Download Circulating histamine and eosinophil cationic

Clinical Science (1992) 83, 227-232 (Printed in Great Britain)
227
Circulating histamine and eosinophil cationic protein
levels in nocturnal asthma
Michael F. FITZPATRICK', Thomas MACKAY', Carol WALTERS, Po-Chun TAP',
Martin K. CHURCH', Stephen T. HOLGATEl and Neil J. DOUGLAS'
'Respiratory Medicine Unit, Department of Medicine (RIE), City Hospital, Edinburgh, U.K.,
ZDepartmentsof Medicine and Pharmacology, University of Southampton, Southampton General
Hospital, Southampton, U.K., and 3Cardiovascular Immunology Research Group, University of
London, St George's Hospital Medical School, London, U.K.
(Received 16 April
1992; accepted I May 1992)
1. To investigate the role of mast cells and eosinophils in the pathogenesis of nocturnal asthma, the
plasma methylhistamine concentration, serum eosinophi1 cationic protein level and peak expiratory flow
rate were measured 2-hourly for 24h in 10 patients
with nocturnal asthma and in 10 healthy control
subjects. Nocturnal asthma was defined as at least
one nocturnal awakening per week due to cough,
wheeze or breathlessness with an average overnight
fall in peak expiratory flow rate of a t least 15%
during a 2-week run-in period.
2. The lowest peak expiratory flow rate occurred at
02.00-04.00 hours in the group with nocturnal
asthma, whose overnight fall in peak expiratory flow
rate was 29+5% in comparison with 5+1% (means
+SEM) in the normal subjects.
3. Plasma methylhistamine levels at night (0.28004.00 hours) were lower than during the day
(10.00-20.00 hours) in both asthmatic patients and
normal subjects (asthmatic patients: day, median
0.22 ng/ml, 95% confidence intervals 0.18-0.34 ng/ml;
night, 0.17 ng/ml, 0.13-0.24 ng/ml; P< 0.01; normal
subjects: day, 0.31 ng/ml, 0.24-0.41 ng/ml; night,
0.24 ng/ml, 0.21-0.33 ng/ml; P< 0.01).
4. The serum eosinophil cationic protein level was
higher by day (30ng/ml, 8-47ng/ml) than by night
(21 ng/ml, 5-34nglml; P<O.O4) in the group with
nocturnal asthma, but did not change significantly
with the time of day in the normal subjects (day:
8 ng/ml, 4-14 ng/ml; night: 8 ng/ml, 5-21 nglml).
5. Peripheral blood eosinophil counts fell in the early
morning in the patients with nocturnal asthma (day:
0.52 x 109/1, 0.14-0.76 x 109/l; night: 0.29 x 109/1,
0.13-0.57 x 109/l;P= 0.03), but did not change significantly in the normal subjects.
6. This study indicates that a rise in plasma histamine concentration is not a prerequisite for nocturnal
asthma.
INTRODUCTION
Asthma tends to be most severe in the early hours
of the morning, with around 90% of asthmatic
patients being affected by nocturnal asthma symptoms [l, 21. The pathogenesis of this nocturnal
airway narrowing is poorly understood. Increased
parasympathetic tone contributes to overnight airway narrowing [3, 41, but is insufficient to account
for all of the observed nocturnal bronchoconstriction 13, 41.
Other factors which may contribute to nocturnal
airway narrowing include increased release of
bronchoconstricting mediators and increased bronchial reactivity to such mediators. It has been
reported that plasma histamine levels rise at night,
and this finding was equated with mast cell involvement in nocturnal bronchoconstriction [ S ] . However, the plasma levels of histamine reported in the
latter study are higher than those that would normally be accepted as physiological [6], suggesting
either basophil contamination or technical limitations in the assay [7]. To clarify the relationship
between nocturnal asthma and plasma levels of
circulating mediators we have measured circadian
changes in plasma histamine and serum eosinophil
cationic protein (ECP) [8] levels in patients with
nocturnal asthma and in normal control subjects.
METHODS
Patients and control subjects
We studied 10 clinically stable patients with nocturnal asthma (three were atopic with positive skin
tests to house dust mite, pollens or animal dander;
four males, six females; mean age 43 years, range
18-60 years) and 10 normal subjects (five males, five
females, mean age 35 years, range 24-54 years).
