1
Supporting Information with highlight
2
Smoking attenuates the age-related decrease in IgE levels and maintains eosinophilic
3
inflammation in patients with asthma
4
5
Tadao Nagasaki1, Hisako Matsumoto1, Hitoshi Nakaji1,2, Akio Niimi1,3, Isao Ito1, Tsuyoshi
6
Oguma1, Shigeo Muro1, Hideki Inoue1, Toshiyuki Iwata1, Tomoko Tajiri1, Yoshihiro
7
Kanemitsu1, Michiaki Mishima1
8
9
1
Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University,
10
Kyoto, Japan
11
2
Department of Respiratory Medicine, Wakayama Red Cross Hospital, Wakayama, Japan
12
3
Division of Respiratory Medicine, Department of Medical Oncology and Immunology,
13
Nagoya City University School of Medical Sciences, Nagoya, Aichi, Japan
14
15
Corresponding author: Hisako Matsumoto, MD, PhD
16
Department of Respiratory Medicine
17
Postgraduate School of Medicine, Kyoto University
18
54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
19
Telephone: +81-75-751-3830; Fax: +81-75-751-4643
20
E-mail: hmatsumo@kuhp.kyoto-u.ac.jp
21
1
22
Supporting Information: Methods
23
Subjects
24
The present study was a cross-sectional study on adult patients with asthma that
25
were newly referred to the Asthma Clinic of Kyoto University Hospital between June 2006
26
and October 2011. Asthma was newly diagnosed according to the American Thoracic Society
27
criteria, which define asthma as a history of recurrent episodes of wheezing and chest
28
tightness, with or without cough, and documented airway reversibility with a bronchodilator
29
or hyper-responsiveness to inhaled methacholine [S1]. The diagnosis of asthma was made
30
independent from this study and the presence of atopy, levels of serum IgE and blood
31
eosinophil counts were not considered at the time smokers were assigned to this study.
32
Patients were not treated with a steroid or leukotriene antagonist, and demonstrated normal
33
chest radiographic findings. Ex-smokers were defined as those who had stopped smoking for
34
at least 1 year. Patients with asthma who had smoked less than 5 pack-years were excluded.
35
Smoking status, the presence of atopic dermatitis, allergic rhinitis, and childhood asthma
36
were evaluated via a self-reported questionnaire. The study protocol was approved by the
37
Ethics Committee of Kyoto University, and written informed consent was obtained from all
38
subjects.
39
40
Measurements
41
Patients underwent a work-up, including a physical examination, blood tests, chest
42
radiographs, fractional exhaled nitric oxide (FeNO) concentration measurements, pulmonary
43
function tests, and sputum induction.
44
Total and specific serum IgE antibody titers were measured via radioimmunosorbent
45
testing (Pharmacia Diagnostics, Uppsala, Sweden). Patients were considered atopic when one
46
or more specific IgE antibodies against grass pollen, mold, weed, house dust mite,
47
Dermatophagoides pteronyssinus, Japanese cedar pollen, cat dander, dog dander, or
2
48
Trichophyton were positive.
49
FeNO at a constant exhalation flow rate of 50 mL/s was measured with a
50
chemiluminescence analyzer (NOA 280, Sievers, Boulder, CO, USA) [S2], according to the
51
current guidelines [S3]. The analyzer was calibrated daily with non-NO-containing gas,
52
which was generated by exposing ambient air to NO scavengers and a standard concentration
53
of 640 ppb NO. The lower detection limit for NO was 2 ppb. The signal output from the NO
54
analyzer was fed to a computer data acquisition program, and concentrations were measured
55
using a data analysis program (NOA Analysis™ Software, Sievers). Seated subjects inhaled
56
orally until total lung capacity was reached, and then inserted a mouthpiece and exhaled
57
immediately against a resistance to achieve a constant exhalation flow rate of 50 ml/s. FeNO
58
measurements were taken from a steady plateau. The average of three measurements was
59
used, and measurements were performed prior to spirometry.
