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Division of Respiratory Medicine, Leicester University Medical School, United Kingdom
Correspondence: Dr. Peter Bradding, Department of Respiratory Medicine, Glenfield Hospital, Groby Rd., Leicester, LE3 9QP UK. E-mail: pbradding{at}hotmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-N-acetylgalactosaminidase, indicating that HLMC
adhere to bronchial epithelial cells via galactose-bearing
carbohydrates expressed on a cell-surface peptide(s).
Key Words: adhesion molecules • galactose
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An interesting pathological observation is that mast cells infiltrate
the bronchial epithelium of asthmatic but not normal subjects, placing
them at the portal of entry of noxious stimuli such as proteolytic
allergenic peptides [3
4
5
6
]. The factors controlling the
migration of mast cells into the bronchial epithelium, and the
mechanism by which they are retained there are unknown. Human lung mast
cells (HLMC) express a number of integrins and immunoglobulin
superfamily adhesion receptors including the ß1 integrins
VLA-4 (4ß1; CD49d/CD29) and VLA-5
(5ß1; CD49e/CD29), the vitronectin
receptor (vß3; CD51/CD61), and ICAM-1
[7
]. In addition, a proportion of human skin and uterine
mast cells express CD18 [8
, 9
]. Bronchial
epithelial cells express a number of integrins, which are important for
maintaining normal epithelial structure [10
]. In
addition, they express ICAM-1, the counter-ligand for LFA-1
(CD11a/CD18), and the human bronchial epithelial cell line, BEAS-2B,
has been reported to express VCAM-1, the counter-ligand for VLA-4
[11
].
Epithelial cells also express the homotypic and homophilic adhesion
molecule E-cadherin, which can also mediate a specific interaction with
the integrin Eß7 (CD103)
[12
13
14
]. This latter integrin is expressed by a subset
of T lymphocytes that home to intraepithelial sites at mucosal surfaces
[15
] and, in mice, mediates their adhesion to epithelial
cells derived from breast and intestine [16
]. The
ability of mouse mast cells to express Eß7
after exposure to transforming growth factor (TGF)-ß or anti-IgE has
been interpreted as the mechanism by which mast cells localize to
epithelial sites [17
, 18
], although there
is no functional data to support this hypothesis. In fact the only
published study investigating the adhesion of mast cells to epithelium
described the adhesion of dog mastocytoma cells to dog tracheal
epithelium [19
]. This study found that approximately
35% of dog mastocytoma cells adhered to tracheal epithelium, but that
this did not require Ca2+ or energy, and was not mediated
via mast cell CD18.
Because mast cells infiltrate the bronchial epithelium in asthma, but are scarce in bronchoalveolar lavage fluid and sputum compared with other cells like eosinophils [20 , 21 ], we hypothesized that HLMC adhere to bronchial epithelial cells through a specific interaction involving adhesion molecules expressed by the two cell types. To test this hypothesis, we have studied the adhesion of purified HLMC and the human mast cell line HMC-1 to both primary cultures of human bronchial epithelium and the human bronchial epithelial cell line BEAS-2B.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eß7) (leukocyte typing workshop 1996);
Ber-Act-8 (Eß7) (Dr. H. Dürkop,
Freie Universität, Berlin, Germany); 29C6 (Fc
RI) (Dr. J.
Hakimi, Hoffmann-LaRoche); HP1/2 (4-chain) (Dr. F.
Sánchez-Madrid, Hospital de la Princesa, Madrid, Spain); Act I
(4ß7) (Dr. A. I. Lazarovits,
University Hospital, Ontario, Canada); 4B9 (VCAM-1) (Dr. R. Lobb,
Biogen, Cambridge, MA); R6.5 F(ab)2 (ICAM-1) (Dr. R.
Rothlein, Boehringer Ingelheim); B2C10 and A3A12 (galectin-3) (Dr. F-T.
Liu, La Jolla Institute for Allergy and Immunology, San Diego, CA).
Polyclonal galectin-1 rabbit antiserum was a generous gift from Dr.
