Eosinophils, major basic protein, and polycationic peptides
augment bovine airway myocyte Ca2+
mobilization
Mark E.
Wylam1,2,
Nesli
Gungor1,
Richard W.
Mitchell2, and
Jason G.
Umans2,3
Departments of 1 Pediatrics and
2 Medicine, and
3 Committee on Clinical
Pharmacology, Division of Biological Sciences, University of
Chicago, Chicago, Illinois 60637
 |
ABSTRACT |
Previous studies in vivo or in
isolated airway preparations have suggested that eosinophil-derived
polycationic proteins enhance airway smooth muscle tone in an
epithelium-dependent manner. We assessed the direct effects of
activated human eosinophil supernatant, major basic protein (MBP), and
polycationic polypeptides on basal and agonist-stimulated intracellular
Ca2+ concentrations
([Ca2+]i)
in cultured bovine tracheal smooth muscle (TSM) cells. A 1-h incubation
of myocytes with activated eosinophil buffer resulted in a doubling of
basal
[Ca2+]i
and increased responsivity to histamine compared with myocytes that
were exposed to sham-activated eosinophil buffer. In addition, concentration-dependent acute transient increases and subsequent 1-h
sustained elevations of basal
[Ca2+]i
were observed immediately after addition of MBP and model polycationic proteins. Finally, both peak and plateau
[Ca2+]i
responses to bradykinin addition were augmented significantly in
cultured myocytes that had been exposed to low concentrations of MBP or
model polycationic proteins but were inhibited at greater concentrations. This elevated
[Ca2+]i
to polycationic proteins was manifest in epithelium-denuded bovine TSM
strips as concentration-dependent increased basal tone. We conclude
that activated eosinophil supernatant, MBP, and other polycationic
proteins have a direct effect on both basal and subsequent agonist-elicited Ca2+ mobilization
in cultured TSM cells; TSM strips in vitro demonstrated, respectively,
augmented and diminished responses to the contractile agonist
acetylcholine. It is possible that alteration in myocyte Ca2+ mobilization induced by these
substances may influence clinical states of altered airway tone, such
as asthma.
polycationic proteins; intracellular calcium; airway smooth
muscle
 |
INTRODUCTION |
IT HAS BEEN SUGGESTED THAT eosinophil-derived cationic
proteins might be involved in the pathogenesis of airway
hyperresponsiveness. For example, in airway epithelial cells, major
basic protein (MBP) increases prostaglandin synthesis (35), including
E2 and
F2
, reduces ciliary motility
(15), and induces cytological damage. Prior investigations in vivo (7)
and in vitro (5, 8, 11, 24) have suggested that the effect of MBP on
smooth muscle cell contraction is indirect and likely mediated via
barrier interruption of the airway epithelium or by altering epithelial
mediator release (34). However, in some cells, cationic proteins may
directly alter cellular Ca2+
homeostasis (4, 14); this suggested the possibility that they may
directly augment smooth muscle contraction. We sought to test
explicitly the hypothesis that products of activated eosinophils, including MBP, and synthetic cationic polypeptides directly mobilize Ca2+ in cultured airway smooth
muscle. Furthermore, using the fura 2 fluorescent dye technique, we
tested the hypothesis that cationic proteins would augment
receptor-coupled Ca2+
mobilization. We found that MBP and other polycationic polypeptides directly mobilize intracellular
Ca2+ in a concentration-dependent
manner and that, after a 1-h incubation at lesser concentrations, these
compounds augment bovine tracheal smooth muscle (TSM) responsiveness to
receptor-coupled agonists. These findings were paralleled in studies
conducted using epithelium-denuded bovine tracheal muscle strips in
vitro in which polycationic proteins increased basal tone and augmented
muscarinic responsiveness. These data suggest that products of
eosinophil activation may directly augment airway smooth muscle
responsiveness in inflammatory disease states such as asthma.
 |
METHODS |
Isolation of human eosinophils and eosinophil
activation. Activated eosinophil supernatant was a gift
from Dr. Steven R. White (University of Chicago, Chicago, IL). Briefly
(28), human eosinophils were isolated from volunteers according to a
protocol approved by the University of Chicago Institutional Review
Board. Whole blood was collected, white blood cells were isolated, and
eosinophils were separated over discontinuous colloidal Percoll
gradients. The interface containing eosinophils
(1.095-1.000 g/ml Percoll) was collected and diluted
in Hanks' balanced salt solution containing 1 mM
Ca2+ and 0.1% gelatin. Eosinophil
purity was >90%. Eosinophils were kept on ice and used on the same
day of activation; 3.8 million eosinophils were incubated with
10
6 M
formyl-methionyl-leucyl-phenylalanine (fMLP) plus 5 mM cytochalasin B (cytB) in polypropylene tubes for 30 min at 37°C.