Key words: asthma, circadian rhythm, mediators.
Abbreviations: ECP, eosinophil cationic protein; PEFR, peak expiratory flow rate.
'Deceased.
Correspondence: Dr Neil 1. Douglas, Respiratory Medicine Unit, Department of Medicine (RIE), City Hospital, Greenbank Drive, Edinburgh El0 SSB, U.K.
228
M. F. Fitzpatrick et al.
None of the patients or normal subjects smoked.
Nocturnal asthma was defined as at least one
awakening from asthma per week in association
with an average overnight fall in peak expiratory
flow rate (PEFR) of at least 15% over a 2-week runin period. Cromoglycate was stopped in the two
patients on this medication for 5 days before the
study. Oral 8,-adrenoceptor agonists and theophylline were withheld for at least 48 h and inhaled
8-adrenoceptor agonists were withheld for 6 h before
the study. Inhaled ipratropium was provided as
‘rescue’ medication throughout the study period and
patients’ other usual maintenance therapy (morning
once daily oral steroids <7.5mg/day in four
patients, twice daily inhaled steroids in 10 patients)
was continued unchanged through the study.
Normal subjects were selected from respondents to
a newspaper advertisement on the basis of a screening questionnaire to exclude those with a history of
current illness or allergy or those taking medication.
Each patient and normal subject gave their
written informed consent to participation in the
study, which had the approval of the Local Ethical
Advisory Committee.
Immediately after the run-in period, each subject
attended our laboratory for one 24h period commencing at 09.00 hours. All measured their PEFR in
triplicate every 2 h throughout the study from 10.00
hours on day 1 to 08.00 hours on day 2. The
highest of three PEFR recordings at each time point
was used. Sleep times were standardized for all
subjects by fixed ‘lights out’ (23.00 hours) and final
awakening (06.00 hours) times.
Blood sampling
A blood sample was taken every 2h, from 10.00
hours on day 1 until 08.00 hours on day 2 of the
study, through an indwelling (teflon 18 G) forearm
venous catheter, kept patent by flushing with saline
(150mmol/l NaCl) containing 1 unit of heparin/ml
after each sample. The first 3ml of blood at each
sample time was discarded and a further 10ml was
aspirated into a pre-cooled plastic syringe. Eight
millilitres of blood were placed in a polypropylene
tube containing crystalline EDTA (potassium salt;
BDH Chemicals Ltd, Poole, Dorset, U.K.) and were
centrifuged at 4°C and 2000g for 5min. The top
1ml of the supernatant plasma was then pipetted off
carefully, so as not to disturb the buffy coat, and
was stored in a plain polypropylene tube at -70°C.
The remaining 2 ml of blood was placed in a plain
polypropylene tube and was allowed to clot for
45 min at room temperature before being centrifuged at 2000g for 5min. The resulting serum was
transferred to a plain 5ml tube for storage at
-70°C. In all patients and control subjects
measurements of plasma histamine concentrations
were made over the 24h period, and in eight
patients and eight control subjects the serum ECP
level was also determined.
Eosinophil counts
Full blood counts were performed and fresh
blood films were prepared at 14.00, 16.00, 02.00 and
04.00 hours. The slides were fixed and were stained
immediately using the ‘Quikdiff methods [Baxter
Dade AG, Dudingen, Switzerland; the fixative is a
solution of Fast Green in methanol (0.002g/l); stain
1 contained 1.22g of eosin G/1 and stain 2 l . l g of
thiazine dye/l]. A differential leucocyte count on 500
leucocytes was carried out under oil immersion
microscopy, at a magnification of 100, at each time
point. The total leucocyte count at each time point
was measured by an automated blood analyser
(Fismex NE 8000; TOA Medical Electronics
Company Ltd, Kobe, Japan).
