Antigen-induced hyperreactivity to histamine: role of the
vagus nerves and eosinophils
Richard W.
Costello1,
Christopher M.
Evans1,
Bethany
L.
Yost1,
Kristen E.
Belmonte1,
Gerald J.
Gleich2,
David B.
Jacoby1,3, and
Allison D.
Fryer1
1 Department of Environmental
Health Sciences, School of Hygiene and Public Health, and
3 Department of Medicine, Johns Hopkins
University, Baltimore, Maryland 21205; and
2 Departments of Immunology and
Medicine, Mayo Clinic, Rochester, Minnesota 55905
 |
ABSTRACT |
M2
muscarinic receptors limit acetylcholine release from the pulmonary
parasympathetic nerves. M2
receptors are dysfunctional in antigen-challenged guinea pigs, causing
increased vagally mediated bronchoconstriction. Dysfunction of these
M2 receptors is
due to eosinophil major basic protein, which is an antagonist for M2 receptors. Histamine-induced
bronchoconstriction is composed of a vagal reflex in addition to its
direct effect on airway smooth muscle. Because hyperreactivity to
histamine is seen in antigen-challenged animals, we hypothesized that
hyperreactivity to histamine may be due to increased vagally mediated
bronchoconstriction caused by dysfunction of
M2 receptors. In anesthetized,
antigen-challenged guinea pigs, histamine-induced bronchoconstriction
was greater than that in control guinea pigs. After vagotomy or
atropine treatment, the response to histamine in antigen-challenged
animals was the same as that in control animals. In antigen-challenged
animals, blockade of eosinophil influx into the airways or
neutralization of eosinophil major basic protein prevented the
development of hyperreactivity to histamine. Thus hyperreactivity to
histamine in antigen-challenged guinea pigs is vagally mediated and
dependent on eosinophil major basic protein.
muscarinic receptors; parasympathetic nerves; inflammation; major
basic protein; adhesion molecules
 |
INTRODUCTION |
STIMULATION of the pulmonary parasympathetic nerves
releases acetylcholine, which causes airway smooth muscle contraction by activation of M3 muscarinic
receptors (28). The release of acetylcholine from the parasympathetic
nerves is inhibited by M2
muscarinic receptors on these nerves (13). Stimulation of the neuronal
M2 muscarinic receptors with
agonists such as pilocarpine decreases the release of
acetylcholine and thus limits vagally induced bronchoconstriction by as
much as 70% (3, 13). Conversely, M2-receptor antagonists such as
gallamine or methoctramine can potentiate vagally induced
bronchoconstriction by as much as fivefold by blocking the negative
feedback control over acetylcholine release that these receptors
provide (3, 13-15, 32). Thus the neuronal M2 muscarinic receptors play a
pivotal role in limiting vagally induced bronchoconstriction.
The function of neuronal M2
muscarinic receptors is decreased in antigen-challenged guinea pigs
(16, 30), mice (20), and rats (2), as well as in some patients with
asthma (1, 25). Inhibition of the influx of eosinophils into the
airways of antigen-challenged guinea pigs with antibodies to either
interleukin-5 or the eosinophil adhesion molecule very late activation
antigen-4 (VLA-4) preserves the function of the
M2 receptor (10, 12). In vitro,
eosinophil major basic protein is an antagonist at these receptors
(18), whereas in vivo, pretreatment with an antibody to eosinophil
major basic protein prevents loss of neuronal
M2 muscarinic-receptor function
and prevents hyperreactivity to vagal nerve stimulation (11, 21).
Histologically, eosinophils are found in association with cholinergic
airway nerves after antigen challenge (7). In addition, the number of
eosinophils per nerve correlates with the degree of
M2-receptor dysfunction (7). In
humans, eosinophils are clustered around and along the airway nerves in
sections of airway from fatal asthmatics. Extracellular eosinophil
major basic protein has also been deposited on the airway nerves (7).
Thus loss of function of neuronal
M2 muscarinic receptors after
antigen challenge of sensitized animals is mediated by eosinophils and
eosinophil major basic protein. Eosinophils and
M2-receptor function may also
contribute to the hyperreactivity associated with asthma.
