Heme Oxygenase-1, a Potential Biomarker of Chronic
Silicosis, Attenuates Silica-induced Lung Injury
Takashi Sato, Mitsuhiro Takeno, Koichi Honma, Hideyuki Yamauchi, Yoshiaki Saito, Takao Sasaki,
Hiroshi Morikubo, Yoji Nagashima, Shigeto Takagi, Kouichi Yamanaka, Takeshi Kaneko, and
Yoshiaki Ishigatsubo
Department of Internal Medicine and Clinical Immunology, and Department of Pathology, Yokohama City University, Yokohama;
Department of Pathology, Dokkyo University School of Medicine; Department of Internal Medicine, and Department of Radiology,
Rosai Hospital for Silicosis, Tochigi; and Seamen’s Insurance AMHTS Clinic, Yokohama, Japan
Rationale: Heme oxygenase-1 (HO-1), a rate-limiting enzyme in
heme catabolism, has antioxidative, antiapoptotic, and antiinflam-
matory activities. We examined whether HO-1 might be involved
in silicosis.
Objectives: To investigate whether HO-1 can reduce silicosis in mice
and humans.
Methods and measurements: Silicosis was studied using a murine
model, and in 46 male patients. Serum HO-1 and 8-hydroxydeoxy-
guanosine (a marker of oxidative stress) were measured by enzyme-
linked immunosorbent assay. Levels of HO-1 were measured by
immunohistochemistry and immunoblotting.
Main results: Serum HO-1 levels were significantly elevated in pa-
tients with silicosis compared with age-matched control subjects
or patients with chronic obstructive pulmonary disease. Serum HO-1
levels also correlated inversely with serum 8-hydroxydeoxyguanos-
ine levels and positively with vital capacity and forced expiratory
volume in one second in patients with silicosis. HO-1 was present
in the lungs of humans and mice with silicosis, especially at sites
of silica particle deposition. In mice, silica exposure was associated
with acute leukocyte infiltration, leading to development of silicotic
lung lesions. The inflammation was suppressed by treatment with
hemin, an inducer of HO-1, and enhanced by zinc protoporphyrin,
an inhibitor of HO-1.
Conclusions: Pulmonary HO-1 expression is increased in silicosis.
HO-1 suppresses reactive oxygen species activity, and subsequent
pathologic changes, thereby attenuating disease progression.
Keywords: antioxidants; occupational diseases; oxidative stress
Inhalation of crystalline silica for prolonged periods can lead to
silicosis, an inflammatory disorder (1, 2). The acute manifesta-
tions of this disease include inflammation and apoptosis, with
attendant destruction of the lung tissue (3), whereas chronic
silicosis is characterized by progressive fibrosis and emphysema-
tous changes (4). Although lung function is severely impaired
in patients with advanced disease, airway obstruction is common
even in silica-exposed workers with no radiological abnormali-
ties (4, 5). Defining the molecular mechanisms triggered by expo-
sure to silica could facilitate early diagnosis by identifying mark-
(Received in original form August 10, 2005; accepted in final form July 18, 2006 )
This work was supported in part by grants from The Yokohama City University
Center of Excellence Program, and grant (No.16590991) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
Correspondence and requests for reprints should be addressed to Professor
Yoshiaki Ishigatsubo, Department of Internal Medicine and Clinical Immunology,
Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004,
Japan. E-mail: ishigats@med.yokohama-cu.ac.jp
This article has an online supplement, which is accessible from this issue’s table
of contents at www.atsjournals.org.
Am J Respir Crit Care Med Vol 174. pp 906–914, 2006
Originally Published in Press as DOI: 10.1164/rccm.200508-1237OC on July 20, 2006
Internet address: www.atsjournals.org
ers of early disease, and could elucidate novel therapeutic
strategies.
Crystalline silica induces the production of reactive oxygen
species (ROS) (2), which play a key role in the development of
silicosis (6) via the mitogen-activated protein kinase (MAPK)
pathway (7). ROS cause direct tissue injury and activation of
alveolar macrophages to undergo apoptosis (8), enhance the
synthesis of proinflammatory cytokines (9), and augment the
induction of inflammatory responses via the MAPK-dependent
pathways (10). Silica-induced oxidative DNA damage, reflected
by an increase in 8-hydroxydeoxyguanosine (8-OHdG), is associ-
ated with an increased risk of carcinogenesis (11). ROS produc-
tion is down-regulated by elements of the extracellular signal-
regulated kinase (ERK) pathway, which trigger increased tran-
scription of antioxidants (12).
One of the antioxidants that protect against ROS-induced
airway inflammation is heme oxygenase (HO)-1, an enzyme
that degrades heme into bilirubin, Fe2⫹, and carbon monoxide
(CO) (13, 14). Lung epithelial cells, smooth muscle cells, macro-
phages, and endothelial cells can all produce HO-1 (15–17). The
protective activity of HO-1 was established in several model
systems. For example, adenovirus-mediated gene transfer of
HO-1 cDNA suppresses lipopolysaccharide-induced lung injury
(18), influenza-induced pneumonia (19), and bleomycin-induced
pulmonary fibrosis (20) in murine models. The current work
explores whether HO-1 can play a role in attenuating silica-
mediated lung injury.
