunohistochemistry and Western blotting
showed that a human cholinergic nerve cell line (IMR-32) expressed
VCAM-1 and ICAM-1. Inhibitory experiments using monoclonal blocking
antibodies to ICAM-1, VCAM-1, or CD11/18 and with the very late
antigen-4 peptide inhibitor ZD-7349 showed that eosinophils adhered to
IMR-32 cells via these adhesion molecules. The protein kinase C
signaling pathway is involved in this process as a specific inhibitor-attenuated adhesion. Eosinophil adhesion to IMR-32 cells was
associated with the release of eosinophil peroxidase and leukotriene C4. Thus eosinophils adhere to cholinergic nerves via
specific adhesion molecules, and this leads to eosinophil activation
and degranulation; this may be part of the mechanism of
eosinophil-induced vagal hyperreactivity.
hyperreactivity; neural inflammation; muscarinic receptors
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INTRODUCTION |
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IN THE LUNGS, pulmonary parasympathetic nerves in the vagus provide the dominant innervation of airway smooth muscle (17). Stimulation of these nerves releases ACh, which binds to M3 muscarinic receptors on the airway smooth muscle, causing its contraction. Control over the release of ACh is mediated by neuronal M2 muscarinic receptors located on the parasympathetic nerves (12). In antigen-challenged animals and in some humans with asthma, M2 muscarinic receptors are dysfunctional, leading to vagally mediated hyperreactivity (13).
In vivo studies in antigen-challenged animals have shown that M2 muscarinic receptor dysfunction is prevented by inhibiting eosinophil localization to the airways (3, 7, 9). The eosinophil protein major basic protein (MBP) is an antagonist at M2 muscarinic receptors in vitro (15); neutralizing MBP prevents both M2 receptor dysfunction and antigen-induced hyperreactivity in vivo (8). Furthermore, eosinophils and extracellular MBP are found closely associated with airway parasympathetic nerves in antigen-challenged guinea pigs and rats as well as in humans with asthma (4). Thus it is likely that the localization of eosinophils to airway nerves and the subsequent release of MBP on M2 muscarinic receptors are mechanisms for vagally mediated hyperreactivity.
Inflammatory cells, such as eosinophils, selectively localize to specific sites within inflamed tissue through interactions between adhesion molecules on their surface and counterligands on tissue structures (21). Eosinophils express the integrin adhesion molecules CD11/18 complex and very late antigen-4 (VLA-4), which interact with intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), respectively (23). It is also recognized that the process of adhesion to some integrin adhesion molecules is important for cell activation (18). One mechanism whereby eosinophils localize to and subsequently release MBP on M2 muscarinic receptors may be that parasympathetic nerves express adhesion molecules recognized by eosinophils. The aim of this study, therefore, was to test the hypothesis that cholinergic nerves express adhesion molecules to which eosinophils adhere and that this subsequently leads to eosinophil degranulation.
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MATERIALS AND METHODS |
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Materials.
Unless otherwise stated, all chemicals were purchased from
Sigma-Aldrich (Poole, UK) or from BDH-Merck (Poole, UK). The IMR-32 cell line was purchased from ECACC (Salisbury, UK). DMEM, normal goat
serum, and FCS were purchased from GIBCO-BRL (Paisley, UK). Medium 199 was obtained from GIBCO-BRL (Rockville, MD). All cell culture plastic
materials were from Becton-Dickinson Labware (Oxford, UK). Mouse
anti-rat ICAM-1 (IgG1-, clone 1A29) and mouse anti-rat CD11/18 (IgG2a1-
, clone WT.1) were purchased from
Pharmingen. The anti-rat VCAM-1 antibody (IgG1, clone 5F10)
was a gift from Biogen (Cambridge, MA). Goat polyclonal anti-human
ICAM-1 (sc-1510), goat polyclonal anti-human VCAM-1 (sc-1504), and
affinity-purified mouse and goat IgG1 were obtained from
Autogen Bioclear (Calne, UK). Nitrocellulose paper,
[51Cr]sodium chromate, and Percoll were obtained from