Exposure of sensitized animals and allergic asthmatics to an antigen
characteristically results in hyperreactivity of the airways.
Hyperreactivity is commonly measured as increased contractile responses
to a variety of agents including histamine (4-6, 29). Histamine
constricts airway smooth muscle by a direct effect on the muscle and
indirectly by stimulating a reflex that releases acetylcholine from the
vagus nerves (8, 19, 24). Because neuronal
M2 muscarinic receptors limit
vagally induced bronchoconstriction, it would be expected that
M2 muscarinic-receptor dysfunction
in antigen-challenged animals may increase a vagally mediated reflex. In these studies, we tested whether hyperreactivity to histamine in
antigen-challenged animals is the result of an exaggerated vagal
reflex. Because antagonism of M2
muscarinic receptors by eosinophil major basic protein may be
responsible for the loss of function of these receptors, we also
investigated the role of eosinophils in antigen-induced hyperreactivity
to histamine.
 |
METHODS |
Specific pathogen-free guinea pigs
(Dunkin-Hartley, 200-250 g) were purchased from
Hilltop (Scottsdale, PA). Guinea pigs were shipped in filtered crates
and housed in laminar flow hoods in clean rooms. All guinea pigs were
handled in accordance with the standards established by the US 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.
Sensitization and challenge. Guinea
pigs were injected intraperitoneally with 10 mg/kg of ovalbumin on
days 1, 3, and
5. Three weeks later, the sensitized
guinea pigs were exposed to an aerosol of 5% ovalbumin for 5 min
either on a single occasion or daily for 4 days. Sensitization was
confirmed by demonstrating that ovalbumin (250 mg/kg iv) administered
at the end of the experiment in some randomly chosen animals caused a
rapid sustained rise in pulmonary inflation pressure
(Ppi). In
contrast, ovalbumin had no effect on
Ppi in
nonsensitized animals.
Eosinophil-blocking antibodies.
Sensitized animals were pretreated with either rabbit polyclonal
antibody to eosinophil major basic protein (1 ml ip) or control rabbit
serum (1 ml ip) 1 h before antigen challenge (11). In other
experiments, sensitized guinea pigs were pretreated with
HP1/2 (4 mg/kg ip), a mouse anti-human antibody to
VLA-4, 1 h before each of the four antigen challenges (12, 33).
Measurement of
Ppi.
The experiments were carried out 18-24 h after the exposure of
sensitized guinea pigs to ovalbumin or for the nonchallenged control
group on day
26. The guinea pigs were anesthetized
with urethan (1.5 mg/kg ip). None of the experiments lasted longer than
3 h, although this dose of urethan produces a deep anesthesia lasting
8-10 h (17). However, because paralyzing agents were used, the
depth of anesthesia was monitored by observing for fluctuations in
heart rate and blood pressure.
Once the guinea pigs were anesthetized, cannulas were placed into both
jugular veins for the administration of drugs. Each animal's
body temperature was maintained at 37°C with a homeothermic heating blanket (Harvard, Cambridge, MA).
The animals were ventilated with a positive-pressure constant-volume
animal ventilator (Harvard) and were paralyzed with succinylcholine chloride (infused at 10 µg · kg
1 · min
1).
Ppi was measured with a pressure
transducer (Spectromed DTX, Oxnard, CA). All signals
were displayed on a Grass polygraph (Quincy, MA).
The baseline
Ppi
of the anesthetized guinea pigs was 70-150
mmH2O. Bronchoconstriction was
measured as the increase in
Ppi over the
baseline Ppi
produced by the ventilator (9, 13). A change in
Ppi probably reflects changes in
both resistance and compliance (3, 9). The sensitivity of the method
was increased by taking the output
Ppi signal from the driver to the
input of the preamplifier of a second channel on the polygraph. Thus
Ppi was recorded on one channel, and increases in
Ppi were recorded on a separate
channel at a greater amplification. With this method, increases in
pressure as small as 2 mmH2O could
be recorded accurately.
Histamine-induced bronchoconstriction.