After activation, the tube was placed on ice and then centrifuged at
4°C and 400 g for 10 min. The
activated supernatant was then separated and stored at
70°C
for subsequent use.
Cell culture. Bovine tracheae were
obtained from a local abattoir and transported to the laboratory in
ice-cold
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline [HBS (in mM): 130 mM NaCl, 5.0 mM KCl, 1.0 mM CaCl2, 1.0 mM
MgSO4, 10 mM dextrose, and 10.0 mM
HEPES; pH 7.4] supplemented with antibiotics (see below). TSM was
dissected from the mucosa, cut into
~1-mm3 pieces, and washed two
times in HBS. Tissue was then incubated in 2 ml of HBS containing 0.2%
collagenase (type IV; Sigma, St. Louis, MO) and 0.05% elastase (type
IV; Sigma) for 30 min at 37°C. Approximately 1 g of dissociated
tissue was filtered through a nylon mesh, washed two times with HBS,
and then resuspended in medium 199 (M199) with 1% fetal bovine serum,
100 µl/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml
amphotericin B. After 24-h incubation, the medium was changed to M199
with 20% FBS, and cells were grown to confluence. Isolated cells were
identified by morphological appearance on light microscopy and by
immunofluorescent staining for
-smooth muscle actin (Sigma). Cells
were maintained at 37°C in a 5%
CO2-95%
O2 environment, passaged every
5-7 days, and grown on eight-well Lab-Tek glass coverslides (Nunc,
Naperville, IL) for use in experiments at 1-3 days postconfluence.
Cells from passages 2-6 were used
in these studies; all responses were stable over these passages.
Measurement of intracellular
Ca2+
concentration.
In each well of the coverslide, incubation medium was replaced for 1 h
with activated eosinophil supernatant (100 µl in 300 µl HBS) or 400 µl of HBS alone. For experiments using polycationic proteins,
individual wells were incubated for 1 h in the presence of
10
9 to
10
5 M MBP,
10
8 to
10
3 M
poly-L-arginine (poly-A),
10
8 to
10
5 M
poly-L-lysine (poly-L), or
10
8 to
10
5 M melittin. After
incubation, the cells were rinsed and incubated in HBS with 0.1%
bovine serum albumin containing 5 µM fura 2-AM for 30 min at room
temperature. Cells were washed two times with fresh buffer and
incubated for an additional 30 min to allow for complete hydrolysis of
the ester. The slide was then transferred to the stage of an inverted
Nikon Diaphot microscope for microspectrofluorimetery using a ×10
objective, with the results being taken from confluent fields
containing 50-80 cells. The microscope was coupled to an air
turbine spectrofluorimeter (Biomedical Instrumentation Group, University of Pennsylvannia, Philadelphia, PA). The fluorimeter filter
wheel contained 340- and 380-nm excitation filters and was adjusted to
spin at 100 ± 10 Hz. The wheel was coupled to the microscope via a
fiber-optic light guide. Emitted fluorescence, gated to the position of
the filter wheel, passed through a dichroic mirror and a 510-nm filter
and was measured by a photomultiplier tube (PMT); the current output of
the PMT was converted to voltage input into an encoding amplifier.
Amplifier output was then digitized and sampled with software
(Lakeshore Technologies, Chicago, IL) at 1 Hz by a microcomputer.
Ca2+ concentration (nM) was
calculated using an in vitro calibration using known free
Ca2+ (0-1.35 µM) and
pentapotassium fura 2 (5 µM). The linear plot of the log of the
Ca2+ concentration versus the log
of the 340- to 380-nm fluorescence ratio [(R
Rmin)/(Rmax
R) × (380min/380max),
where R is ratio, Rmin is the
minimum ratio, Rmax is maximum
ratio, 380min is minimum fluorescence at 0 Ca2+, and
380max is the maximum fluorescence
at 1.35 µM Ca2+] was plotted to
determine the dissociation constant
(Kd) after subtraction of unloaded cell and system background at each wavelength. Total field intracellular Ca2+ was
calculated from experimental ratios by the equation of Grynkiewicz et
al. (12) using Rmax,
Rmin, and
Kd from the
calibration plot; selected standards were run daily. In all cells,
Ca2+ responses were acquired for
20 s to establish average baseline intracellular
Ca2+ and for 300 s to characterize
peak and plateau responses after the acute addition of activated
eosinophil supernatant, MBP, and model cationic proteins and the
subsequent addition of bradykinin (BK;
10
5 M) or histamine (Hist;
10
4 M) after the 1-h
incubation period (see above).