60
Pre-bronchodilator forced vital capacity (FVC), FEV1, and mid-forced expiratory
61
flow25-75% (FEF25-75%) were tested using a ChestGraph HI-701 spirometer (Chest MI Corp,
62
Tokyo, Japan), according to the guidelines of the American Thoracic Society [S4].
63
Sputum induction and processing were performed according to the slightly modified
64
methodology of Pin et al [S5]. Briefly, subjects inhaled a hypertonic (3%) saline solution
65
from an ultrasonic nebulizer (MU-32, Azwell Inc., Osaka, Japan) for 15 min, and adequate
66
plugs of sputum were separated from the saliva. After treatment with 0.1% dithiothreitol
67
(OXOID Ltd., Hampshire, UK), the sample was cytocentrifuged and cells were stained using
68
the May-Grünwald-Giemsa method. Cell differentials were determined by counting at least
69
400 non-squamous cells on each sputum slide [E5]. Supernatants of the sputum were stored at
70
-20°C for later use. TSLP concentrations in sputum supernatants were measured via an
71
enzyme-linked immunosorbent assay kit (R&D Systems, Inc., MN, USA), according to the
72
manufacturer’s instructions. The detection limit of this assay was 3.46 pg/mL, and values
73
below this threshold were assigned values of 0 pg/mL. A spike-back analysis using
3
74
exogenous TSLP resulted in greater than 70% recovery.
75
76
77
Statistical analysis
Statistical analyses were performed with JMP system version 8 (SAS Institute Inc.,
78
Cary, NC, USA). Data are expressed as means ± standard deviation. Serum IgE levels, blood
79
cell counts or proportions, and FeNO levels were log-transformed to achieve normal
80
distributions. Two or more groups were compared using the Wilcoxon rank-sum test,
81
Kruskal-Wallis test, analysis of variance (ANOVA), or χ2 test, where appropriate. The
82
Spearman or Pearson correlation coefficients were used to analyze the relationships among
83
data, where appropriate. Stepwise multivariate regression analysis was performed to
84
determine variables predictive of serum IgE levels, blood eosinophil counts, and FeNO levels,
85
including gender, age, smoking status, atopic status, and second order interactions between
86
explanatory variables. In the multivariate regression analysis for blood eosinophil counts and
87
FeNO levels, serum IgE levels were also included as explanatory variables. Sputum
88
eosinophil proportions were not entered in the multivariate analysis due to the limited sample
89
numbers. Analysis of covariance (ANCOVA) was used to analyze associations of serum IgE
90
levels, blood eosinophil counts, and FeNO levels with smoking status and age. Post hoc
91
analyses using the Bonferroni correction for ANOVA and ANCOVA were conducted using
92
StatView software 5.0 (SAS Institute Inc., Cary, NC, USA). Current smokers were excluded
93
when analyzing the contributing factors to FeNO. A p value of <0.05 was considered
94
significant.
95
96
4
97
Supporting Information: References
98
S1.
99
diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and
asthma. This official statement of the American Thoracic Society was adopted by the ATS
Board of Directors, November 1986. Am Rev Respir Dis 1987; 136: 225-44.
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
Dantzker D PS, Pierce J, Niewoehner D, Thurlbeck W, Buist A. Standards for the
S2.
Matsumoto H, Niimi A, Jinnai M, Nakaji H, Takeda T, Oguma T, Otsuka K, Inoue H,
Yamaguchi M, Matsuoka H, Ito I, Hirai T, Chin K, Mishima M. Association of alveolar nitric
oxide levels with pulmonary function and its reversibility in stable asthma. Respiration 2011;
81: 311-7.
S3.
American Thoracic Society ERS. ATS/ERS recommendations for standardized
procedures for the online and offline measurement of exhaled lower respiratory nitric oxide
and nasal nitric oxide, 2005. Am J Respir Crit Care Med 2005; 171: 912-30.
S4.
Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, Crapo R,
Enright P, van der Grinten CP, Gustafsson P, Jensen R, Johnson DC, MacIntyre N, McKay R,
Navajas D, Pedersen OF, Pellegrino R, Viegi G, Wanger J. Standardisation of spirometry.