All animals were pretreated with guanethidine (10 mg/kg iv), and 30 min
later, increasing doses of histamine sulfate (1-20 nmol/kg iv)
were administered. There was an interval of at least 5 min before each
dose of histamine. The rise in Ppi
above baseline in response to histamine was recorded and compared
between groups of animals.
Animals served as their own controls, and bronchoconstriction to
histamine was compared before and after vagotomy. Experiments were also
performed in animals in which the vagi were intact and the
bronchoconstriction to histamine was compared with histamine-induced bronchoconstriction in animals studied only after vagotomy.
Reagents. Atropine, guanethidine,
histamine, pilocarpine, normal rabbit serum, succinylcholine chloride,
and urethan were all purchased from Sigma (St. Louis, MO). Ovalbumin
(grade II) was also purchased from
Sigma. Methoctramine was purchased from ICN Chemicals
(Aurora, OH). The antibody to VLA-4 was a generous gift from Dr. R. Lobb (Biogen, Cambridge, MA). The antibody to eosinophil major basic
protein was isolated as previously described (22, 31).
Statistics. Differences in baseline
pulmonary inflation and responses to methoctramine were compared
between groups with an analysis of variance. Differences in the
increase in Ppi between groups of
animals were compared with an analysis of variance for repeated measures.
 |
RESULTS |
With the vagus nerves intact, the baseline
Ppi was 99.2 ± 6.7 mmH2O in control animals, 97.8 ± 4.9 mmH2O in single
antigen-challenged animals, and 107.5 ± 7.5 mmH2O in repeatedly challenged
animals. In animals studied only after the vagus nerves were cut, the
Ppi was 100.8 ± 7.3 mmH2O in control animals, 107.5 ± 6.7 mmH2O single antigen-challenged animals, and 105 ± 9.2 mmH2O in repeatedly challenged
animals. None of the baseline
Ppi values
were significantly different from each other.
Effect of histamine on
Ppi.
Preliminary studies indicated that when the vagus nerves were intact,
doses of histamine > 20 nmol/kg frequently caused a fatal bronchoconstriction, in particular in
antigen-challenged animals. Thus, in the experiments reported here,
the maximum dose of histamine was 20 nmol/kg iv.
With the vagus nerves intact, histamine (1-20 nmol/kg iv) induced
a dose-dependent increase in
Ppi in
control animals (Fig. 1,
). Vagotomy did not alter the response to
histamine in these control animals (Fig. 1,
). In
antigen-sensitized guinea pigs, histamine-induced bronchoconstriction
was significantly increased after either a single antigen challenge
(P = 0.0001; Fig. 1,
) or repeated antigen challenges
(P = 0.0001; Fig.
2,
)
compared with their respective control animals. There was no difference in the response to histamine between control and antigen-challenged guinea pigs after vagotomy (Fig. 1).

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Fig. 1.
Hyperreactivity to histamine is vagally mediated in sensitized guinea
pigs studied 24 h after a single antigen challenge. Histamine
(1-20 nmol/kg iv) induces a dose-dependent bronchoconstriction,
measured as a rise in pulmonary inflation pressure
(Ppi), in control and
antigen-challenged guinea pigs. Values are means ± SE. With vagus
nerves intact, response to histamine is significantly increased in
antigen-challenged guinea pigs (n = 6)
compared with control guinea pigs (n = 6). After vagotomy, there is no difference in response to histamine
between control and challenged animals.
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Fig. 2.
Hyperreactivity to histamine is inhibited by daily pretreatment with
antibody to very late activation antigen-4 (AbVLA-4) in sensitized
guinea pigs studied 24 h after repeated antigen challenge. Values are
means ± SE. Histamine (1-20 nmol/kg iv) induced a
dose-dependent bronchoconstriction, measured as a rise in
Ppi in antigen-challenged guinea
pigs (n = 6). Response to histamine in
antibody-pretreated, antigen-challenged guinea pigs
(n = 4) with vagus nerves intact was
same as that seen in control guinea pigs
(n = 5).
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|
In the above experiments, each animal served as its own control, with
dose-response curves being performed before and after vagotomy.