Preparation of TSM strips. Bovine
tracheae were obtained from a local abattoir. The tracheae were placed
in 4°C Krebs-Henseleit (KH) solution of the following composition
(in mM): 115 NaCl, 25 NaHCO3, 1.38 NaH2PO4,
2.51 KCl, 2.46 MgSO4 · 7H2O,
1.92 CaCl2, and 11.2 dextrose. The
KH solution was gassed with a mixture of 95%
O2-5%
CO2 gas to maintain a pH of
7.35-7.45 (19, 20). A 10-cm midtracheal segment was cleaned of
overlying connective tissue. The posterior aspect of the tracheal
cartilage was clipped away, and the connective tissue lying between the
cartilage rings and the smooth muscle was cut. The opened posterior
aspect of the TSM was cleaned of overlying connective tissue by careful dissection, and the ends of the rings were cut away. The major portion
of the ventral cartilage rings was cut away, and the remaining segment
of trachea was pinned epithelium side up to a dissecting tray. Under
cold KH, the epithelium was dissected away from the smooth muscle; care
was taken not to overstretch the underlying smooth muscle layer. Eight
tracheal preparations (~2 mm wide and 10 mm in length) were
dissected. Each tracheal ring preparation was tethered at the junctions
of the posterior membrane and adjacent cartilage with 3-0 silk
thread (Deknatel). Each TSM preparation was randomly assigned to one of
the treatment groups described below.
Tissue perfusion. The TSM strips were
mounted in 15-ml glass tissue baths (Easterbrooke, Winnipeg, MB)
containing 10 ml of continuously gassed KH at 37°C. One end of the
TSM strip was attached with a loop of 3-0 braided silk suture to a
rigidly held glass rod within the bath. The upper end was fastened to a
Grass model FT.03 force displacement transducer. This transducer was
mounted on a rack-and-pinion system to enable the muscle preparation to be stretched to optimal length (20). Isometric tension was recorded digitally with a microcomputer. An initial resting
load (isoproterenol insensitive) of 1.0-2.0 g was
applied to each TSM strip; this resting load stretched bovine TSM
preparations to approximately optimal length to allow for optimal
contractile responses to electrical field stimulation (EFS) and
acetylcholine (ACh).
Active equilibration with EFS. To
establish maximal and reproducible isometric contraction, tissues were
equilibrated initially for 60 min as follows (20, 29). Each TSM strip
received supramaximal EFS (60-Hz alternating current, 10 V,
10-s duration) applied through platinum wire electrodes placed on
either side and parallel to the TSM preparation. Strips were stimulated
every 15 min and rinsed with fresh KH every 15 min (20, 29). During
this time, a limited length-tension study for each strip was conducted
to determine optimal length and response to EFS. All subsequent data
were normalized to this maximal contractile response to EFS (20).
Response to model polycationic
proteins. Bovine TSM strips were randomly assigned to
receive no polycationic protein (control), a concentration of poly-A
(10
6 to
10
4 M), or a concentration
of poly-L (10
7 to
10
5 M). These
concentrations were determined based on the
Ca2+ studies. Tissues were
incubated for 1 h in the presence or absence of protein. Resting tone
was noted before and after polycationic protein exposure. All TSM
preparations were then washed with fresh KH buffer.
Concentration-response curves to ACh.
After incubation with polycationic protein, each bovine TSM strip
(n = 80 preparations from 10 animals)
was subjected to an ACh concentration-response study. Cumulative
concentration-response curves were generated with
10
9 to
10
3 M ACh. The next greater
concentration of ACh was not added until either 5 min had elapsed
and/or a plateau response to the previous concentration had
been achieved.
Reagents. MBP was a gift from Dr.