Eur Respir J 2005; 26: 319-38.
S5.
Matsumoto H, Niimi A, Takemura M, Ueda T, Minakuchi M, Tabuena R, Chin K,
Mio T, Ito Y, Muro S, Hirai T, Morita S, Fukuhara S, Mishima M. Relationship of airway
wall thickening to an imbalance between matrix metalloproteinase-9 and its inhibitor in
asthma. Thorax 2005; 60: 277-81.
117
5
118
Supporting Information: Figure legends
119
Figure S1. Relationships between log-transformed serum IgE levels and smoking status when
120
data were separately analyzed in the elderly patients (≥64 yr) (p = 0.003 using the Kruskal–
121
Wallis test) and younger patients (<64 yr) with asthma. *using the Wilcoxon rank-sum test
122
123
Figure S2. Relationships between log-transformed blood eosinophil counts and smoking
124
status when data were separately analyzed in the elderly patients (p = 0.005 using the
125
Kruskal–Wallis test) and younger patients with asthma (p = 0.050 using the Kruskal–Wallis
126
test). *using the Wilcoxon rank-sum test
127
128
Figure S3. Relationships between log-transformed fractional exhaled nitric oxide (FeNO)
129
levels and smoking status when data were separately analyzed in the elderly and younger
130
patients with asthma. *using the Wilcoxon rank-sum test
131
6
132
Supporting Information: Table
133
Table S1: Multivariate regression analyses for predictors of log-transformed fractional
exhaled nitric oxide levels in atopic and non-atopic patients.
Estimate (SE)
p value
134
Atopic (n=185)
*Serum IgE, IU/mL
0.19 (0.05)
<.0001
Smoking
0.14 (0.07)
0.0498
-0.005 (0.002)
0.035
0.23 (0.06)
0.0003
Interaction between age and *serum IgE
Non-atopic (n=76)
*Serum IgE, IU/mL
135
136
Smoking (ex-smoking = 1, never-smoking = 0)
* log-transformed, current smokers were excluded from the analysis
137
138
7
139
Supporting Information without highlight
140
Smoking attenuates the age-related decrease in IgE levels and maintains eosinophilic
141
inflammation in patients with asthma
142
143
Tadao Nagasaki1, Hisako Matsumoto1, Hitoshi Nakaji1,2, Akio Niimi1,3, Isao Ito1, Tsuyoshi
144
Oguma1, Shigeo Muro1, Hideki Inoue1, Toshiyuki Iwata1, Tomoko Tajiri1, Yoshihiro
145
Kanemitsu1, Michiaki Mishima1
146
147
1
148
Kyoto, Japan
149
2
Department of Respiratory Medicine, Wakayama Red Cross Hospital, Wakayama, Japan
150
3
Division of Respiratory Medicine, Department of Medical Oncology and Immunology,
151
Nagoya City University School of Medical Sciences, Nagoya, Aichi, Japan
Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University,
152
153
Corresponding author: Hisako Matsumoto, MD, PhD
154
Department of Respiratory Medicine
155
Postgraduate School of Medicine, Kyoto University
156
54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
157
Telephone: +81-75-751-3830; Fax: +81-75-751-4643
158
E-mail: hmatsumo@kuhp.kyoto-u.ac.jp
159
8
160
Supporting Information: Methods
161
Subjects
162
The present study was a cross-sectional study on adult patients with asthma that
163
were newly referred to the Asthma Clinic of Kyoto University Hospital between June 2006
164
and October 2011. Asthma was newly diagnosed according to the American Thoracic Society
165
criteria, which define asthma as a history of recurrent episodes of wheezing and chest
166
tightness, with or without cough, and documented airway reversibility with a bronchodilator
167
or hyper-responsiveness to inhaled methacholine [S1]. The diagnosis of asthma was made
168
independent from this study and the presence of atopy, levels of serum IgE and blood
169
eosinophil counts were not considered at the time smokers were assigned to this study.
170
Patients were not treated with a steroid or leukotriene antagonist, and demonstrated normal
171
chest radiographic findings. Ex-smokers were defined as those who had stopped smoking for
172
at least 1 year. Patients with asthma who had smoked less than 5 pack-years were excluded.