L. G. Baum (UCLA School of Medicine, Los Angeles, CA).
Cytokines and reagents
Stem cell factor (SCF), TGF-ß, tumor necrosis factor
(TNF-), IL-1ß, and interferon-
(IFN-) were purchased from R
& D; pronase, heparinase I (heparinase, from Flavobacterium
heparinum), heparinase III (heparatinase I from
Flavobacterium heparinum), ß-galactosidase (from bovine
testis), hyaluronidase type I-S (from bovine testis),
endo--N-acetylgalactosaminidase (from Diplococcus
pneumoniae), collagenase type IA (from Clostridium
histolyticum), hyaluronic acid (from bovine trachea), EDTA, EGTA,
manganese, paraformaldehyde, pertussis toxin, cholera toxin,
cytochalasin D, genistein, sodium azide, deoxyglucose, phorbol
myristate acetate (PMA), galactose, mannose, mannose-6-phosphate, and
fucoidin were purchased from Sigma (Poole, Dorset, UK); neuraminidase
(from Arthrobacter ureafaciens), and human myeloma IgE were
purchased from Calbiochem-Novabiochem (Nottingham, UK); sheep
polyclonal anti-human IgE was purchased from Serotec (Kidlington,
Oxford, UK). Histamine and
S-adenosyl-L-[methyl-3H]methionine
was purchased from Amersham Life Sciences (Little Chalfont, Bucks, UK);
rat kidney histamine methyl transferase was a generous gift from Dr. S.
Harper (Astra Charnwood, Loughborough, UK).
Cell lines and culture media
The BEAS-2B epithelial cell line was purchased from the European
Collection of Animal Cell Cultures (Porton Down, Wiltshire, UK);
primary bronchial epithelial cells (normal human bronchial epithelial
cells, NHBE) were purchased from TCS Biologicals (Buckingham, UK). The
human mast cell line HMC-1 was a kind gift from Dr. J. H.
Butterfield (Mayo Clinic, Rochester, MN). Small airway growth medium
(SAGM) was purchased from BioWhittaker (Wokingham, UK); Iscove’s
medium, iron-supplemented FCS, RPMI 1640/Glutamax/HEPES,
antibiotic/antimycotic solution, minimum essential medium (MEM)
nonessential amino acids, and fetal calf serum (FCS) were purchased
from Life Technologies (Paisley, Scotland, UK); bovine serum albumin
(BSA), human plasma fibronectin, and monothioglycerol were purchased
from Sigma.
Human lung mast cell purification
HLMC were dispersed from macroscopically normal lung
(n = 45 donors) obtained within 1 h of resection
for lung cancer. The study was approved by the Leicestershire Research
Ethics Committee. The enzymatic dispersal procedure used was as
described previously [22
], with the exception that cells
were sensitized with human myeloma IgE (3 µg/mL) during the blocking
stage with human IgG. Mast cells were then purified using
immunomagnetic affinity selection with the mouse anti-c-kit
mAb YB5.B8 (5 µg/mL) and anti-mouse IgG1 magnetic beads (Dynal,
Wirral, UK) as previously described [22
]. In latter
experiments we used the Dynal rat anti-mouse IgG1 Cellection kit in
accordance with the manufacturer’s instructions, permitting detachment
of mast cells from beads using DNAse digestion. Final mast cell purity
was 99.4 ± 0.4%, and viability 98.7 ± 0.6%.
Mast cell culture
After purification, HLMC were cultured overnight on 1%
BSA-coated plastic (to prevent adhesion), in RPMI 1640/Glutamax/HEPES
containing antibiotic/antimycotic solution, nonessential amino acids,
10% FCS, and 10 ng/mL SCF. Cells were activated overnight with sheep
anti-human IgE as required. HMC-1 cells were cultured in Iscove’s
medium containing 10% iron-supplemented FCS and 1.2 mM
monothioglycerol.
Bronchial epithelial cell culture
The BEAS-2B cell line (passages 44–64) and NHBE (up to the
fifth passage) were both grown in SAGM. BEAS-2B were seeded at 2 x 105 cells/mL in human plasma fibronectin-coated 96-well
tissue culture plates and grown to confluence before use in adhesion
assays. Monolayers were stimulated for 18–24 h as required with a
cocktail of cytokines (50 ng/mL each of human IL-1ß, TNF-, and
IFN-) and gently washed twice before use.
Eosinophil purification and culture
Eosinophils were purified from human peripheral blood by
negative selection using the MACS system (Miltenyi Biotec, Bisley,
Surrey, UK) as described previously [23
]. Final
eosinophil purity and viability were >98 and >99%, respectively.