Gerald J. Gliech (Mayo Clinic and Mayo Foundation, Rochester, MN) and
was isolated as described previously (1). BK, Hist, poly-A HCl (mean
mol wt = 10,800), poly-L hydrobromide (poly-L; mean mol
wt = 123,900), and melittin (purity 85%, phospholipase
A2 impurity <5 U/mg; mol wt = 2,848) were dissolved in HBS and were obtained from Sigma. Fura 2-AM and Ca2+ standards were obtained
from Molecular Probes (Eugene, OR).
Statistical analysis. Data are
expressed as group means ± SE where n is the number of
fields of 50-80 cells analyzed. Group comparisons were by ANOVA
with Bonferroni correction for multiple comparisons;
P values
0.05 were considered
significant.
 |
RESULTS |
Acute and 1-h exposure to eosinophil-activated buffer on basal and
Hist-elicited
Ca2+
mobilization.
The acute addition of fMLP, cytB, or eosinophil-activated buffer did
not immediately alter basal intracellular
Ca2+ concentration
([Ca2+]i).
There were no effects on basal or agonist-stimulated
[Ca2+]i
after a 1-h incubation of myocytes with fMLP or cytB. However, a 1-h
incubation of myocytes with eosinophil-activated buffer resulted in an
increased basal level (Fig. 1) compared
with myocytes exposed to sham-activated buffer. In preliminary studies,
BK alone (10
5 M) elicited
an increase in peak
[Ca2+]i
above basal level of 477 ± 79 nM, which was not significantly different from the effect of
10
5 M BK in cells incubated
in eosinophil-activated buffer (475 ± 88 nM,
n = 4). However, eosinophil-activated
buffer incubation significantly increased peak
[Ca2+]i
elicited by 10
4 M Hist
(Fig. 1).

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Fig. 1.
Influence of 1-h incubation with either sham (open bars) or
eosinophil-activated (filled bars) buffer on basal and histamine
(10 4 M)-elicited
incremental increase in peak intracellular
Ca2+ concentration
([Ca2+]i).
Values ± SE indicate the increase in
[Ca2+]i
(n = 4 in each group).
* P < 0.05 and
P < 0.001 compared with
respective control.
|
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Effect of acute exposure to MBP and cationic polypeptides on
Ca2+ transients.
After the acute addition of HBS (100-µl control addition),
[Ca2+]i
increased maximally 5 ± 1 nM above resting values of 45 ± 2 nM
(n = 35). Low concentrations of
(10
7 M) poly-A, MBP, and
melittin led to significant rapid, although transient, increases in
[Ca2+]i
compared with buffer. Peak increases were 20 ± 13 nM
(n = 5) for poly-A, 18 ± 8 nM
(n = 6) for MBP, and 26 ± 9 nM
(n = 4) for melitin
(P < 0.007, 0.002, and 0.0001 vs.
control, respectively; Fig. 2; sample
traces 10
7 and
10
5 M MBP). Poly-L
(10
7 M) did not elicit an
acute increase in basal
[Ca2+]i.
However, greater concentrations
(10
5 M) of poly-L, poly-A,
and MBP elicited significant increases in basal
[Ca2+]i
compared with control myocytes [poly-A, 18 ± 5 nM
(n = 7); poly-L, 128 ± 27 nM (n = 7); MBP, 78 ± 28 nM
(n = 5);
P < 0.0002, 0.0001, and 0.0001 vs.
control, respectively]. Moreover, melittin (10
5 M) elicited increases
in basal
[Ca2+]i
by >1 µM, an amount that exceeds precise quantification using fura
2 (P < 0.00001 vs. control,
n = 13), and was associated with morphological cellular injury.

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Fig. 2.
Sample traces of
[Ca2+]i
vs. time, showing the transient effect of
10 7 M
(A) and
10 5 M
(B) major basic protein (MBP; arrow)
in confluent bovine tracheal smooth muscle cells on
[Ca2+]i.
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Effect of 1-h incubation with MBP and cationic polypeptides on basal
[Ca2+]i.
In control cells incubated for 1 h with HBS, basal
[Ca2+]i
remained unchanged (Figs. 3 and
4; control). However, after incubation for
1 h, poly-A, poly-L, and melittin elicited, at sufficient concentration, an increase in basal
[Ca2+]i.
This increase was concentration dependent and significant at
concentrations of
10
5 M
for poly-A and MBP, and 10
6
M for poly-L and melittin (Fig. 4).

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Fig. 3.
Sample traces of effect of addition (first arrow) of either control
buffer or MBP (10 9 M to
10 5 M) on
[Ca2+]i.