Amersham Pharmacia Biotech (Little Chalfont, UK). Chemiluminescent
substrate stable peroxidase solution and chemiluminescent substrate
luminol/enhancer solution were obtained from Pierce (Rockford, IL). The
biotinylated mouse anti-goat and horse anti-mouse secondary antibodies,
avidin-biotinylated horseradish peroxidase complex, chromagen Slate
Gray (SG) peroxidase substrate, Novo Red, and 3-amino-9-ethylcarbazole
(AEC) staining kits were purchased from Vector Laboratories
(Peterborough, UK). CD16 MicroBeads and MACS VS+ columns were obtained
from Miltenyi Biotech (Bisley, UK). Tumor necrosis factor-
(TNF-
), interferon-
(IFN-
), and interleukin-1
(IL-1
)
were purchased from PeproTech (London, UK). The cyclic peptide VLA-4
inhibitor (ZD-7349),
cyclo-(MePhe-Leu-Asp-Val-D-Arg-D-Arg) acetate
salt, an equipotent nonbasic VLA-4 peptide inhibitor
cyclo-(MePhe-Leu-Asp-Val-D-Ala-D-Ala), and an
inactive control cyclic peptide cyclo-(Ile-Leu-Asp-Val-
-Ala) were
generously provided by Dr. Duncan Haworth (Astra Zeneca
Pharmaceuticals, Macclesfield, UK). The neutralizing anti-human CD11/18
monoclonal antibody (Med Cla285) was obtained from Accurate Scientific
Chemicals. The neutralizing mouse monoclonal anti-human antibody to
ICAM-1 (RR1/1.1.1) was a kind gift from Dr. Robert Rothlein (Boehringer Ingelheim Pharmaceuticals). The neutralizing monoclonal anti-human VCAM-1 antibody (B-K9, isotype IgG1) was obtained from Lab
Vision. The protein kinase C (PKC) inhibitor rottlerin was purchased
from Affiniti Research Products (Mamhead, UK). Peroxidase-conjugated immunoglobulin standard was obtained from Dako (High Wycombe, UK). The
leukotreine C4 (LTC4) EIA kit came from Cayman
Chemicals (Ann Arbor, MI).
Culture of tracheal parasympathetic nerves. Two different species of animal were used because of the current availability of appropriate antibodies. Specific pathogen-free female Dunkin-Hartley guinea pigs and Sprague-Dawley rats (both 180-200 g) were housed in high-efficiency particulate-filtered air and were fed a normal diet. Animals were handled in accordance with the standards established by the United States Animal Welfare Acts set forth in the National Institutes of Health guidelines and the Policy and Procedures manual published by the Johns Hopkins University School of Hygiene and Public Health Animal Care and Use Committee. The animals were killed with a lethal intraperitoneal dose of pentobarbital sodium. The technique of cell culture was essentially the same for the two species, and the methods have been described previously (10). Briefly, the trachealis muscle was excised, cut into small sections, and suspended in 0.2% (wt/vol) collagenase solution at 4°C overnight. The pellet was resuspended in DMEM containing 10% (vol/vol) FCS and penicillin (100 U/ml), plated on a petri dish, and incubated overnight at 37°C in 5% CO2-95% air. Nonadherent cells were collected, resuspended in serum-free medium, and plated on poly-L-lysine- and Matrigel-coated four-well glass Lab-Tek chamber slides. After 24 h, the medium was replaced with serum-free medium containing 5 µM cytosine arabinoside. In prior studies, it had been shown that after 7 days these cells have extensive neurite outgrowth and demonstrate a cholinergic phenotype (10).
Culture of IMR-32 human neuroblastoma cells. The human cholinergic neuroblastoma cell line IMR-32 was cultured in 25-ml flasks in DMEM with 10% (vol/vol) FCS, 100 U/ml (wt/vol) penicillin, 0.1 mg/ml (wt/vol) streptomycin, and 10 µg/ml (wt/vol) gentamicin at 37°C in 5% CO2-95% air. Confluent, adherent cells were removed by application of medium, centrifuged at 100 g for 10 min, and resuspended in fresh medium. IMR-32 cells were plated on 48-well flat-bottom plates at a density of 2 × 104 cells/well in 400 µl of the above medium. After 24 h, the medium was removed and replaced with serum-free medium containing 1 mM dibutyryl-cAMP (DBcAMP). They were then incubated for 7 days to allow differentiation and neurite outgrowth. In preliminary studies, it was shown that these cells develop neurites and develop a cholinergic phenotype including expression of M2 muscarinic receptors, hemicholinium-dependent uptake of choline, and release of ACh in response to chemical and electrical stimuli.