FACS analysis for adhesion receptor expression on BEAS-2B and human
mast cells
Expression of cell-surface adhesion receptors on epithelial and
mast cells were analyzed by FACS. Briefly, cells were incubated with
optimal dilutions of primary mouse mAb for 15 min at 4°C, washed,
then labeled with fluorescein isothiocyanate (FITC)-conjugated
F(ab’)2 fragments of rabbit anti-mouse Ig. Cells were
analyzed with a FACScan (Becton Dickinson, Cowley, Oxford, UK).
Controls were performed with an unrelated mouse IgG of appropriate
isotype.
Mast cell-epithelial cell adhesion assay
Epithelial cells were grown to confluence in 96-well tissue
culture plates. Bead-free HLMC were obtained either by allowing
spontaneous detachment in overnight culture and separation with a
magnet (MPC-1, Dynal; 
50% detachment), or in latter experiments
using the Dynal Cellection kit (there was no difference in the level of
HLMC adhesion between the two methods). HLMC (1 x
104) or HMC-1 (3 x 104), suspended in 100
µL of mast cell medium, were added to each well. After initial
time-course studies, mast cells were allowed to adhere for 1 h at
37°C. After 1 h, 10 µL of supernatant was gently removed and
stored for later histamine analysis in order to monitor release during
the assay. The wells were then filled completely with medium, sealed
with Mylar sealing tape (Sigma, Poole, UK), inverted, and then
subjected to centrifugation at 15 g for 5 min to remove
nonadherent cells. All medium was then removed, and the remaining
adherent cells lysed in 100 µL sterile water. Plates were frozen at
-80°C until later histamine analysis. HLMC (104) or
HMC-1 (3 x 104) were lysed in sterile water and
stored for measurement of total histamine content. All experiments were
performed in triplicate. Percentage adhesion was calculated as the
amount of histamine remaining in a well divided by the total amount of
histamine added originally.
Analysis of adhesion using radiolabeling was also performed with HMC-1 cells for comparison with the histamine-based assay.
Adhesion to fibronectin
Human plasma fibronectin (40 µg/mL) was used to coat 96-well
plates for 1 h at 37°C, and wells were washed gently with
sterile phosphate-buffered saline (PBS). To block nonspecific sites,
1% BSA was allowed to bind for 1 h at 37°C, and the wells were
washed gently again. HLMC were added for 1 h at 37°C, and the
adhesion assay carried out as before, including relevant controls.
Eosinophil-epithelial cell adhesion assay
Eosinophils were radiolabeled with 51Cr, then washed
to remove free 51Cr. The assay was then performed as
described for mast cells above, except that 1 x 105
eosinophils were added per well, and adhesion was calculated as the
percentage of radioactivity of total cells added.
Histamine assay
Histamine was measured by sensitive radioenzymic assay based on
the conversion of histamine to [3H]methylhistamine in the
presence of the enzyme histamine-N-methyl transferase
[24
]. Briefly, samples and control standards were
incubated with rat kidney histamine methyl transferase and
S-adenosyl-L-[methyl-3H]methionine.
After organic extraction in a 4:1 mixture of toluene/isoamyl alcohol,
the samples were read in a Tri-carb liquid scintillation analyzer
(model 1500, Packard, Pangbourne, Berks, UK). Histamine concentrations
were calculated from a standard curve that was run in triplicate in the
range 500–7500 pg. This assay has a lower detection limit of 50 pg
histamine, and is linear throughout the range 50 to 7500 pg. Intra- and
interassay coefficients of variation are 2 and 3%, respectively.
Adhesion modulation assays
Adhesion-blocking mAb
The following well-characterized adhesion-blocking antibodies
were incubated with the epithelial cell monolayers at room temperature
for 15 min before the adhesion assay: R6.5 (ICAM-1), 15 µg/mL
[25
]; 4B9 (VCAM-1), 20 µg/mL [26
,
27
]; HECD-1 (E-cadherin), 100 µg/mL
[28
]; SHE78-7 (E-cadherin), 1 µg/mL
[29
], B2C10 (galectin-3) 20 and 100 µg/mL
[30
]; polyclonal anti-SCF, 10 µg/mL. The following
adhesion-blocking mAb were incubated with HLMC for 15 min at room
temperature before the adhesion assay: BBIG-I1 (ICAM-1), 10 µg/mL;
IB4 (CD18), 5 µg/mL [31
]; HP1/2 (4
chain), supernatant diluted 1:2 [26
]; Ber-Act-8
(Eß7), supernatant diluted 1:40
[16
]; LM609 (CD51/61, vitronectin receptor), 10 µg/mL
[32
]; P4C10 (ß1 chain), 20 µg/mL
[27
]; B2C10 (galectin-3), 20 and 100 µg/mL. Controls
were performed with IgG control up to 100 µg/mL. All of the above
antibodies remained present during the adhesion assay.