Subsequent to 1-h incubation (axis break), the influence of increasing
concentrations of MBP are noted upon addition (second arrow) of
bradykinin (BK; 10 5 M).
A: control;
B:
10 9 M MBP;
C:
10 8 M MBP;
D:
10 7 M MBP;
E:
10 6 M MBP;
F:
10 5 M MBP.
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Fig. 4.
Influence of 1-h incubation with poly-L-arginine (poly-A;
A), poly-L-lysine (poly-L; B), MBP
(C), or melittin (D) on basal
[Ca2+]i.
Values indicate the increase above basal
[Ca2+]i
(n = 5-11 for poly-A, 4-15
for poly-L, 6-22 for MBP, and 6-15 for melittin).
* P < 0.05, ** P < 0.001, and
P < 0.0001 compared with
respective control.
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Effect of 1-h incubation with MBP and cationic polypeptides on
BK-elicited peak and plateau
Ca2+
mobilization.
A 1-h incubation of bovine airway myocytes with polycationic proteins
produced a biphasic response to subsequent BK-elicited Ca2+ mobilization (Fig.
5). In control cells, BK
(10
5 M) elicited a peak
[Ca2+]i
of 336 ± 19 nM (n = 20). At
relatively lower concentrations of poly-A
(10
8 to
10
7 M), poly-L
(10
7 to
10
6 M), and MBP
(10
9 to
10
8 M), BK-elicited peak
[Ca2+]i
was significantly augmented compared with control cells (Fig. 4).
However, at relatively high concentrations of each agent (poly-A
10
4 M, poly-L
10
5 M, and MBP
10
5 M), the magnitude of
BK-elicited peak
[Ca2+]i
was substantially attenuated compared with control cells (Fig. 5). At relatively lower concentrations of
melittin (10
8 to
10
7 M), there was a
tendency for BK-elicited peak
[Ca2+]i
to be enhanced in some cells compared with controls; however, the
response was widely variable. In addition, at concentrations greater
than 10
7 M, melittin caused
a direct and irreversible release of
Ca2+ such that there was no
discernible response to the subsequent addition of BK. In control cells
300 s after the addition of BK, the sustained plateau elevation of
[Ca2+]i
[68 ± 3 nM (n = 43)]
was significantly greater than basal level [45 ± 2 (n = 62);
P < 0.0001]. This
value was concentration dependently augmented by poly-L, increased
significantly only at the highest concentrations of poly-A and MBP
tested, and was concentration dependently increased at low
concentrations (10
8 and
10
7 M) of melittin, with no
response obtained at higher concentrations (Fig. 6).

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Fig. 5.
Effect of 1-h incubation with poly-A
(A), poly-L
(B), MBP
(C), or melittin
(D) on the incremental increase in
peak
[Ca2+]i
elicited by 10 5 M BK.
Values indicate the increase above basal
[Ca2+]i
(n = 4-11 for poly-A, 4-11
for poly-L, 8-22 for MBP, and 6-13 for melittin).
* P < 0.05, ** P < 0.001, and
P < 0.0001 compared with
respective control.
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Fig. 6.
Effect of 1-h incubation with poly-A
(A), poly-L
(B), MBP
(C), or melittin
(D) on the incremental increase in
the plateau value of
[Ca2+]i
elicited by 10 5 M BK
(n = 5-20 for control, 4-16
for poly-A, 6-13 for poly-L, 8-24, and 6-10 for
melittin). * P < 0.05, ** P < 0.001, and
P < 0.0001 compared with
respective control.
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Response of TSM strips to model polycationic
proteins. Over the incubation period of 1 h, basal tone
of bovine TSM strips increased in response to polycationic proteins
(Fig. 7); control tissues maintained
baseline resting tone (Fig. 8). The
increase in basal tone was dependent on the exposure concentration of
polycationic protein. For
10
7 M poly-L, resting tone
did not increase significantly (4.4 ± 4.3% EFS). However, for
10
5 M poly-L, resting tone
increased to 28.5 ± 9.9% EFS (P < 0.001 vs. initial; P < 0.05 vs.
10
7 M poly-L). For
10
6 M poly-A, resting tone
increased to 9.9 ± 4.1% EFS (P < 0.01); for 10
4 M
poly-A, resting tone increased to 36.3 ± 8.9% EFS
(P < 0.0005 vs. initial;
P = 0.01 vs.
10
6 M poly-A).

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Fig. 7.