Methylhistamine assay
Histamine was measured as its primary methylated metabolite, N-methylhistamine, using a commercial r i a . kit (Pharmacia, Milton Keynes, Bucks,
U.K.) with a 80% selectivity for N-methylhistamine
over histamine. Methylhistamine was measured in
preference to histamine because of its longer half-life
in biological fluids. In four subjects the levels of
native histamine were also measured using a commercial r i a . kit specific for histamine (Serotec,
Oxford, U.K.). Standard curves were constructed for
both assays with linear portions between 0.1 and
10ng/ml for the N-methylhistamine assay and 0.0515ng/ml for the histamine assay. The assays were
tested with two spiked plasma samples of known
histamine concentration. With the 0.44 ng/ml
sample, the N-methylhistamine assay gave a mean
concentration of 0.43 ng/ml with an inter-day coefficient of variation of 8% (n= 16), and the histamine
assay gave a mean level of 0.43ng/ml and an interday coefficient of variation of 12% (n=16). A
0.25 ng/ml sample was tested using the N-methylhistamine assay to give a mean concentration of
0.22 ng/ml with an inter-day coefficient of variation
of 5% (n=31). The intra-day result with the
0.25 mg/ml sample using the N-methylhistamine
assay gave a mean concentration of 0.24ng/ml and
a coefficient of variation of 17% (n=8). The mean
values obtained for methylhistamine and histamine
respectively in the four subjects in whom both
assays were performed in all samples were
0.17 f0.03 ng/ml and 0.20 f0.02 ng/ml (means
+SEM). These values were not significantly different.
ECP assay
Serum ECP level was measured by an e.1.i.s.a.
based on the method of Tai et al. [9]. The method
used immobilized monoclonal antibody EG1 to
capture ECP on to the surface of 96-well e.1.i.s.a.
plates. The quantity of bound ECP was then
assayed using an alkaline-phosphatase-linked anti-
229
Circulating histamine and eosinophil cationic protein in nocturnal asthma
Table I. Characteristics of the asthmatic patients. Abbreviations:
B, inhaled 8-adrenoceptor agonirt; I, inhaled ipratropium; C, inhaled cromoglycate; S, inhaled steroid, followed by daily dose in pg; OB, oral padrenoceptor agonist, T, oral theophylline, P, oral prednirolone,followed by daily
dose in mg.
Patient
no.
Sex
Usual medications
Maximum
PEFR (% of
predicted)
Diurnal
change in
PEFR (%)
B, S loo0
B, S 1600, OB, T
B, S 200
B, S 1oo0, OB, T, P 5
B, I, S 1600, C, T. P 7.5
B, I, S 1600, T, P 7.5
B, S 200, OB, T
B, S 1600, C, T
B, I, S 1600, OB, T
B, S 1600, OB, T, P 5
95
89
78
56
25
31
31
64
90
68
40
I
2
3
4
5
6
7
8
9
10
F
F
F
F
M
M
M
M
F
F
52
38
18
39
42
56
60
46
32
43
38
85
97
61
4
2 300
200
I
100 I
33
48
33
65
56
body to ECP (antibody EG2) which binds to a
second determinant on ECP. Unknown samples
were assayed at four concentrations in duplicate.
Controls with known concentrations of ECP were
assayed at the same time. The absorbance at 405nm
of the reaction product developed from the alkaline
phosphatase substrate nitrophenyl phosphate was
plotted against log [ECP]. Calculations of ECP
concentrations in ng/ml were made, with corrections, from the linear part of the graph. The
sensitivity of the ECP assay was 5ng/ml, and the
coefficient of variation of repeated assays was 7%.
Statistical analysis
Results are expressed as meansfSEM for normally distributed data, and as the median and 95%
confidence interval for non-normally distributed
data. The Mann-Whitney U-test was used to compare results between normal subjects and asthmatic
patients. Wilcoxon signed ranks tests were used to
examine changes within each group. The appropriate Bonferroni correction factor was applied where
multiple comparisons were made.
RESULTS
One patient, a 38-year-old female, was withdrawn
on the evening of the study because she required
nebulized P,-adrenoceptor agonists for relief of asthmatic symptoms. Thus, her daytime data only were
included in the analysis below.