Proteolytic digestion
HLMC were pretreated with pronase at concentrations of 1, 2.5,
7.5, and 10 µg/mL for 20 min at 37°C in serum-free medium. After 20
min, an equal volume of medium containing 10% FCS was added, and the
cells then washed once.
Carbohydrates and proteoglycans
To determine whether cell-surface carbohydrates or proteoglycans
are involved in the adhesion of mast cells to epithelial cells, HLMC
and HMC-1 were pretreated with the following at room temperature for 30
min: heparin (1 mg/mL), hyaluronic acid (1 mg/mL), galactose (50 mM),
lactose (50 mM and 200 mM, for 30 min and 2 h), mannose (50 mM),
mannose-6-phosphate (3.3 mM), and fucoidin (1 mg/mL). BEAS-2B were
incubated with these sugars (with the exception of mannose-6-phosphate)
under the same conditions. These carbohydrates, which bind
competitively [33
, 34
] remained present
during the adhesion assay. The following enzymes were used to pretreat
mast cells at 37°C for 30 min: heparinase I (2 U/mL), heparinase III
(2 U/mL), hyaluronidase (300 U/mL), neuraminidase (1 U/mL), ß(1–3,
4, 6)-galactosidase (0.5 U/mL),
endo--N-acetylgalactosaminidase (0.3 U/mL). Mast cells
exposed to enzymes were washed before the adhesion assay.
ß-Galactosidase was also used on BEAS-2B monolayers under the same
conditions. The concentrations of these carbohydrates and enzymes used
were based on previously published data of our own and others
[33
, 35
36
37
38
39
40
41
].
Modulation of cell signaling and metabolism
HMC-1 and HLMC were pretreated with the following agents at
37°C: genistein 5 µM for 30 min; pertussis toxin 0.1 µg/mL for
2 h; cholera toxin 0.1 µg/mL for 2 h; sodium azide 0.01 M
for 1 h; deoxyglucose/sodium azide 0.01 M/0.01 M for 1 h;
cytochalasin D 12 µM for 30 min; PMA 0.05 µg/mL for 10 min.
Requirement for divalent cations
The Ca2+ and Mg2+ chelating agents EDTA
(5 mM) and EGTA (5 mM) were added to HLMC at 37°C for 10 min before
the assay. Because they cause detachment of the epithelial monolayer,
the epithelial cells were fixed in 0.4 and 4.0% paraformaldehyde for 5
min at room temperature, then washed twice before use. Mast cells were
also incubated with Mn2+ (5 mM) for 10 min at 37°C to
assess the effect of integrin activation on adhesion.
Mast cell activation
HLMC were incubated overnight with sheep anti-human IgE with or
without the tryptase inhibitor leupeptin (10 µg/mL), and with the
calcium ionophore A23187 (500 nM). Cells were washed three times before
the adhesion assay. Controls were performed with sheep serum (1%). To
assess the effects of mast cell contents on mast cell adhesion, 1 x 106 mast cells were lysed in 900 µL sterile water,
frozen, and then thawed. The lysate was then reconstituted with 100
µL 10x PBS. After centrifugation (11,500 rpm, 8 min), the lysate was
incubated at various dilutions with mast cells for 20 min at 37°C
before the adhesion assay.
To allow for histamine release from mast cells in response to the above experimental procedures (e.g., anti-IgE, A23187), appropriate aliquots of treated cells were kept for measurement of total histamine content. All experiments and procedures were performed at 37°C unless otherwise stated, and all experiments were performed in triplicate.
Cell viability
Mast cell viability was monitored before and after the above
experiments by exclusion of trypan blue. No treatment was cytotoxic and
mast cell viability was >97% in all experiments.