Representative myogram of the effect of
10 5 M poly-L on bovine
tracheal smooth muscle (TSM) responsiveness. After equilibration and
excitation by electrical field stimulation (EFS),
10 5 M poly-L was added, and
basal tone was monitored for 1 h. After 5 min, basal tone (1.5 g) began
to increase; after 60 min, tone was elevated to 5.9 g. The bovine TSM
strip was rinsed with buffer 3 times, and responses to increasing
concentrations of ACh were elicited.
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Fig. 8.
Effect of polycationic protein exposure on basal tone of bovine TSM.
Basal tone (expressed a percent of each tissue's maximal response to
EFS; %EFS) increased in response to exposure to both poly-L and poly-A
in concentration-dependent manners for bovine TSM strips. Control
tissues did not demonstrate significant changes in resting tone over
the 1-h incubation period (n = 6-10 for each group). * P < 0.05 and ** P < 0.001 compared with control.
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Subsequent response to ACh. Lesser
concentrations of polycationic proteins augmented and greater
concentrations reduced maximal contractile responses of bovine TSM
preparations to ACh (Fig. 9). Tissues
incubated with 10
7 M poly-L
demonstrated a maximal contractile response to
10
3 M ACh of 194.0 ± 6.2% EFS compared with 165.3 ± 9.3% EFS
(P = 0.04) for control TSM strips,
respectively. However, greater concentrations (10
5 M) of poly-L reversed
this augmentation (Fig. 9).

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Fig. 9.
Effect of polycationic protein exposure on bovine TSM response to ACh.
Lesser concentrations of poly-L and poly-A augmented and greater
concentrations reduced contractile responses of bovine TSM preparations
to ACh compared with control tissues
(n = 6-10 for each group).
* P < 0.05 and
** P < 0.01 vs. preincubation
values for basal tone.
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Tissues incubated with 10
6
M poly-A demonstrated a similar trend as
10
7 M poly-L in that a
maximal response to 10
3 M
ACh (181.7 ± 10.4% EFS) was augmented; however, this increase in
contraction was not significantly different from the control responses
(Fig. 8). However, preparations incubated with
10
5 M poly-A (126.1 ± 12.6% EFS, P < 0.05 vs. control;
P = 0.01 vs. 10
6 M poly-A; Fig. 9)
demonstrated a reduction in the maximal contractile response.
 |
DISCUSSION |
Evidence of increased epithelial permeability (36), cytotoxicity (18),
and/or bronchoconstrictor mediator release (27, 28, 30, 34)
clearly supports an indirect effect of polycations, favoring smooth
muscle contraction in intact airway preparations. By contrast, we
sought to determine the direct effects of products of activated
eosinophils, graded concentrations of MBP, and other model polycations
on cultured bovine tracheal myocyte
Ca2+ mobilization. The synthetic
polycations poly-A and poly-L are of similar size and cationic charge
as eosinophil MBP, and in past studies (7), the indirect changes in
airway hyperresponsiveness have been similar for proteins of similar
charge, although the precise mechanism(s) of action of MBP and the role
of its cationic charge remain to be defined. In addition, we compared
the above results with the direct effect of the amphiphilic peptide
melittin, which is known to increase the activity of phosphoinositide
phospholipase through a cationic interaction (22). A further objective
was to assess the effects of these substances on agonist-elicited Ca2+ mobilization and airway
smooth muscle contraction. We used cultured airway smooth muscle cells
so that responses could be studied independent of other airway cell
types, particularly epithelium and neuronal tissue. We also used
epithelium-denuded bovine TSM strips to assess the direct effects of
model polycationic proteins on these tissue preparations. Organ bath
volumes and availability of MBP precluded the use of this
eosinophil-derived polycationic protein in studies on bovine TSM
strips.
Activated eosinophils applied to guinea pig tracheal epithelium
increase basal tone and muscarinic responsiveness (13, 28). Because
this effect is blocked by pretreatment with a 5-lipoxygenase inhibitor,
epithelial synthesis of leukotriene
C4 may mediate the enhanced airway
responsiveness in this species. In contrast, when A-23187-stimulated
eosinophils are added either luminally or adventially to intact bovine
bronchial segments, there is no effect on either basal tone or ACh
responsiveness (24). Because the mode of eosinophil activation may
alter its effects on smooth muscle (28), we used human peripheral blood
eosinophils stimulated by both fMLP and cytB. This method of eosinophil
activation stimulates production of eosinophil peroxidase and leads to
increases in both basal tone and muscarinic responsiveness for
epithelium-intact tissues in guinea pigs (28). A new finding of this
investigation is a direct effect of activated eosinophil supernatant on
cultured airway myocytes. One hour of incubation of cultured bovine
tracheal myocytes with this activated eosinophil supernatant not only
doubled resting myocyte
[Ca2+]i
but also augmented Hist-elicited
Ca2+ mobilization. However,
subsequent myocyte responses to BK were not affected by incubation with
eosinophil supernatant; this differs from results obtained with MBP
(see below). Dissimilar effects on the agonist
Ca2+ response elicited by BK vs.