Immunohistochemical detection of the adhesion molecules VCAM-1
and ICAM-1 on tracheal parasympathetic nerves.
Primary cultures of tracheal parasympathetic nerves were cultured on
four-well Lab-Tek chamber slides as described above for 7 days. In some
cases, cells were incubated with the cytokines TNF- (2 ng/ml) and
IFN-
(1,000 U/ml) for 48 h (days 5-7). The cells were fixed in methanol-acetone (50:50 vol/vol) for 5 min, washed
three times with PBS, and preincubated for 30 min with 5% (vol/vol)
mouse serum. The rat tracheal cells were incubated with either mouse
anti-rat VCAM-1 antibody (mAb5F10) or an IgG1 mouse
antibody (both at 1:50 for 30 min), whereas the guinea pigs were
pretreated with a mouse anti-rat ICAM-1 antibody or an IgG1 mouse antibody (both at 1:100 for 2 h). After a wash with PBS, the
cells were incubated for 30 min at room temperature with a biotinylated
horse anti-mouse secondary antibody added for 30 min. The cells were
further washed with PBS before the addition of the avidin-biotinylated
horseradish peroxidase complex (ABC Elite) for 30 min. Immunoreactivity
was detected with a peroxidase-dependent chromagen, either SG or AEC.
Isolation of guinea pig eosinophils. Female Dunkin-Hartley guinea pigs (0.75-1 kg) were pretreated with a solution of 1% horse serum weekly for a period of 3 wk to induce peritoneal eosinophilia. After the last treatment (24 h), animals were anesthetized with xylazine (10 mg/kg im) and ketamine (40 mg/kg im). A cannula was inserted in the peritoneal cavity, and lavage with sterile PBS was performed. The eosinophils were purified by centrifugation through a Percoll gradient (specific gravity 1,090 g/ml), and any red blood cells were removed by hypotonic lysis. Cells were used if this technique yielded a population of eosinophils >90% pure and >95% viable.
Coculture of guinea pig eosinophils and tracheal parasympathetic nerves. Eosinophils were then coincubated with the cytokine-stimulated parasympathetic cholinergic nerves for 30 min (5 × 104 eosinophils/well). In some experiments the eosinophils were also incubated with a mouse monoclonal anti-rat antibody to CD11/18 (1:1,000 dilution). Nonadherent eosinophils were removed by washing the cocultured cells three times with medium 199. The cells were then fixed with 3.7% (wt/vol) formaldehyde, and the eosinophils were detected using their endogenous peroxidase to catalyze a reaction, which in the presence of exogenous hydrogen peroxide yielded the red chromagen Vector Novo Red. Nerves were counterstained with dilute hematoxylin.
Immunohistochemical detection of the adhesion molecules VCAM-1 and ICAM-1 on IMR-32 cells. IMR-32 cells cultured on four-well Lab-Tek chamber slides were fixed in 4% (wt/vol) paraformaldehyde and incubated in 3% (vol/vol) hydrogen peroxide for 10 min to quench endogenous peroxidase activity. The cells were then incubated in 5% goat serum (wt/vol) for 30 min at room temperature and then with a 1:100 dilution of goat polyclonal anti-human ICAM-1 antibody for 1 h at room temperature, goat polyclonal anti-human VCAM-1 antibody (1:50 dilution for 30 min at room temperature), or a goat IgG1 at 1:50 dilution. After a wash with PBS, the cells were incubated for 30 min at room temperature with a biotinylated mouse anti-goat secondary antibody added for 30 min. The cells were washed further with PBS before the addition of the avidin-biotinylated horseradish peroxidase complex (ABC Elite) for 30 min. Immunoreactivity was detected with a peroxidase-dependent chromagen, either SG or AEC.