We evaluated HO-1 protein levels in sera from patients with
silicosis and in a murine model of that disease. To clarify the
role of HO-1, we assessed the effects of hemin and zinc protopor-
phyrin (ZnPP) (an inducer and an inhibitor of HO-1, respec-
tively) in murine silicosis. Abbreviated results from this study
were previously reported in abstract form (21, 22).
METHODS
For a detailed description of the methods, see the online supplement.
Patients
Forty-six male patients (age range, 56–83 yr) were diagnosed as having
silicosis on the basis of their occupational history and the International
Labor Organization (ILO) International Classification of Radiographs
of Pneumoconioses (23). Radiographic opacities were graded on a 12-
point scale using the ILO system (score of 1 for 0/⫺ and 12 for 3/⫹)
(24). More details are shown in the online supplement. Twenty-seven
male patients with chronic obstructive pulmonary disease (COPD) (age
range, 60–87 yr) and 42 healthy male volunteers (age range, 55–80 yr)
served as age-matched disease and normal control subjects, respectively.
Subjects were defined as smokers if they had smoked at least 1 pack-
year, and as ex-smokers if they had stopped smoking for at least 1 yr (25).
Serum HO-1 and 8-OHdG were measured by enzyme-linked immuno-
sorbent assay (ELISA). C-reactive protein (CRP) in serum was analyzed
by immunonephelometry (26). Spirometry was performed using standard
Sato, Takeno, Honma, et al.: The Role of HO-1 in Silicosis 907

TABLE 1. DEMOGRAPHIC AND CLINICAL CHARACTERISTICS TABLE 3. CORRELATION BETWEEN SERUM HEME
OF THE STUDY GROUPS OXYGENASE-1 AND VARIABLES FROM THE PATIENTS
WITH SILICOSIS
Silicosis (n ⫽ 46) COPD (n ⫽ 27) Normal (n ⫽ 42)
Univariate Model
Age, yr 72.8 ⫾ 0.8 71.8 ⫾ 1.4 71.6 ⫾ 1.0
Height, m 1.61 ⫾ 0.01 1.60 ⫾ 0.02 1.63 ⫾ 0.01 Characteristics Correlation (r) p Value
Weight, kg 57.9 ⫾ 1.6* 58.0 ⫾ 2.4 62.8 ⫾ 1.2
Smoking status Serum parameter
Never smoker, n (%) 3 (7) 0 (0) 19 (45) 8-OHdG, ng/ml 0.28 ⫾ 0.02 ⫺0.311 0.035
Ex-smoker, n (%) 41 (89) 22 (81) 17 (41) Pulmonary function
Current smoker, n (%) 2 (4) 5 (19) 6 (14) VC, L 2.54 ⫾ 0.11 ⫹0.296 0.046
Pack-years 39.8 ⫾ 3.6 †‡
71.6 ⫾ 9.2‡ 15.9 ⫾ 4.0 FEV1, L 1.49 ⫾ 0.08 ⫹0.390 0.008
Pulmonary function
Definition of abbreviations: 8-OHdG ⫽ 8-hydroxydeoxyguanosine; FEV1 ⫽ forced
VC, L 2.54 ⫾ 0.11* 2.70 ⫾ 0.15 2.96 ⫾ 0.11
expiratory volume in one second; VC ⫽ vital capacity.
VC, %predicted 80.4 ⫾ 3.0§ 86.7 ⫾ 4.4 92.7 ⫾ 3.0
Values are mean ⫾ SEM. The relationships between serum heme oxygenase-1
FEV1, L 1.49 ⫾ 0.08‡ 1.51 ⫾ 0.16‡ 2.31 ⫾ 0.09
and other laboratory data were analyzed by using linear regression analysis with
FEV1, %predicted 69.6 ⫾ 3.5‡
71.8 ⫾ 6.7‡ 103.9 ⫾ 3.5
the method of least squares.
Serum CRP, mg/L 5.39 ⫾ 1.51‡ 2.56 ⫾ 0.83 0.66 ⫾ 0.09
Serum HO-1, ng/ml 6.23 ⫾ 0.46‡¶ 1.77 ⫾ 0.21 2.75 ⫾ 0.22
Serum 8-OHdG, ng/ml 0.280 ⫾ 0.018‡¶ 0.189 ⫾ 0.006 0.185 ⫾ 0.007

Definition of abbreviations: 8-OHdG ⫽ 8-hydroxydeoxyguanosine; COPD ⫽
chronic obstructive pulmonary disease; CRP ⫽ C-reactive protein; FEV1 ⫽ forced
expiratory volume in one second; HO-1 ⫽ heme oxygenase-1; VC ⫽ vital capacity.
Values are mean ⫾ SEM. Comparisons between study groups were made by
using one-way analysis of variance (ANOVA) followed by post hoc analysis with
the Bonferroni test.