Preliminary studies showed that with no intervention there was no
difference in the magnitude of the response to three repeated
dose-response curves to histamine. To further control for tachyphylaxis
as an explanation for the differences in the response to histamine
before and after vagotomy, experiments were performed in separate
groups of animals where histamine dose-response curves were carried out
only once. In these experiments, the response to histamine was
identical to that seen in Fig. 1.
Effect of muscarinic-receptor antagonists and agonists
on histamine-induced bronchoconstriction. Pretreatment
of nonvagotomized antigen-challenged animals with the nonselective
muscarinic-receptor antagonist atropine (1 mg/kg iv) completely
reversed histamine hyperreactivity compared with
antigen-challenged-only animals (P = 0.001; Fig. 3,
).

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Fig. 3.
Hyperreactivity to histamine is vagally mediated in sensitized guinea
pigs studied 24 h after a single antigen challenge. Values are means ± SE. Histamine (1-20 nmol/kg iv) induced a dose-dependent
bronchoconstriction, measured as a rise in
Ppi in antigen-challenged guinea
pigs. Response to histamine after atropine administration (1 mg/kg iv)
in antigen-challenged animals (n = 5)
with vagus nerves intact was same as that seen in control animals
(n = 6).
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|
Selective blockade of neuronal M2
muscarinic receptors with methoctramine (1 mg/kg iv) in control
nonvagotomized animals potentiated histamine-induced
bronchoconstriction threefold (P = 0.02); this effect was completely blocked by atropine
(bronchoconstriction in the presence of atropine is not significantly
different from controls; Fig.
4).

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Fig. 4.
Histamine (10 nmol/kg iv)-induced bronchoconstriction is significantly
potentiated by M2
muscarinic-receptor antagonist methoctramine (1 mg/kg iv) in control
nonvagotomized animals. This effect of methoctramine was completely
inhibited by pretreatment with atropine (1 mg/kg iv). Values are means ± SE; n = 5 animals.
* P = 0.02 compared with control value.
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|
The effect of the muscarinic agonist pilocarpine (10 µg/kg iv) on
histamine-induced bronchoconstriction in control and antigen-challenged animals was also compared. In these studies, pilocarpine inhibited the
bronchoconstrictor response to histamine (10 nmol/kg iv) by 40 ± 5% in control animals (n = 3). In
contrast, pilocarpine did not inhibit histamine-induced
bronchoconstriction in antigen-challenged animals
(n = 3; data not shown).
Pretreatment of antigen-sensitized animals with an
antibody to VLA-4 before antigen challenge.
Pretreatment with the antibody to VLA-4 1 h before antigen challenge
did not inhibit antigen-induced hyperreactivity in animals studied 24 h
after a single antigen challenge (n = 2; data not shown). However, pretreatment of antigen-sensitized animals
with the antibody to the adhesion molecule VLA-4 daily 1 h before each
antigen challenge (5 min/day on 4 consecutive days) significantly
inhibited antigen-induced hyperreactivity (P = 0.01;
n = 4 animals; Fig. 2,
). When the
vagus nerves were cut, there were no differences in the response to
histamine among any of the groups of animals (data not shown).
Pretreatment with an antibody to eosinophil major
basic protein. The effect of histamine on
Ppi was tested in
antigen-sensitized guinea pigs pretreated with the antibody to
eosinophil major basic protein before a single antigen challenge
(n = 5; Fig.
5,
). When the vagus nerves were intact,
the antibody to major basic protein completely attenuated histamine
hyperreactivity in antigen-challenged animals (Fig. 5,
) compared
with that in antibody-pretreated antigen-challenged animals
(P = 0.01; Fig. 5,
).

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Fig. 5.
Hyperreactivity to histamine is inhibited by pretreatment with antibody
to major basic protein (AbMBP) in sensitized guinea pigs studied 24 h
after a single antigen challenge. Values are means ± SE.
Histamine (1-20 nmol/kg iv) induced a dose-dependent
bronchoconstriction, measured as a rise in
Ppi in
antigen-challenged guinea pigs (n = 6). Response to histamine in antibody-pretreated, antigen-challenged
guinea pigs (n = 5) with vagus nerves
intact was same as that seen in control guinea pigs
(n = 6).