Gel electrophoresis and Western blotting to detect the adhesion molecules VCAM-1 and ICAM-1 on IMR-32 cells. IMR-32 cells were lysed with Tris-buffered saline (TBS) containing 10 mM EDTA, 0.5% (vol/vol) Nonidet, 0.5% (wt/vol) deoxycholate, 8 M urea, 2% (wt/vol) SDS, and 1% (wt/vol) bromphenol blue (pH 7.1) at a concentration of 2 × 108 cells/ml. Samples were boiled for 5 min, and 2-mercaptoethanol was added to a final concentration of 5% (vol/vol). The samples were then separated on 8% SDS-polyacrylamide gels with a 4% polyacrylamide stacking gel, and the proteins were transferred to nitrocellulose paper, as described previously (20). The resulting Western blots were incubated with 5% (wt/vol) BSA for 1 h and then incubated overnight at 4°C with a 1:1,000 dilution of goat polyclonal anti-human ICAM-1 or VCAM-1 antibody. Controls, to which no antibody was added, were maintained in 5% (wt/vol) BSA. Blots were then washed three times with 0.05% Tween 20 in TBS and incubated with 1:10,000 dilution of peroxidase-conjugated sheep anti-goat immunoglobulin for 2 h at ambient temperature. After further washing, the chemiluminescent substrate stable peroxidase and substrate luminol/enhancer solutions were added. Blots were then applied to Kodak X-Omat film, and immunoreactive proteins were visualized on the developed film.
Isolation of human eosinophils. Eosinophils were isolated with the VarioMACS magnetic cell separation system (MACS; Miltenyi Biotech). Peripheral venous blood (60 ml), donated from healthy volunteers, was suspended in an equal volume of PBS containing 1,000 U/ml heparin sulfate, layered over 12 ml of Percoll solution (specific gravity 1.090 g/ml), and centrifuged at 400 g for 20 min at room temperature. The resulting supernatant and monocyte layer were carefully aspirated and discarded, and red blood cells were removed by hypotonic water lysis. The remaining granulocytes were washed in 10% (wt/vol) PIPES (pH 7.4), 1% (wt/vol) glucose, and 0.0003% (wt/vol) human serum albumin. The cells were then coincubated with anti-human CD16 microbeads (1 µl/106 cells) and an equal volume of MACS buffer [0.5% (wt/vol) BSA and 2 mM EDTA in PBS] at 6°C for 45 min. The eosinophils were then separated from contaminating neutrophils by passing the solution through the VarioMACS magnetic separation apparatus. Cell viability was determined by Trypan blue exclusion, and eosinophil purity was determined by Diffquick staining. Only populations of eosinophils that were >98% pure and >95% viable were used.
Eosinophil adhesion assay.
Freshly isolated eosinophils, suspended in IMR-32 culture medium (see
above), were incubated with [51Cr]sodium chromate (37 kBq/ml; sp act >9.25 GBq/mg chromium) for 1 h at 37°C. The
eosinophils were then washed three times with culture medium.
Eosinophils (5 × 104/well) were then coincubated on
48-well plates with differentiated IMR-32 cells (2 × 104 cells/well) at 37°C for 30 min. In some experiments,
the eosinophils were pretreated with antibodies to CD11/18 at 4°C or
with the VLA-4 inhibitor ZD-7349 with an equipotent concentration of
the nonbasic VLA-4 peptide inhibitor
cyclo-(MePhe-Leu-Asp-Val-D-Ala-D-Ala), with the
inactive control cyclic peptide cyclo-(Ile-Leu-Asp-Val--Ala), or
with the specific PKC inhibitor rottlerin at 37°C for 30 min. Alternatively, the differentiated IMR-32 cells were pretreated with
antibodies to VCAM-1 or ICAM-1 for 30 min or with TNF-
and/or IL-1
for 24 h at 37°C (see RESULTS for drug
concentrations and antibody dilutions). In some experiments after
coincubation with IMR-32 cells, eosinophils were stained with the
Vector AEC stain for 5 min followed by three washes with medium and
were viewed by light microscopy.
Assessment of the activation and degranulation of human
eosinophils.
The release of eosinophil peroxidase (EPO) and LTC4 was
used as an index of degranulation and activation, respectively. After incubation with [51Cr]sodium chromate for simultaneous
cell adhesion assays (see above), isolated eosinophils were resuspended
in IMR-32 culture medium and 5 × 104 eosinophils
incubated with 2 × 104 IMR-32 cells, which had been
differentiated for 7 days with DBcAMP on 48-well culture plates.