Data presentation and analysis
The percentage of cells adhering is expressed as mean ±
SEM. N represents the number of mast cell donors
studied or the number of times an experiment was performed with
different passages of the HMC-1 cell line. Data comparing mast cell and
eosinophil adhesion was compared using Student’s unpaired t
test (two-tailed), and maneuvers aimed at modulating mast cell adhesion
were analyzed using Student’s paired t test (two-tailed).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mast cell adherence to bronchial epithelium under basal conditions
A high proportion of both HLMC and HMC-1 adhered to both BEAS-2B
and NHBE cells (Fig. 1
); this was in marked contrast to the relative paucity of
eosinophil adhesion. Thus a mean 60.1 ± 2.5 and 68.4 ±
7.7% HLMC adhered to BEAS-2B (n = 45) and NHBE
(n = 3), respectively, and a mean 52.0 ± 5.7%
HMC-1 adhered to BEAS-2B (n = 13). In contrast, only a
mean 10.3 ± 1.3% peripheral blood eosinophils adhered to BEAS-2B
(n = 7; P = 0.0007 compared to both
HLMC and HMC-1). HLMC were routinely cultured overnight in the presence
of SCF before the adhesion assay, but omitting this from the culture
conditions did not affect adhesion (adhesion without SCF 60.7 ±
13.2%, adhesion with SCF for 16 h 51.1 ± 10.5%,
n = 3, P = 0.09). The histamine-based
adhesion assay and 51Cr-based assay were compared using
HMC-1 and gave comparable results (adherence 47.0 vs. 45.3%,
respectively, n = 1).
|
, TNF-,
and IL-1ß (50 ng/mL each) did not influence mast cell adhesion
(Table 1
), in spite of the observation that this combination of cytokines
induces a marked increase in the epithelial expression of ICAM-1 as
demonstrated by FACS (not shown).
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Mast cell adhesion to bronchial epithelium is not modulated by
adhesion-blocking antibodies, integrin activation, or cations
There are several potential mast cell-epithelial cell
integrin-immunoglobulin superfamily interactions that could mediate the
adhesive process. Expression of several integrins and ICAM-1 on mast
cells as analyzed by FACS is summarized in Table 2
, and a representative experiment is shown in Figure 3
. In contrast to previous reports [7
,
42
], we found that a small but consistent number of HLMC
expressed CD18, with smaller proportions expressing CD11b and CD11c.
However, mast cell adhesion was not modulated by preincubation of mast
cells with blocking anti-CD18, anti-VLA-4, anti-CD51/61 (vitronectin
receptor) or anti-ICAM-1 mAb, or preincubation of epithelial cells with
blocking anti-ICAM-1 or anti-VCAM-1 mAb (Fig. 4
). Integrin activation on mast cells with Mn2+ was also
without any effect (Table 1)
, and integrin-blocking mAbs still had no
effect after Mn2+ exposure, supporting the observation that
adhesion is not integrin-mediated (data not shown). The
ß1-blocking mAb P4C10 completely inhibited adhesion of
HLMC cells to plasma fibronectin (control adhesion 49.4 ± 2.7%,
anti-ß1 adhesion 2.6 ± 1.9%, n =
2) but had no effect on adhesion to epithelium (control adhesion
30.5%, anti-ß1 adhesion 42.3%, n = 1),
indicating that mast cells were not adhering to the fibronectin
required for epithelial cell anchorage.
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Eß7 on HLMC or
HMC-1 either at rest or after 24 h culture with TGF-ß (5 ng/mL)
or anti-IgE (1%; Table 2
). Eß7 was not
destroyed by the enzymatic dispersion procedure because it was clearly
detectable on T cells in these preparations (data not shown).
Furthermore, overnight culture with TGF-ß (5 ng/mL) had no effect on
mast cell adhesion (mean adherence in unstimulated cells 41.8 ±
9.8 vs. 45.3 ± 11.9 in TGF-ß-treated cells, n =
3, P = 0.26). In addition, there was no inhibition of
adhesion after incubation of HLMC with the
Eß7 function-blocking mAb Ber-Act-8 nor
with two E-cadherin mAb known to block homotypic adhesion between
E-cadherin molecules (not shown). Integrin- and C-type lectin-mediated adhesion generally requires the presence of calcium [43 ]. Because epithelial monolayers detach in the presence of EDTA and EGTA, they were fixed in 0.4 and 4% paraformaldehyde before addition of EDTA/EGTA-treated mast cells. With 0.4% paraformaldehyde fixation, there was a small but nonsignificant fall in adhesion, and adhesion was not reduced further after chelation of cations with EDTA or EGTA (5 mM; n = 6; Table 1 ). Four percent paraformaldehyde fixation reduced adhesion from 61.3 ± 2.1 to 25.7 ± 2.5% (n = 3, P = 0.0016), and the remaining adhesion was also not altered by EDTA or EGTA. These observations further suggest that an integrin-mediated adhesive interaction is not operating.