Hist may be due to differing signal transduction mechanisms or
competitive signalling due to other smooth muscle modifying mediators
found in activated eosinophil supernatant.
Second, we found that both MBP and model polycations led to
concentration-dependent acute transient increases and a subsequent 1-h
sustained elevation in basal
[Ca2+]i
in these same airway myocytes (Figs. 1-3). We also found that peak
and plateau values for
[Ca2+]i
in response to BK were significantly greater in cultured myocytes exposed to relatively low concentrations of MBP and other polycationic proteins (Figs. 4 and 5). Incubation with greater concentrations of MBP
and polycationic proteins increased basal
[Ca2+]i
to the extent that subsequent responses to BK were attenuated (Fig. 4).
As with melittin, greater concentrations of MBP and analogs may
adversely affect cellular and Ca2+
homeostasis. In the absence of other agonists, MBP and other model
polycations led to concentration-dependent acute and sustained Ca2+ mobilization in these
cultured cells.
A third finding of our study was that the elevated
[Ca2+]i
could be manifest functionally by increased basal tone in
epithelium-denuded bovine TSM preparations (Fig. 6). Parallel to our
findings for [Ca2+]i,
basal tone increased with exposure to model polycationic proteins in a
concentration-dependent manner (Fig. 7). Also, low concentrations of
poly-L augmented and high concentrations of poly-A reduced subsequent
responses to ACh (Fig. 8).
These findings contrast with conclusions of prior investigations
carried out in intact tissue models that suggest an intact airway
epithelium is required for the effect of MBP on enhanced airway
responsiveness (5, 11, 34). Several studies have examined the effect of
MBP on airway responsiveness only in epithelium-intact preparations (7,
30). Others have clearly demonstrated an epithelium dependence to MBP
enhancement of airway responsiveness in some species. Flavahan et al.
(11) in vitro and White et al. (34) in situ noted that, in
epithelium-denuded preparations, MBP did not directly affect the tone
of guinea pig trachea and did not increase sensitivity to contractile
agonist. Similarly, Brofman et al. (5) noted that, although the
intraepithelial administration of MBP augmented contraction of
underlying canine TSM, this effect was absent after epithelial removal
or direct injection of MBP into trachealis muscle. However, none of
these previous studies was able to assess the direct
effects of MBP or polycationic proteins on airway
smooth muscle myocytes, and conclusions drawn were limited by the
potential effects of these substances on neuronal tissue and other
cells, such as mast cells, in the whole airway preparation.
The synthetic cationic proteins poly-A, poly-L, and melittin are of
similar size and cationic charge as eosinophil-derived MBP and have
been shown to augment muscarinic airway hyperresponsiveness (7, 32), an
effect that may be related to cationic charge density. However, as with
MBP, previous studies have either examined only epithelium-intact
tissues (27, 30, 32) or determined that model cationic proteins
influence airway hyperresponsivenss in an epithelium-dependent manner
(8, 24). In addition to an epithelium-mediated effect, Spina and Goldie
(27) found that poly-A caused tracheal contraction via the release of
ACh from parasympathetic nerves and/or by the release of mast
cell-derived serotonin. Similarly, Strek et al. (30) determined that
model cationic proteins may mediate airway contraction via the
cyclooxygenase pathway (30). Although the present results do not
diminish the contributing effects of epithelium-dependent and
neuron-dependent influences, they point to a significant possibility
for an additional direct effect of eosinophil-derived cationic proteins
on airway myocyte stability and activity in vivo.
Eosinophil-derived cationic proteins appear to be released continually
into the blood of patients with stable persistent asthma (6), and
eosinophil infiltration may be found in the lamina propria and smooth
muscle layer of subjects with asthma (10, 16, 23). Thus MBP may be
continually released in high concentrations by eosinophils interspersed
and migrating among airway myocytes or in adjacent subepithelium.