Eosinophils were also cultured alone (5 × 104
cells/well) to determine background levels of degranulation. Aliquots
of medium were then removed from the wells at various time periods
after eosinophil adhesion, and degranulation was terminated by
centrifugation of the aliquots at 5,400 g for 5 min. The
supernatants were stored at 80°C until assayed.
Statistics. All values are shown as means ± SE from the number of experiments indicated. The effects of agents inducing or inhibiting eosinophil adhesion or degranulation were compared with control values using ANOVA with Dunnett's post hoc test.
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RESULTS |
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Experiments in primary cultures of parasympathetic nerve cells.
Immunohistochemical staining of rat tracheal nerve cells demonstrated
that VCAM-1 was expressed by these cells (Fig.
1A), and this expression was
not altered by pretreatment with the cytokines TNF- and IFN-
(Fig. 1B); no staining was detected when an isotype control
antibody was used (Fig. 1C). In contrast, staining of untreated cultured guinea pig nerve cells did not reveal evidence of
expression of ICAM-1 (Fig.
2A). However, when these nerve
cells were preincubated with the cytokines TNF-
and IFN-
,
immunostaining of ICAM-1 (Fig. 2B) was detected on the cell
bodies and along the neurites. In contrast, there was no staining of
nerve cells incubated in the presence of cytokines when an
isotype-matched mouse IgG1 antibody was used in place of
the anti-ICAM-1 antibody (Fig. 2C). Coincubation of guinea
pig eosinophils with primary cultures of parasympathetic nerves
revealed that eosinophils only adhered to these cells after
preincubation with the cytokines TNF-
and IFN-
(compare untreated
neurones in Fig. 2E with cytokine-pretreated neurones Fig.
2D). Eosinophil adhesion was attenuated by pretreatment with
an antibody to eosinophil CD11/18 (Fig. 2F).
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Expression of VCAM-1 and ICAM-1 by IMR-32 cells.
Immunohistochemical staining of IMR-32 neuroblastoma cells demonstrated
the expression of both VCAM-1 (Fig.
3A) and ICAM-1 (Fig.
3C) on the cell bodies, and along the neurites of these cells no staining was apparent when an isotype-matched antibody was
used. The expression of these adhesion molecules was detected in the
absence of cytokine pretreatment. Western blotting studies also
demonstrated that IMR-32 cells constitutively expressed both VCAM-1 and
ICAM-1 cell adhesion molecules (Fig. 4).
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Adhesion of eosinophils to cholinergic cells in culture: dependence
on cell adhesion molecules.
When freshly isolated eosinophils were cocultured with IMR-32 cells,
eosinophils adhered to the cells even after extensive washing to remove
nonadherent cells (Fig. 5A).
This adhesion was prevented when the experiments were performed on ice
or in the presence of 10 mM EDTA (results not shown). The number of
eosinophils that adhered to IMR-32 cells depended on the duration of
incubation. Because adhesion was well established, but not maximal,
after 30 min, this time point was used for subsequent studies to
investigate the factors that inhibited or augmented adhesion (Fig.
5B). After treatment of differentiated IMR-32 cells with
either 10 nM TNF- or 10 nM IL-1
for 24 h, there was a
significant increase in the number of eosinophils adhering to the cells
compared with control nonstimulated IMR-32 cells (Fig.
6).
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Eosinophil degranulation followed adhesion to IMR-32 cells.
To determine the role of eosinophil adhesion to differentiated IMR-32
cells on the process of eosinophil activation and degranulation, EPO
release and LTC4 generation were measured. The time course of EPO release showed that degranulation occurred in parallel with
eosinophil adhesion, with maximal secretion of EPO after 1 h of
coincubation (0.060 ± 0.006 units of peroxidase activity; P < 0.01; n = 4; Fig.
11). Spontaneous secretion of EPO from
control eosinophil cultures (i.e., in the absence of IMR-32 cells)
remained low at that time point (0.010 ± 0.004 units of
peroxidase activity) and throughout the assay period. When adhesion, as
opposed to close contact, was prevented by placing Transwell inserts
between the IMR-32 cells, eosinophil EPO release was completely
inhibited (data not shown). The released EPO represented between 15 and 20% of the total cellular EPO content.