Mouse mast cells are reported to adhere to mouse fibroblasts through an SCF receptor-SCF interaction [44 ]. Epithelial cells are also able to produce SCF [45 , 46 ], although whether or not they express the membrane-bound form like fibroblasts is not known. Incubation of epithelial monolayers with a neutralizing polyclonal goat anti-human SCF antibody did not reduce HLMC (n = 1) or HMC-1 adhesion (n = 3; data not shown).
Mast cell-epithelial adhesion is pronase-sensitive
Pre-incubation of HLMC with pronase demonstrated a clear
dose-related reduction in adhesion, with a mean baseline value of
50.2 ± 8.7% falling to 6.5 ± 1.4% at 10 µg/mL
(n = 3, P = 0.028; Fig. 5
). Expression of CD9, CD18, ICAM-1, and CD69 on HMC-1 (analyzed by
FACS) were markedly reduced after incubation with pronase (10 µg/mL)
for 20 min (data not shown), confirming proteolytic activity under
these conditions.
|
3, HMC
n = 1) [33
34
35
36
37
, 39
,
40
]. No significant effect on adhesion was seen with any
of these carbohydrates (Table 1)
. Next we used a panel of glycosidases
to digest carbohydrates expressed on the mast cell surface.
ß-Galactosidase reduced HLMC adhesion from 57.8 ± 6.4 to
15.0 ± 2.1% (n = 3, P = 0.031),
and reduced adhesion of HMC-1 from 37.0 ± 1.8 to 7.8 ±
7.6% (n = 3, P = 0.045; Fig. 6
). Endo--N-acetylgalactosaminidase was used only on
HLMC and reduced adhesion from 51.1 ± 13.1% at baseline to
26.5 ± 8.1% (n = 3, P = 0.04;
Fig. 6
). ß-Galactosidase and
endo--N-acetylgalactosaminidase (which is certified as
protease-free) did not express proteolytic activity as analyzed by
FACS. Heparinase I had a small but statistically significant effect in
reducing HLMC adhesion (Table 1)
, but no effect on HMC-1 adhesion
(control adhesion 37.0 ± 1.8, heparinase I adhesion 34.4 ±
2.3, P = 0.58, n = 3). Heparinase III,
hyaluronidase, and neuraminidase were without effect on HLMC (Table 1)
or HMC-1 (data not shown). Incubating BEAS-2B with ß-galactosidase
did not alter HLMC adhesion (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-N-acetylgalactosaminidase. However, there was no
evidence for involvement of integrins in the experiments conducted.
Mast cell adhesion to the BEAS-2B cell line demonstrated similar
characteristics as adhesion to primary bronchial epithelial cells,
suggesting that this cell line is a suitable model for studying this
adhesion process. Both these cell types are typical of basal epithelial
cells rather than pseudostratified columnar cells morphologically, and
in that they divide, do not have cilia, and do not produce mucus in
culture. Because mast cells are found between basal cells and between
basal cells and columnar cells in the bronchial epithelium, we believe
that the adhesion assay employed in this study is physiologically
relevant. BEAS-2B express both ICAM-1 and VCAM-1 [11
],
the counterligands for which include CD11/CD18 and VLA-4
(4ß1), respectively. HLMC are known to
express VLA-4 (4ß1), and in the present
study we were able to demonstrate that about 20% of HLMC expressed
CD18 as analyzed by FACS. This integrin has not been found on HLMC
previously [7
, 42
]. However, we were able
to confirm its presence on HLMC by using immunomagnetic affinity
selection on enzymatically dispersed lung cells (data not shown). The
reason for this discrepancy with previous studies is not clear, but it
is of note that human uterine mast cells do express CD18, and up to
50% of mast cells in inflamed skin also express CD18 [8
,
9
]. Taking the above observations together, it might be
predicted that HLMC would adhere to bronchial epithelium via a
CD18/ICAM-1 and/or VLA-4/VCAM-1 pathway. However, it was clear from our
studies that these molecules were not involved because appropriate
function-blocking mAb did not inhibit adhesion, and adhesion did not
require the presence of Ca2+ or Mg2+, and was
not enhanced in the presence of manganese.