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|
In contrast, pretreatment with control (normal) rabbit serum had no
inhibitory effect on hyperreactivity to histamine in antigen-challenged guinea pigs (1-20 nmol histamine/kg; data not shown). In these experiments, the maximum increase in
Ppi in
response to 20 nmol/kg of histamine was 295 ± 29 mmH2O in rabbit serum-treated
guinea pigs (n = 3) compared with 287 ± 23 mmH2O in
non-serum-treated antigen-challenged guinea pigs (Fig. 5,
).
 |
DISCUSSION |
In antigen-challenged animals, the response to histamine was
significantly greater than that in control animals when the vagus nerves were left intact. When vagal reflexes were eliminated either by
vagotomy (Fig. 1) or by pretreatment with atropine (Fig. 3), there was
no difference in the response to histamine between control and
antigen-challenged animals even at the highest dose tolerated by the
challenged animals (20 nmol/kg iv). With the vagus nerves cut, there
were no differences between control and antigen-challenged animals,
indicating that the increased reactivity to histamine in
antigen-challenged guinea pigs is not due to an effect on airway smooth
muscle. Thus, in guinea pigs, antigen-induced hyperreactivity to
histamine is vagally mediated.
Experiments in control and antigen-challenged animals were performed by
assessing the response to histamine before and after vagotomy, with
each animal serving as its own control. This control overcomes bias
introduced by the variability in the response to antigen among guinea
pigs. The response to histamine was also compared between animals that
were only administered histamine once, either before or after vagotomy.
Because there were no differences in the results of the experiments
performed with either protocol, it is unlikely that the differences in
the response to histamine before and after vagotomy were due to
tachyphylaxis to histamine, confirming a previous
report in guinea pigs (35).
Under normal circumstances, neuronal
M2 muscarinic receptors limit
acetylcholine release from the vagus nerves. These
M2 muscarinic receptors are
dysfunctional after antigen challenge in guinea pigs (16, 30), mice
(20), and rats (2) as well as in some humans with asthma (1, 25). The
presence of functional M2
muscarinic receptors in control animals limits the magnitude of the
vagal reflex response. When these receptors are stimulated with
pilocarpine, histamine-induced bronchoconstriction is inhibited. Conversely, when these receptors are blocked with an
M2-selective antagonist such as methoctramine, the response to
histamine is potentiated. It is likely that methoctramine is
potentiating the reflex portion of the histamine response because the
potentiation is blocked with atropine. The presence of functional,
inhibitory M2 receptors may
explain why there is no significant vagally mediated response to
histamine in control animals.
In antigen-sensitized and -challenged guinea pigs, eosinophils are
selectively recruited to cholinergic nerves (7). The influx of
eosinophils into the lungs of antigen-challenged guinea pigs can be
inhibited by pretreatment with an antibody to VLA-4 (12, 26, 33). In
antigen-challenged animals, inhibiting the influx of eosinophils into
the airways prevents loss of neuronal M2 muscarinic-receptor function
and prevents the development of hyperreactivity (10, 12, 26). In vitro,
eosinophil major basic protein is an antagonist for
M2 muscarinic receptors (18). In
vivo, neutralizing major basic protein with heparin (12) or with an
antibody to major basic protein (11) also prevents loss of
M2 receptor function in
antigen-challenged guinea pigs. The antibody to major basic protein
does not inhibit recruitment of eosinophils to the nerves (11); it acts
by neutralizing the eosinophil product, major basic protein (22, 31).
These studies demonstrate that loss of neuronal
M2 muscarinic-receptor function in
antigen-challenged guinea pigs is due to blockade of
M2 receptors by eosinophil major
basic protein.
Because antigen-induced hyperreactivity to histamine is vagally
mediated, the role of eosinophil major basic protein in antigen-induced hyperreactivity was tested. Pretreatment of single antigen-challenged guinea pigs with the antibody to eosinophil major basic protein, but
not with control rabbit serum, completely prevented hyperreactivity 24 h later. These data demonstrate that hyperreactivity to histamine 24 h
after antigen challenge is mediated by eosinophil major basic protein.