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DISCUSSION |
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In previous studies, we have shown that, both in animal models of hyperreactivity and in humans with asthma, eosinophils and MBP are seen in close association with airway cholinergic nerves (4). Furthermore, in vivo, preventing the localization of eosinophils to airway nerves prevents antigen-induced hyperreactivity (9). In this study, we have investigated the mechanism of this localization of eosinophils to airway parasympathetic nerves.
The results of this study show that primary cultures of airway
parasympathetic nerves expressed the adhesion molecule VCAM-1, whereas
ICAM-1 was expressed only after pretreatment of these cells with
TNF- and IFN-
. Furthermore, after cytokine stimulation, eosinophils adhered to these cells via a CD11/18-dependent mechanism. The human cholinergic cell line IMR-32 constitutively expressed VCAM-1
and ICAM-1. Human eosinophils adhered to these cells via these adhesion
molecules in a time-dependent manner, and this adhesion led to
eosinophil activation and degranulation.
For practical reasons, including the limited number of reagents available and the relatively small number of neurones obtained by primary culture of guinea pig and rat tracheal parasympathetic nerves, further studies examining the interactions of eosinophils with neural adhesion molecules were performed using human IMR-32 cells and human eosinophils. In preliminary studies (results not shown), we demonstrated that these cells develop a cholinergic phenotype on differentiation in DBcAMP, with the ability to release ACh in response to electrical stimulation and to express M2 muscarinic receptors. In keeping with the observations in primary cultures of tracheal parasympathetic nerves, IMR-32 cells also expressed VCAM-1 and ICAM-1; however, the expression of these adhesion molecules did not require the addition of cytokines.
Using IMR-32 cells, we then investigated whether eosinophils adhered to
these cells and whether this was mediated by VCAM-1 or ICAM-1. In these
studies, eosinophils adhered to IMR-32 cells in a time-dependent
manner. Adhesion appeared to be an active process, since it was
completely inhibited in the presence of EDTA or the experiments were
performed at low temperature. Although cytokine stimulation was not
necessary for expression of ICAM-1 or VCAM-1, pretreatment with the
cytokines TNF- and IL-1
increased eosinophil adhesion to IMR-32
cells that had been differentiated for 7 days with DBcAMP.
The adhesion molecules mediating adhesion were investigated by means of
specific blocking antibodies and inhibitors. Eosinophil CD11/18 was
inhibited by blocking the common -family of integrin molecules with
a specific antibody, and the dependence of the adhesion on ICAM-1 was
investigated with an anti-ICAM-1 antibody. In both cases, eosinophil
adhesion to IMR-32 cells was inhibited, and an isotype-matched control
antibody had no such inhibitory effect.
The VLA-4 peptide inhibitor ZD-7349, a related cyclic peptide
(14), and a specific antibody to VCAM-1 also prevented
eosinophil adhesion to IMR-32 cells. We can therefore conclude that
eosinophil adhesion involves both ICAM-1 and VCAM-1. It appears from
quantitative analysis of the extent of inhibition in each case that the
contributions of the two cell adhesion molecules were not additive,
since >50% inhibition occurred in the presence of inhibitors of
either molecule. A synergistic interaction between various integrins
and adhesion molecules has been reported with other inflammatory cells;
for example, binding of VLA-4 on T cells to VCAM-1 strengthens CD11a/18 adhesion to ICAM-1, mediated by an increase in avidity in the 2-integrin (2). A mechanism for this was
proposed by Nagel et al. (19), who showed that
binding to VLA-4 integrins releases cytohesin-1, which binds to the
cytoplasmic tail of the
2-integrin and promotes
2-integrin clustering. Thus inhibition of the
interaction between one set of integrin/cell adhesion molecules can
reduce the ability of another set to support adhesion. Our data suggest a possible intracellular link via PKC, which is a known secondary messenger in the CD11/18 signaling pathway (22), since the
inhibitor rottlerin inhibited the adhesion of eosinophils to the IMR-32 cells to near baseline levels.