Eß7 (CD103) is an integrin expressed on
the surface of the intraepithelial subset of mucosal T cells
[15
], a ligand for which is E-cadherin expressed on the
surface of epithelial cells [14
]. In vitro,
the binding of Eß7 to E-cadherin mediates
the adhesion of intraepithelial T cells to epithelial cells derived
from a variety of sources including gut and breast [16
],
suggesting that this is the epithelial homing mechanism for this
population of lymphocytes. Eß7 is also
expressed by mouse mast cells after in vitro activation with
TGF-ß (5 ng/mL) or anti-IgE [17
]. As a result of this,
it has become the widely held view that this is also the mechanism
through which mast cells localize to epithelial sites
[17
, 18
]. In the present study, however, in
spite of demonstrating high levels of mast cell adhesion to bronchial
epithelium, we have been unable to detect
Eß7 on HLMC or HMC-1 either at rest, which
is in keeping with previous studies [7
], or after
overnight incubation with anti-IgE (vide infra) or TGF-ß.
TGF-ß did not increase HLMC adhesion, and adhesion was not blocked by
a function-blocking anti-Eß7 mAb. This
reiterates the important phenotypic differences that exist between
mouse and human mast cells, and shows that caution is required in
extrapolating mast cell data from mouse to humans.
There are many potential carbohydrate interactions between different
cell types, the mechanics of which are still poorly understood
[47
]. To explore the potential role of mast cell-surface
carbohydrates as adhesion molecules, we used neuraminidase to remove
cell-surface carbohydrates bearing sialic acid residues,
ß-galactosidase to remove terminal ß-linked galactose residues, and
endo--N-acetylgalactosaminidase. This latter enzyme
specifically cleaves Galß(1–3)GalNAc disaccharides attached to
the serine or threonine residues of glycoproteins [41
],
typical of the O-linked glycans present in mucin-like molecules. It is
interesting that ß-galactosidase treatment of both HLMC and HMC-1,
but not epithelium, produced a marked reduction in mast cell adhesion,
suggesting that mast cell-expressed ß-galactosides are responsible
for a large proportion of the adhesion seen. In addition,
endo--N-acetylgalactosaminidase produced approximately a
40% reduction in adhesion, suggesting that mucin-like molecules are
involved. Because endo--N-acetylgalactosaminidase
specifically cleaves unsubstituted Galß(1–3)GalNAc disaccharides
linked to core proteins, and the terminal galactose residue of these is
also susceptible to ß-galactosidase (our enzyme preferentially
cleaves galß1–3 linkages), it is possible that these two enzymes are
active on the same carbohydrate molecules. Heparinase I had a small but
statistically significant effect at reducing adhesion of HLMC. However,
there was no effect on HMC-1, and heparin did not inhibit adhesion, so
the biological significance of this observation is uncertain. In
contrast, neuraminidase was clearly without effect, indicating that
sialic acid residues are not required for adhesion.
Lectins are carbohydrate-binding peptides often expressed on cell
surfaces, and are classified into C-type (e.g., selectins), P-type
(e.g., mannose-6-phosphate receptor), S-type (e.g., galectins), I-type
(e.g., CD22), and others including heparin-binding proteins and
pentraxins [47
]. C-type lectin-dependent adhesion
requires the presence of calcium, and can thus be excluded as a mast
cell-epithelial cell adhesion mechanism in this study. P-type lectins
bind mannose-6-phosphate, whereas S-type lectins (e.g., galectins)
typically bind ß-galactosides. The inhibitory effect of
ß-galactosidase treatment therefore suggests that a member of the
galectin family is a candidate adhesion molecule [for review see ref. 48
]. Adhesion mediated by galectins and other lectin subtypes can
often be competitively and specifically inhibited by various mono- and
polysaccharides. Using a variety of sugars at concentrations reported
to inhibit lectin-mediated adhesion in other systems
[33
34
35
36
37
, 40
, 49
], we were
unable to modulate adhesion. Lactose is particularly useful for
inhibiting galectin interactions, and although it makes it unlikely
that a galectin is involved, the lack of effect with lactose in our
experiments does not completely rule out galectins because the affinity
for lactose could be much lower than for a physiological ligand. We
can, however, exclude a role for galectins-1 and -3 because our HLMC,
HMC-1, and BEAS-2B did not express these molecules when analyzed by
FACS, and an adhesion-blocking galectin-3 mAb was without effect. Taken
together, our data suggest that HLMC and HMC-1 adhere to bronchial
epithelial cells via mast cell-surface ß-galactosides including the
unsubstituted Galß(1–3)GalNAc disaccharide linked to a core
peptide, but that this does not involve a recognized
carbohydrate-receptor interaction.