173
Smoking status, the presence of atopic dermatitis, allergic rhinitis, and childhood asthma
174
were evaluated via a self-reported questionnaire. The study protocol was approved by the
175
Ethics Committee of Kyoto University, and written informed consent was obtained from all
176
subjects.
177
178
Measurements
179
Patients underwent a work-up, including a physical examination, blood tests, chest
180
radiographs, fractional exhaled nitric oxide (FeNO) concentration measurements, pulmonary
181
function tests, and sputum induction.
182
Total and specific serum IgE antibody titers were measured via radioimmunosorbent
183
testing (Pharmacia Diagnostics, Uppsala, Sweden). Patients were considered atopic when one
184
or more specific IgE antibodies against grass pollen, mold, weed, house dust mite,
185
Dermatophagoides pteronyssinus, Japanese cedar pollen, cat dander, dog dander, or
9
186
Trichophyton were positive.
187
FeNO at a constant exhalation flow rate of 50 mL/s was measured with a
188
chemiluminescence analyzer (NOA 280, Sievers, Boulder, CO, USA) [S2], according to the
189
current guidelines [S3]. The analyzer was calibrated daily with non-NO-containing gas,
190
which was generated by exposing ambient air to NO scavengers and a standard concentration
191
of 640 ppb NO. The lower detection limit for NO was 2 ppb. The signal output from the NO
192
analyzer was fed to a computer data acquisition program, and concentrations were measured
193
using a data analysis program (NOA Analysis™ Software, Sievers). Seated subjects inhaled
194
orally until total lung capacity was reached, and then inserted a mouthpiece and exhaled
195
immediately against a resistance to achieve a constant exhalation flow rate of 50 ml/s. FeNO
196
measurements were taken from a steady plateau. The average of three measurements was
197
used, and measurements were performed prior to spirometry.
198
Pre-bronchodilator forced vital capacity (FVC), FEV1, and mid-forced expiratory
199
flow25-75% (FEF25-75%) were tested using a ChestGraph HI-701 spirometer (Chest MI Corp,
200
Tokyo, Japan), according to the guidelines of the American Thoracic Society [S4].
201
Sputum induction and processing were performed according to the slightly modified
202
methodology of Pin et al [S5]. Briefly, subjects inhaled a hypertonic (3%) saline solution
203
from an ultrasonic nebulizer (MU-32, Azwell Inc., Osaka, Japan) for 15 min, and adequate
204
plugs of sputum were separated from the saliva. After treatment with 0.1% dithiothreitol
205
(OXOID Ltd., Hampshire, UK), the sample was cytocentrifuged and cells were stained using
206
the May-Grünwald-Giemsa method. Cell differentials were determined by counting at least
207
400 non-squamous cells on each sputum slide [E5]. Supernatants of the sputum were stored at
208
-20°C for later use. TSLP concentrations in sputum supernatants were measured via an
209
enzyme-linked immunosorbent assay kit (R&D Systems, Inc., MN, USA), according to the
210
manufacturer’s instructions. The detection limit of this assay was 3.46 pg/mL, and values
211
below this threshold were assigned values of 0 pg/mL. A spike-back analysis using
10
212
exogenous TSLP resulted in greater than 70% recovery.