Previous studies in both animal models and in humans with asthma have shown a benefit of heparin in ameliorating antigen-induced hyperreactivity (1, 5, 11). This is thought to be because of the ability of this compound to displace the cationic protein MBP from M2 muscarinic receptors (6). In addition, it has been suggested that heparin may also be of benefit in the treatment of a number of other conditions, including inflammatory bowel disease, because of an anti-inflammatory mechanism of action (6). In these studies, it was shown that pretreatment of IMR-32 cells with heparin completely inhibited eosinophil adhesion to these cells. These findings suggest that some of heparin's anti-inflammatory properties and benefits in asthma may relate to an inhibition of adhesion of inflammatory cells to nerves.
Adhesion of eosinophils to nerve cells would not in itself constitute a mechanism for parasympathetic nerve hyperreactivity. Therefore, it was necessary to demonstrate that the nerve cells influenced eosinophil function. The release of the eosinophil mediator EPO was increased after incubation with IMR-32 nerve cells under conditions in which adhesion took place. This was not simply an effect on peroxidase activity, since similar enhancement of LTC4 release occurred under the same conditions. Because it is known that eosinophil granule proteins mediate the loss of function of neuronal M2 muscarinic receptors (8), the process of nerve cell-mediated eosinophil degranulation may be critical to this dysfunction. The process of nerve cell-mediated eosinophil degranulation may also account for the apparent reduction in adhesion that follows several hours of incubation of eosinophils with nerves that is seen in Fig. 5. The precise mechanism of how nerve cells lead to eosinophil degranulation is the subject of further current work in our laboratory.
In summary, this study suggests that the expression of the adhesion molecules VCAM-1 and ICAM-1 by airway parasympathetic nerves contributes to the process whereby eosinophils localize to airway nerves. Furthermore, the results suggest that eosinophil adhesion to airway nerves may be central to the development of antigen-induced hyperreactivity since eosinophil degranulation follows adhesion.
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ACKNOWLEDGEMENTS |
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This work was supported by the Wellcome Trust (059037), the British Lung Foundation (P97/5), an American Heart Association Award (to A. D. Fryer), and by a Samuel Crossley-Barnes studentship (to D. A. Sawatzky).
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. W. Costello, Dept. of Medicine, RCSI, Beaumont Hospital, Dublin 9, Ireland (E-mail: rcostello{at}rcsi.ie).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 25, 2002;10.1152/ajplung.00279.2001
Received 24 July 2001; accepted in final form 7 January 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bendstrup, KE,
and
Jensen JI.
Inhaled heparin is effective in exacerbations of asthma.
Respir Med
94:
174-175,
2000[ISI][Medline].
2.
Chan, JR,
Hyduk SJ,
and
Cybulsky MI.
Alpha 4 beta 1 integrin/VCAM-1 interaction activates alpha L beta 2 integrin-mediated adhesion to ICAM-1 in human T cells.
J Immunol
164:
746-753,
2000
3.
Costello, RW,
Evans CM,
Yost BL,
Belmonte KE,
Gleich GJ,
Jacoby DB,
and
Fryer AD.
Antigen-induced hyperreactivity to histamine: role of the vagus nerves and eosinophils.
Am J Physiol Lung Cell Mol Physiol
276:
L709-L714,
1999
4.
Costello, RW,
Schofield BH,
Kephart GM,
Gleich GJ,
Jacoby DB,
and
Fryer AD.
Localization of eosinophils to airway nerves and effect on neuronal M2 muscarinic receptor function.
Am J Physiol Lung Cell Mol Physiol
273:
L93-L103,
1997
5.
Diamant, Z,
Timmers MC,
van der Veen H,
Page CP,
van der Meer FJ,
and
Sterk PJ.
Effect of inhaled heparin on allergen-induced early and late asthmatic responses in patients with atopic asthma.
Am J Respir Crit Care Med
153:
1790-1795,
1996[Abstract].
6.
Dwarakanath, AD,
Yu LG,
Brookes C,
Pryce D,
and
Rhodes JM.
"Sticky" neutrophils, pathergic arthritis, and response to heparin in pyoderma gangrenosum complicating ulcerative colitis.
Gut
37:
585-588,
1995[Abstract].
7.
Elbon, CL,
Jacoby DB,
and
Fryer AD.
Pretreatment with an antibody to interleukin-5 prevents loss of pulmonary M2 muscarinic receptor function in antigen-challenged guinea pigs.
Am J Respir Cell Mol Biol
12:
320-328,
1995[Abstract].