1
10.w 14.w 18.w nw
m.w 06.w 10.w
Time of day (hours)
,
,
,
,
,
,
,
I0.W 14.00 18.00 2LW Ol.WO6.W I0.W
Time of day (hours)
Fig. I. PEFR (top), plasma methylhistamine concentration (middle)
and serum ECP concentration (bottom) over the 24h period in
normal subjects (left) and patients with nocturnal asthma (right).
Data are displayed as means SEM.
in the asthmatic group (29+5%) than in the normal
group ( 5 f 1%, P<O.OOl). The minimum overnight
PEFR in the asthmatic patients (47+5% of predicted) occurred at 02.00-04.00 hours (in seven out
of nine patients). Thus, results obtained at 02.0004.00 hours are used to illustrate changes at the
time of maximal airway narrowing in the asthmatic
patients.
Plasma methylhistamine levels
The daytime plasma methylhistamine level in the
asthmatic patients did not differ significantly from
that in the normal subjects (P=O.lO, Table 2, Fig.
24, but the mean night-time plasma methylhistamine level was lower in the asthmatic patients
than in the normal subjects (P<0.04). The mean
02.0&04.00 hours plasma methylhistamine levels
were lower than the daytime values in both groups
( P 0.01), with similar percentage falls from the
daytime values (asthmatic patients, 21 +6%; normal
subjects, 24 f5%; P =0.60).
-=
PEFR measurements
Both asthmatic patients and normal subjects had
significant overnight drops in PEFR (Table 1, Fig.
l), with the percentage overnight fall in PEFR
[(PEFR at 22.00 hours-lowest recorded PEFR
overnight) x 100/(PEFR at 22.00 hours] being larger
Serum ECP levels
Mean serum ECP levels were not significantly
different in the asthmatic patients and normal subjects either by day (P=O.O8, Table 2, Fig. 2b) or by
night (P=0.22). The absolute serum ECP levels at
M. F. Fitzpatrick et al.
230
Table 2. PEFR, plasma methylhistamine concentration, serum ECP concentration and eosinophil count in normal
subjects and asthmatic patients during day-time and night-time. Values are means with 95% confidence intervals. Statistical
significance: *P<0.05, **P<0.01 compared with normal subjects at the same time; tP<O.OS, ttP<O.OI compared with daytime in
the same eroup.
Normal subjects
PEFR (% of predicted)
Plasma methylhistamine concn. (ng/ml)
Serum ECP concn. (nglml)
x Eosinophil count (I-')
Asthmatic patients
Daytime
Night-time
Daytime
Night-time
113 (105-119)
0.31 (0.24-0.41)
8 (414)
0.12 (0.05429)
106 (98-1 13)
0.24 (0.21433)
8 (5-21)
0.14 (0.1-0.26)
73 (56-83)**
0.22 (0.18-0.34)
30 (8-47)
0.52 (0.16-0.76)
54 (29-57)**ft
0.17 (O.IM.24)*t
21 (5-34)t
0.29 (0. I M . 5 7 ) t
90
70
E
I
B
=
0.2
0.1
Day
Night
Asthmatic patients
20
I
Day
Night
Normal subjects
Day
Night
Asthmatic patients
0 Day
Night
Asthmatic patients
Day
Night
Normal subjects
Day
Night
Normal subjects
Fig. 2. Results in individual asthmatic patients and normal subjects for plasma methylhistamine concentration (a), serum ECP concentration (6) and
hours)
peripheral blood eosinophil count (c) by day (1O.W-20.00 hours) and by night (02.W.00
02.00-04.00 hours were significantly lower than the
daytime baseline levels in the asthmatic patients, but
not in the normal subjects (P=O.36), with a greater
percentage fall in serum ECP levels at 02.00-04.00
hours, from the daytime baseline, in the asthmatic
patients (43 & 10%) than in the normal subjects
(6&9%; Pc0.01).
Eosinophil counts
Eosinophil counts were higher in the asthmatic
patients than in the normal subjects during the
afternoon and early morning (Table 2, Fig. 2c).
There was a fall in eosinophil count in the early
morning in the asthmatic patients ( P = 0.03), but not
in the normal subjects ( P = 0.60).