Therefore, the acute experimental addition of MBP or model cationic
proteins to intact tissue preparations may fail to permeate myocytes
sufficiently to produce a direct effect on myocyte contractility.
In addition to direct effects on airway myocyte
Ca2+ and contraction, we also
found a biphasic effect of MBP and cationic proteins on
receptor-stimulated Ca2+
mobilization; each agent tested augmented peak BK-elicited
Ca2+ responses. By contrast,
progressively increasing concentrations of these substances inhibited
peak Ca2+ mobilization in response
to BK while continuing to elicit sustained elevation in myocyte
Ca2+ concentration. In the case of
melittin, this was associated with morphologically evident cell injury.
In a variety of cells, MBP and polycations may interact with
cell-surface anions and integral membrane proteins and ultimately may
lead to cytotoxicity. Like our results, Ayars et al. (3) found that MBP
effects, in their case epithelial cytotoxicity after high doses,
occurred only after a significant time delay. Similarly, we noted that
persisting MBP effects on myocyte
Ca2+ metabolism required at least
a 1-h exposure to 10
7 M. Perhaps continuous exposure to even lesser concentrations of MBP at the
level of the individual myocyte in vivo may alter airway contractility.
Cationic proteins may induce myoepithelial cellular contraction by
histochemical alteration of actin filaments as has been shown in canine
kidney epithelial cells (25). These effects are often associated with
receptor inhibition or synthesis and release of other mediators. Others
have shown that MBP or cationic proteins cause prostaglandin synthesis
in cultured guinea pig tracheal epithelium (35), rat glomerular
mesangial cells (2), fibroblasts (26), and endothelial cells (21). Thus it is possible that these agents stimulated eicosanoid
synthesis/release in our cultured airway myocytes, perhaps through a
Ca2+- and phospholipase
C-dependent mechanism (17). As well, polycations may specifically
inhibit Ca2+-ATPase (4), inhibit
sarcolemmal
Na+-Ca2+
exchange (22), stimulate the activity of phophatidylinositol 4-kinase
(33), or activate phospholipase
A2- and glibenclamide-sensitive potassium channels (9). In addition, MBP may activate a pertussis toxin-sensitive G protein (31). All of these potential mechanisms would
lead to augmented contractile responses in airway smooth muscle cells.
In summary, we found that eosinophil supernatant, MBP, and model
polycations cause elevation in basal airway myocyte
Ca2+. Moreover, in a biphasic
manner, low concentrations of these agents augmented receptor-coupled
Ca2+ mobilization, whereas high
concentrations inhibited these events, and, in the case of melittin,
induced cytotoxic damage. These changes in
Ca2+ mobilization to MBP were
manifest functionally in bovine TSM strips by
1) concentration-dependent,
increased basal tone with exposure to model polycationic proteins,
2) augmentation of contractile response to low concentrations of polycationic protein, and
3) reduced contractile response to
ACh after exposure to high concentrations of poly-L or poly-A. In
airways, these events could lead to direct contraction of airway smooth
muscle. Although these events may, in other preparations, be influenced
by epithelial or neuronal factors, they do not depend exclusively on
contributions by these other cells. Moreover, the in vivo
time-dependent effects of continuous or repeated exposure of myocytes
to cationic proteins remain unknown. However, our data demonstrate the
potential for products of eosinophil inflammation to directly alter
airway smooth muscle tone in disease states such as asthma.
 |
ACKNOWLEDGEMENTS |
We thank Claire Buchanan who performed preliminary studies on the
effect of activated eosinophil supernatant on cultured bovine tracheal
myocyte Ca2+ mobilization. We
thank Gerald J. Gleich, Department of Immunology, Mayo Clinic,
Rochester, MN, for the purified major basic protein used in this study.
 |
FOOTNOTES |
This research was supported in part by the Ralph S. Zitnik Clinical
Investigatorship Award from the Chicago Heart Association (to M. E. Wylam), by a career development award in clinical pharmacology from the
Pharmaceutical Research and Manufacturers of America Foundation (to J. G. Umans), and by National Heart, Lung, and Blood Institute Grant
HL-48302 (to J. G. Umans).
Address for reprint requests and present address of M. E. Wylam: Mayo
Clinic and Mayo Foundation, 200 First St., SW, Rochester, MN
55905-0001.
Received 15 April 1997; accepted in final form 18 February
1998.
 |
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