8.
Evans, CM,
Fryer AD,
Jacoby DB,
Gleich GJ,
and
Costello RW.
Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.
J Clin Invest
100:
2254-2262,
1997
9.
Fryer, AD,
Costello RW,
Yost BL,
Lobb RR,
Tedder TF,
Steeber DA,
and
Bochner BS.
Antibody to VLA-4, but not to L-selectin, protects neuronal M2 muscarinic receptors in antigen-challenged guinea pig airways.
J Clin Invest
99:
2036-2044,
1997
10.
Fryer, AD,
Elbon CL,
Kim AL,
Xiao HQ,
Levey AI,
and
Jacoby DB.
Cultures of airway parasympathetic nerves express functional M2 muscarinic receptors.
Am J Respir Cell Mol Biol
15:
716-725,
1996[Abstract].
11.
Fryer, AD,
and
Jacoby DB.
Function of pulmonary M2 muscarinic receptors in antigen-challenged guinea pigs is restored by heparin and poly-L-glutamate.
J Clin Invest
90:
2292-2298,
1992[ISI][Medline].
12.
Fryer, AD,
and
Maclagan J.
Muscarinic inhibitory receptors in pulmonary parasympathetic nerves in the guinea-pig.
Br J Pharmacol
83:
973-978,
1984[Abstract].
13.
Fryer, AD,
and
Wills-Karp M.
Dysfunction of M2-muscarinic receptors in pulmonary parasympathetic nerves after antigen challenge.
J Appl Physiol
71:
2255-2261,
1991
14.
Haworth, D,
Rees A,
Alcock PJ,
Wood LJ,
Dutta AS,
Gormley JJ,
Jones HB,
Jamieson A,
and
Reilly CF.
Anti-inflammatory activity of c(ILDV-NH(CH2)5CO), a novel, selective, cyclic peptide inhibitor of VLA-4-mediated cell adhesion.
Br J Pharmacol
126:
1751-1760,
1999
15.
Jacoby, DB,
Gleich GJ,
and
Fryer AD.
Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.
J Clin Invest
91:
1314-1318,
1993[ISI][Medline].
16.
Menegazzi, R,
Zabucchi G,
Zuccato P,
Cramer R,
Piccinini C,
and
Patriarca P.
Oxidation of homovanillic acid as a selective assay for eosinophil peroxidase in eosinophil peroxidase-myeloperoxidase mixtures and its use in the detection of human eosinophil peroxidase deficiency.
J Immunol Methods
137:
55-63,
1991[ISI][Medline].
17.
Nadel, JA,
and
Barnes PJ.
Autonomic regulation of the airways.
Annu Rev Med
35:
451-467,
1984[ISI][Medline].
18.
Nagata, M,
Sedgwick JB,
Bates ME,
Kita H,
and
Busse WW.
Eosinophil adhesion to vascular cell adhesion molecule-1 activates superoxide anion generation.
J Immunol
155:
2194-2202,
1995[Abstract].
19.
Nagel, W,
Zeitlmann L,
Schilcher P,
Geiger C,
Kolanus J,
and
Kolanus W.
Phosphoinositide 3-OH kinase activates the beta2 integrin adhesion pathway and induces membrane recruitment of cytohesin-1.
J Biol Chem
273:
14853-14861,
1998
20.
Roberts, RE,
and
McLean WG.
Protein kinase C isozyme expression in sciatic nerves and spinal cords of experimentally diabetic rats.
Brain Res
754:
147-156,
1997[ISI][Medline].
21.
Schleimer, RP,
and
Bochner BS.
The role of adhesion molecules in allergic inflammation and their suitability as targets of antiallergic therapy.
Clin Exp Allergy
28, Suppl3:
15-23,
1998[ISI][Medline].
22.
Steadman, R,
Petersen MM,
and
Williams JD.
CD11b/CD18-dependent stimulation of leukotriene B4 synthesis by human neutrophils (PMN) is synergistically enhanced by tumour necrosis factor alpha and low dose diacylglycerol.
Int J Biochem Cell Biol
28:
771-76,
1996[ISI][Medline].
23.
Von Andrian, UH,
and
Mackay CR.
T-cell function and migration. Two sides of the same coin.
N Engl J Med
343:
1020-1934,
2000