DISCUSSION
Our results show that plasma histamine levels fall
during early-morning airway narrowing in both
normal subjects and patients with nocturnal asthma,
and that there is no difference in the percentage
change in plasma histamine levels at night between
normal subjects and asthmatic patients, despite a
much more profound fall in PEFR in the asthmatic
group. We have also demonstrated lower serum
ECP levels in the early morning in patients with
nocturnal asthma than in normal subjects, a fall in
Circulating histamine and eosinophil cationic protein in nocturnal asthma
serum ECP level during early-morning bronchoconstriction in the asthmatic patients but not in
normal subjects, and a significant fall in peripheral
eosinophil numbers at the time of maximal
bronchoconstriction in the asthmatic patients.
Our findings conflict with those of an earlier
study which demonstrated a rise in plasma histamine concentration coincident with maximal
bronchoconstriction at night in patients with nocturnal asthma [S]. However, there are inconsistencies which must be addressed when interpreting
data from the latter study. Free plasma histamine
levels greater than lng/ml are accompanied by
facial flushing, increased skin temperature, a fall in
diastolic blood pressure and tachycardia in both
asthmatic patients and normal subjects [6,7]. Similar findings were not documented by Barnes et al.
[S], despite reported plasma histamine levels well in
excess of 1ng/ml in their patients with nocturnal
asthma. Their reliance on an enzymic (histamine
methyltransferase) assay, which is less reliable than
r.i.a. techniques for the measurement of histamine
[lo, 111, may have led to inaccuracies. Also, only
about 0.5% of the histamine present in blood exists
as free histamine, the rest being stored in basophils.
Therefore, any basophil contamination of plasma
samples will lead to artificially high estimates of
plasma histamine levels. This may have been an
additional source of error in the estimates of plasma
histamine levels by Barnes et al. [S]. We were
careful to avoid this problem by observing a strict
protocol while handling the blood samples.
Two studies performed simultaneously with the
present study have also examined histamine levels in
nocturnal asthma. Van Aalderen et al. [12] found
that nine children with nocturnal asthma had higher
24 h urinary N-methylhistamine levels than nine
patients without nocturnal asthma, with a trend to
higher histamine levels occurring at night in the
patients with nocturnal asthma. However, it is not
clear whether this trend was statistically significant.
Szefler et al. [13] recently found no significant
change in the plasma histamine level at 04.00 hours
compared with at 16.00 hours in normal subjects or
in patients with nocturnal asthma, although again
there was a trend to higher plasma histamine levels
at night. Our study involved a larger number of
patients with nocturnal asthma and found significant decreases in plasma histamine levels in the
early morning. In addition, in contrast with the
study by Szefler et al. [13], we measured plasma
histamine levels 2-hourly throughout the day and
not just at two time points. Another difference is
that all of our patients were on inhaled steroids and
four were on oral steroids. Steroids modify the
inflammatory response in the airway [14], with a
reduction in mast cell and eosinophil populations in
bronchial biopsies, and this may conceivably have
affected our results. Indeed, this could have allowed
dissociation of the temporally, but not causally,
related circadian changes in plasma histamine level
231
and PEFR reported in other studies. However, the
circadian pattern of plasma histamine levels in our
normal subjects who were not on steroids was
identical with that in the asthmatic patients.
An increase in plasma histamine level is seen after
antigen challenge in asthmatic patients coincident
with the early asthmatic response, suggesting the
involvement of mast cells in this response [lS, 161.
Our study does not exclude the possibility that
small amounts of histamine released into the airway
could still contribute to nocturnal bronchoconstriction. However, the similarity of plasma histamine levels in our asthmatic patients and normal
subjects suggests that nocturnal bronchoconstriction
is not an early asthmatic reaction. This is supported
by the observation that cromoglycate is ineffective
in treating nocturnal asthma [17, 181.
ECP is the most potent tissue-damaging protein
released by eosinophils and is a measure of eosinophil activation [S]. ECP levels are raised in the
bronchoalveolar lavage fluid of patients with
chronic asthma [19] and rise further during late
asthmatic responses [20]. Serum ECP levels tend to
be similar in patients with asthma and normal
subjects [21, 221, but fall during the late asthmatic
response [22, 231 in association with the transient
fall in peripheral blood eosinophil counts [23, 241.