213
214
215
Statistical analysis
Statistical analyses were performed with JMP system version 8 (SAS Institute Inc.,
216
Cary, NC, USA). Data are expressed as means ± standard deviation. Serum IgE levels, blood
217
cell counts or proportions, and FeNO levels were log-transformed to achieve normal
218
distributions. Two or more groups were compared using the Wilcoxon rank-sum test,
219
Kruskal-Wallis test, analysis of variance (ANOVA), or χ2 test, where appropriate. The
220
Spearman or Pearson correlation coefficients were used to analyze the relationships among
221
data, where appropriate. Stepwise multivariate regression analysis was performed to
222
determine variables predictive of serum IgE levels, blood eosinophil counts, and FeNO levels,
223
including gender, age, smoking status, atopic status, and second order interactions between
224
explanatory variables. In the multivariate regression analysis for blood eosinophil counts and
225
FeNO levels, serum IgE levels were also included as explanatory variables. Sputum
226
eosinophil proportions were not entered in the multivariate analysis due to the limited sample
227
numbers. Analysis of covariance (ANCOVA) was used to analyze associations of serum IgE
228
levels, blood eosinophil counts, and FeNO levels with smoking status and age. Post hoc
229
analyses using the Bonferroni correction for ANOVA and ANCOVA were conducted using
230
StatView software 5.0 (SAS Institute Inc., Cary, NC, USA). Current smokers were excluded
231
when analyzing the contributing factors to FeNO. A p value of <0.05 was considered
232
significant.
233
234
11
235
Supporting Information: References
236
S1.
237
diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and
asthma. This official statement of the American Thoracic Society was adopted by the ATS
Board of Directors, November 1986. Am Rev Respir Dis 1987; 136: 225-44.
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
Dantzker D PS, Pierce J, Niewoehner D, Thurlbeck W, Buist A. Standards for the
S2.
Matsumoto H, Niimi A, Jinnai M, Nakaji H, Takeda T, Oguma T, Otsuka K, Inoue H,
Yamaguchi M, Matsuoka H, Ito I, Hirai T, Chin K, Mishima M. Association of alveolar nitric
oxide levels with pulmonary function and its reversibility in stable asthma. Respiration 2011;
81: 311-7.
S3.
American Thoracic Society ERS. ATS/ERS recommendations for standardized
procedures for the online and offline measurement of exhaled lower respiratory nitric oxide
and nasal nitric oxide, 2005. Am J Respir Crit Care Med 2005; 171: 912-30.
S4.
Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, Crapo R,
Enright P, van der Grinten CP, Gustafsson P, Jensen R, Johnson DC, MacIntyre N, McKay R,
Navajas D, Pedersen OF, Pellegrino R, Viegi G, Wanger J. Standardisation of spirometry.
Eur Respir J 2005; 26: 319-38.
S5.
Matsumoto H, Niimi A, Takemura M, Ueda T, Minakuchi M, Tabuena R, Chin K,
Mio T, Ito Y, Muro S, Hirai T, Morita S, Fukuhara S, Mishima M. Relationship of airway
wall thickening to an imbalance between matrix metalloproteinase-9 and its inhibitor in
asthma. Thorax 2005; 60: 277-81.
255
12
256
Supporting Information: Figure legends
257
Figure S1. Relationships between log-transformed serum IgE levels and smoking status when
258
data were separately analyzed in the elderly patients (≥64 yr) (p = 0.003 using the Kruskal–
259
Wallis test) and younger patients (<64 yr) with asthma. *using the Wilcoxon rank-sum test
260
261
Figure S2. Relationships between log-transformed blood eosinophil counts and smoking
262
status when data were separately analyzed in the elderly patients (p = 0.005 using the
263
Kruskal–Wallis test) and younger patients with asthma (p = 0.050 using the Kruskal–Wallis
264
test). *using the Wilcoxon rank-sum test
265
266
Figure S3. Relationships between log-transformed fractional exhaled nitric oxide (FeNO)
267
levels and smoking status when data were separately analyzed in the elderly and younger
268
patients with asthma. *using the Wilcoxon rank-sum test
269
13
270
Supporting Information: Table
271
Table S1: Multivariate regression analyses for predictors of log-transformed fractional
exhaled nitric oxide levels in atopic and non-atopic patients.
Estimate (SE)
p value
272
Atopic (n=185)
*Serum IgE, IU/mL
0.19 (0.05)
<.0001
Smoking
0.14 (0.07)
0.0498
-0.005 (0.002)
0.035
0.23 (0.06)
0.0003
Interaction between age and *serum IgE
Non-atopic (n=76)
*Serum IgE, IU/mL
273
274
Smoking (ex-smoking = 1, never-smoking = 0)
* log-transformed, current smokers were excluded from the analysis
275
276
277
14