However, inhibition of eosinophil influx into the airways with the
antibody to VLA-4 did not prevent hyperreactivity to histamine 24 h
after antigen challenge. Thus, although eosinophil major basic protein
is critical to developing hyperreactivity, recruitment of eosinophils
into the lungs is not required, suggesting that the major basic protein
must have come from resident eosinophils.
In contrast, when sensitized animals were pretreated with the antibody
to VLA-4 and challenged repeatedly with antigen over 4 days,
hyperreactivity to histamine after antigen challenge was prevented. One
explanation for these findings may be that degranulation of resident
eosinophils in the guinea pig mediates the hyperreactivity seen 24 h
after a single antigen challenge, whereas maintenance of
hyperreactivity to histamine requires recruitment of additional eosinophils from the peripheral circulation to the airway nerves.
In control nonsensitized animals, including guinea pigs (24), rabbits
(19), and dogs (23, 36), histamine has been shown to cause
bronchoconstriction by a direct effect on airway smooth muscle in
addition to a vagal reflex response because sectioning the vagus nerves
inhibited the histamine-induced bronchoconstriction by up to 50%. In
contrast, sectioning the vagus in our control animals did not alter the
histamine-induced bronchoconstriction. However, in previous studies
(13, 15), it is noteworthy that the animals had been paralyzed with
gallamine, which is a selective antagonist for
M2 muscarinic receptors. When we
blocked the neuronal M2 receptors
with methoctramine (Fig. 4), there was a considerable vagal response to
histamine in control animals.
In summary, histamine-induced bronchoconstriction is mediated by a
direct effect on airway smooth muscle in control animals, although
there is a vagal component when the neuronal
M2 receptors are inhibited. In
antigen-challenged guinea pigs, hyperreactivity to histamine is vagally
mediated. Furthermore, this vagally mediated hyperreactivity is
dependent on release of major basic protein from resident eosinophils.
Because in antigen-challenged guinea pigs there is loss of function of
the neuronal M2 muscarinic
receptors, which is also eosinophil major basic protein mediated, the
results of this study suggest that histamine hyperreactivity seen after antigen challenge is due to antagonism of neuronal
M2 muscarinic receptors by
eosinophil major basic protein.
In humans, the response to histamine in vivo does not correlate with in
vitro responses, suggesting that the hyperresponsiveness does not
reflect an intrinsic abnormality of the airway smooth muscle (27, 34).
In some humans with asthma, the function of the neuronal
M2 muscarinic receptors is
impaired while histologically eosinophils are localized to airway
nerves (7). Thus antagonism of M2
muscarinic receptors by eosinophil major basic protein may also be a
mechanism for the hyperreactivity to agents such as histamine in
patients with asthma.
 |
ACKNOWLEDGEMENTS |
We acknowledge the generous donation of the antibody to very late
activation antigen-4 from Dr. R. Lobb (Biogen, Cambridge, MA). We also
thank Dr. George Jakab for the use of his inhalation facilities,
supported by National Institute of Environmental Health Sciences Grant
ES-03819.
 |
FOOTNOTES |
This work was funded by National Heart, Lung, and Blood Institute
Grants HL-44727, HL-55543 (both to A. D. Fryer), HL-54659 (to D. B. Jacoby), and HL-09389 (to R. W. Costello); National Institute of
Allergy and Infectious Diseases Grants AI-37163 (to D. B. Jacoby),
AI-09728 and AI-34577 (both to G. J. Gleich); the Foundation for
Fellows in Asthma Research Award; the British Lung Foundation (R. W. Costello); the Center for Indoor Air Research; and the American Heart
Association (D. B. Jacoby and A. D. Fryer).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. D. Fryer,
Dept. of Environmental Health Sciences, School of Hygiene and Public
Health, Johns Hopkins Univ., 615 N. Wolfe St., Baltimore, MD 21205 (E-mail: afryer{at}jhsph.edu).
Received 8 July 1998; accepted in final form 29 January 1999.
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