Thus, the falls in both serum ECP level and eosinophil count in the present study are compatible with,
but by no means prove, the involvement of a late
asthmatic reaction in the pathogenesis of nocturnal
asthma. Alternatively, the overnight fall in serum
ECP level in the asthmatic patients might not be
causally related to nocturnal asthma and could
reflect hormonal changes or therapy.
The potential importance of inflammation in the
pathogenesis of nocturnal asthma has been strengthened by the recent observations that asthmatic
patients exhibit late asthmatic reactions more commonly and more markedly at night than during the
day [24], and that the bronchoalveolar lavage fluid
obtained from patients with nocturnal asthma at
04.00 hours has increased numbers of inflammatory
cells, including eosinophils, as compared with
samples taken at 16.00 hours from the same patients
[25, 261. Nevertheless, there is strong evidence that
nocturnal airway narrowing is associated with
increased parasympathetic [4, 271 and decreased
non-adrenergic, non-cholinergic [28] nervous
activity at night.
This was not designed to be a study of untreated
nocturnal asthma, which anyway would have been
ethically dubious as all our patients had troublesome nocturnal symptoms. All of the patients were
on inhaled steroids and four were on oral prednisolone. Although such therapy modifies airway inflammatory responses [14], most patients with clinically
troublesome nocturnal asthma will be on such
treatment, at least in this country. It is significant
that severe nocturnal asthma may persist, despite
such treatment, and our study shows that in this
232
M. F.
Fitzpatrick et at.
situation, plasma histamine levels do not rise and
eosinophil numbers and serum ECP levels fall in
association with nocturnal airway narrowing.
ACKNOWLEDGMENTS
We thank Professor Christopher Spry, St
George’s Hospital, London, for organizing the
measurement of serum ECP levels in his laboratory
and for helpful comments on the manuscript, and
Dr Rob Elton of Edinburgh University for statistical advice. M.F.F. was supported by grant no. 89
from the National Asthma Campaign.
REFERENCES
I. Turner-Warwick, M. Nocturnal asthma. A study in general practice. J. R. SOC.
Gen. Pract. 1989; 39, 239-43.
2. Fitzpatrick, M.F., Martin, K., Peck, D., Shapiro, C.M. & Douglas, N.J.
A community based survey of subjective sleep quality in asthmatic patients
and snorers [Abstract]. Thorax 1990; 45, 789.
3. Morrison, J.F.J., Teale, C., Pearson, S.B. et al. Adrenaline and nocturnal
asthma. Br. Med. J. 1990; 301, 473-6.
4. Catterall, J.R., Rhind, G.B., Whyte, K.F., Shapiro, C.M. & Douglas, N.J.
Is nocturnal asthma caused by changes in airway cholinergic activity?
Thorax 1988; 43,7204.
5. Barnes, P., Fitzgerald. G., Brown, M.J. & Dollery, C. Nocturnal asthma and
changes in circulating epinephrine, histamine and COrtiSOl. N. Engl. J. Med.
1980; 303, 263-7.
6. Ind, P.W., Brown, M.J., Lhoste. F.J.M., Macquin, I.M. & Dollery, C.T.
Concentration effect relationships of infused histamine in normal volunteers.
Agents Actions 1982; 12, 12-16.
7. Ind, P.W.. Barnes, P.J., Brown, M.J., Causon, R. & Dollery, C.T. Measurement
of plasma histamine in asthma. Clin. Allergy 1983; 13, 61-7.
8. Fredens, K., Dahl. R. & Venge, P. Eosinophils and cellular injury: the Gordon
phenomenon as a model. Allergy Proc. 1985; 6, 346-51.
9. Tai, P.C., Capron, M., Bakes, D.M., Barkans, J. & Spry, C.J. Monmlonal
antibodies t o eosinophil membrane antigens enhance the secretion of
eosinophil cationic protein. Clin. Exp. Immunol. 1986; 63, 72B-37.
10. Mwdley, I., Zhong, N.S., Morgan, D.J.R. & Davies, R.J. A comparison of the
available methods for measurement of histamine in sputum. Clin. Allergy
1984; 14, 153-63.