To our knowledge, the only previous investigation of mast cell adhesion to epithelium reported adhesion of dog mastocytoma cells to dog tracheal epithelium [19 ]. The results of this study were remarkably similar to ours, in that the authors found high baseline adhesion in the order of about 35%, but that adhesion was not calcium-dependent, did not require energy because it was not blocked by sodium azide, but was sensitive to proteolytic digestion with pronase. We too found that pronase markedly reduced adhesion. Thus a mast cell-expressed peptide or peptides are also required for mast cells to adhere to bronchial epithelium, and are likely to be the scaffold for adhesion-mediating galactose-bearing carbohydrates. Most cell-surface proteins are decorated with carbohydrates to some extent, so it is not possible to speculate with any conviction as to whether any currently recognized molecules are involved.
A further study has described the adhesion of HMC-1 cells and human skin mast cells to human fibroblasts. Again, high baseline adhesion was observed, on the order of 95% for HMC-1 cells and 60% for skin mast cells. Adhesion of HMC-1 cells was studied in more detail and was not mediated by a number of known adhesion receptors and sugars, was only partially sensitive to calcium depletion, and was reduced by about 50% with trypsin and pronase [50 ]. These findings are not dissimilar to ours and suggest that human mast cells may adhere to fibroblasts and epithelial cells through a similar mechanism.
IgE-dependent activation of HLMC attenuated mast cell adhesion to
epithelium in a dose-dependent manner. This was an interesting
observation because it is hypothesized in mice that IgE-dependent mast
cell activation will increase adhesion to epithelium through the
up-regulation of Eß7 as discussed above
[17
, 18
]. Also, mouse mast cell adhesion to
laminin is increased after IgE-dependent activation
[51
]. However, adhesion of human skin mast cells to
laminin clearly decreases after incubation with anti-IgE, demonstrating
again important differences between mice and humans
[52
]. The mechanism(s) underlying the
adhesion-modulating effect of IgE-dependent mast cell activation in the
present study is not clear, but could include dilution of a membrane
receptor after granule fusion with the plasma membrane, cleavage of a
surface receptor by a mast cell protease, or FcRI-mediated
down-regulation of a cell-surface receptor. Degranulation in response
to calcium ionophore had no effect on adhesion, suggesting that the
first two mechanisms are unlikely. Furthermore, lysates of HMC-1 or
HLMC were without effect on mast cell adhesion. Interpretation of the
effect of the tryptase inhibitor leupeptin was complicated by the fact
that baseline adhesion in the presence of leupeptin was reduced,
although adhesion in the presence of leupeptin was never higher than
with anti-IgE. Taking the data together, we believe that a
FcRI-mediated down-regulation of a cell-surface receptor is the most
likely explanation for these observations. The biological significance
of this finding can only be speculated upon, but could facilitate mast
cell migration toward the epithelial surface in response to perceived
noxious stimuli, or migration to regional lymph nodes after antigen
exposure [53
].
Finally, it is interesting that mast cells show much greater adhesion to epithelium than eosinophils. This may explain the observation that in spite of there being similar numbers of mast cells and eosinophils co-habiting the bronchial epithelium in asthma [6 ], far greater numbers of eosinophils are found in BAL fluid and sputum than mast cells [20 , 21 ]. This would suggest that there is continuous movement of eosinophils from the submucosa through the epithelium and into the bronchial lumen, whereas mast cells are retained in the epithelium and perhaps direct the traffic.
In summary, we have demonstrated that human mast cells adhere avidly to human bronchial epithelial cells in vitro, and provide evidence that this is mediated by a potentially novel mast cell-carbohydrate-dependent interaction with epithelium.
|
ACKNOWLEDGEMENTS |
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Received November 15, 1999; revised January 26, 2000; accepted January 28, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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