I I . Gleich, G.J. & Hull, W.M. Measurement of histamine: a quality control study.
J. Allergy Clin. Immunol. 1980; 66,295-8.
12. van Aalderen, W.M.C., Postma, D.S., Koeter, G.H. & Knol, K. Nocturnal
airflow obstruction, histamine, and the autonomic central nervous system in
children with allergic asthma. Thorax 1991; 46, 366-71.
13. Szefler, S.J., Ando, R., Cicutto, L.C.. Sun. W.. Hill, M.R. & Martin, R.J.
Plasma histamine, epinephrine, cortisol, and leukocyte fi-adrenergic receptors
in nocturnal asthma. Clin. Pharmacol. Ther. 1991; 49, 59-68.
14. Djukanovic, R., Walls, A.F., Wilson, J.W. et al. The effect of inhaled
beclomethasone dipropionate (BDP) on airway mast cells, histamine and
tryptase in atopic asthma. Am. Rev. Respir. Dis. 1991; 143, A627.
IS. White, M.V., Slater, J.E. & Kaliner, M.A. Histamine and asthma. Am. Rev.
Respir. Dis. 1987; 135, 1165-76.
16. Morgan, A.D., Connaughton, J.J.,Catterall, J.R., Shapiro, C.M., Douglas, N.J.
& Flenley, D.C. Sodium cromoglycate in nocturnal asthma. Thorax 1986; 41,
39-41.
17. Hetzel, M.R., Clarke, T.J.H., Gillem, S.J., Isaac. P. & Perkins, M. Is sodium
cromoglycate effective in nocturnal asthma?Thorax 1985; 40, 793-4.
18. De Monchy. J.G.R., Kauffman, H.F., Venge. P. et al. Bronchoalveolar
eosinophilia during allergen-induced late asthmatic reactions. Am. Rev. Respir.
Dis. 1985; 131, 373-6.
19. Venge, P., Dahl, R., Zetterstrom, 0. & Roxin, L.E. Low levels of eosinophil
cationic proteins in patients with asthma. Lancet 1 9 v i, 373-5.
20. Venge, P., Dahl, R. & Peterson, C.G.B. Eosinophil granule proteins in serum
after allergen challenge of asthmatic patients and the effects of anti-asthmatic
medication. Int. Arch. Allergy Appl. Immunol. 1988; 87, 30612.
21. Dahl, R., Venge, P. & Olsson, I. Variations of blood eosinophils and eosinophil
cationic protein in serum in patients with bronchial asthma. Studies during
inhalation challenge tests. Allergy 1978; 33, 21 1-5.
22. Cookson. W.O.. Craddock, C.F., Benson, M.K. & Durham, S.R. Falls in
peripheral eosinophil counts parallel the late asthmatic response. Am. Rev.
Respir. Dis. 1989; 139, 45&62.
23 Metzger, W.J., Zavala, D., Richerson, H.B. et al. Local allergen challenge and
bronchoalveolar lavage of allergic asthmatic dogs. Description of the model
and local airway inflammation. Am. Rev. Resoir. Dis. 1987: 135. 433-40.
24. Martin, R.J. & Mohiuddin. A.A. Circadian b d s of the late asthmatic response.
Am. Rev. Respir. Dis. 1990; 142, 1153-7.
25. Martin, R.J., Ciccuto, L.C., Smith, HA., Ballard, R.D. & Szefler, S.J. Airway
inflammation in nocturnal asthma. Am. Rev. Respir. Dis. 1991; 143, 351-7.
26. Mackay, T.W.. Brown, P., Wallace, W. et al. Does inflammation play a role in
nocturnal asthma? (Abstract). Am. Rev. Respir. Dis. 1992; 145, A22.
27. Morrison, J.F., Pearson, S.B. & Dean, H.G. Parasympathetic nervous system in
nocturnal asthma. Br. Med. J. 1988; 296, 1427-9.
28. Mackay, T.W., Fitzpatrick, M.F. & Douglas, N.J. Non-adrenergic,
non-cholinergic nervous system and overnight airway calibre in asthmatic and
normal subjects. Lancet 1991; 338, 1289-92.