* p ⬍ 0.01, significant difference compared with normal.
†
p ⬍ 0.001, significant difference compared with COPD.
‡
p ⬍ 0.0001, significant difference compared with normal.
§
p ⬍ 0.001, significant difference compared with normal.
¶
p ⬍ 0.0001, significant difference compared with COPD.
protocols according to the American Thoracic Society recommendations
(27). Local HO-1 expression was examined by immunohistochemistry in
the lungs from 10 patients with silicosis (8 autopsies and 2 surgical
specimens) and from 17 control autopsy cases with no occupational
lung diseases. In silicosis samples, HO-1–positive cells were counted
microscopically in 50 high-power fields (HPF) (approximately equiva-
lent to 10 mm2) containing areas of silicotic fibrosis; in control samples,
this was done in 50 randomly selected HPF. The study was approved
by the local Institutional Review Board. All patient studies were per-
formed after obtaining written informed consent, except for the studies
on autopsy samples, for which the need for informed consent was
waived.
Murine Silicosis Model
Six-week-old male BALB/c mice were used for all animal experiments.
All studies were approved by the Institutional Animal Care and Use
Committee at Yokohama City University. Under anesthesia with keta-
mine (80 mg/kg; Sigma-Aldrich, St. Louis, MO) and xylazine (10 mg/
kg; Sigma), 100 mg/kg of sterilized crystalline silica (Min-U-Sil-5, U.S.
Silica, Berkeley Springs, WV) in 100 ␮l of sterile saline were instilled
into the trachea (28). In some experiments, 100 ␮mol/kg hemin (Sigma)
or ZnPP (Porphyrin Products, Logan, UT) were administered intraperi-
toneally at 0.5–48 h before silica administration (18). Cell differentials
were determined in bronchoalveolar lavage fluid (BALF), obtained by
tracheal cannulation using five washes with 0.8-ml aliquots of PBS/wash
(29). Plasma 8-OHdG was measured by ELISA. HO-1 in the lung was
monitored using immunohistopathologic and immunoblotting techniques.

Statistics
Values were expressed as mean ⫾ SEM. In the mouse studies, only
nonparametric methods were used for statistical analysis. Comparisons
TABLE 2. CHARACTERISTICS OF PATIENTS WITH SILICOSIS
between multiple independent groups were made by Kruskal Wallis test
Characteristics n (% )

Occupation
Miner 22 (48)
Stone mason 14 (30)
TABLE 4. DEMOGRAPHIC DATA OF SURGERY AND
AUTOPSY SUBJECTS
Tunnel worker 10 (22)
Duration of exposure, yr* 23.4 ⫾ 1.8 Silicosis Group Control Group
Periods since first exposure, yr* 50.5 ⫾ 1.2 (n ⫽ 10) (n ⫽ 17)
Radiographic category
Small opacities category Male:female 10:0 8:9
p 19 Surgery:autopsy 2:8 0:17
q 17 Age, yr* 63 ⫾ 3.3 67 ⫾ 3.9
r 7 Diagnosis
No small opacities 3 Silicosis (simple:complicated) 10 (3:7) 0
Profusion score * 8.28 ⫾ 0.46 Abdominal malignancy 0 6
Large opacities category Cardiac failure 0 6
A 6 Chronic pancreatitis 0 1
B 8 Pseudomembranous colitis 0 1
C 4 Systemic lupus erythematosus 0 1
No large opacities 28 Polycythemic vera 0 1
Thrombotic thrombocytopenic purpura 0 1
Data represent the number of patients (parentheses show percentages) except Immunohistochemistry
where indicated. Profusion score was derived from the ILO classification (23, 24). HO-1–positive cell number/cm2* 409 ⫾ 72† 39 ⫾ 12
Rounded small opacities are classified into category p (up to 1.5 mm), q (1.5–3
mm), and r (3–10 mm) by chest radiographic findings. Large opacities are classified Definition of abbreviation: HO-1 ⫽ heme oxygenase-1.
into category A (for one or more large opacities not exceeding a combined Data represent the number of patients except where indicated. For details
diameter of 5 cm), B (large opacities with combined diameter ⬎ 5 cm but does concerning the immunohistochemical scoring system, please refer to METHODS.
not exceed the equivalent of the right upper zone), and C (bigger than B). * Values are mean ⫾ SEM.
* Values are mean ⫾ SEM. †
p ⬍ 0.05 vs. control group.
908 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 174 2006
followed by Mann-Whitney U test. In the human studies, comparisons
between study groups were made by using one-way ANOVA followed
by post hoc analysis with the Bonferroni test. The relationships between
serum HO-1 and other laboratory data were examined by using linear
regression analysis with the method of least squares. Probability values
of less than 0.05 were considered statistically significant.
RESULTS
Serum HO-1 Levels Are Increased in Patients with Mild Silicosis
The characteristics of the 46 patients with silicosis, 27 patients
with COPD, and 42 normal control subjects are shown in Tables 1
and 2. The patients with silicosis and COPD had longer smoking
histories than did control subjects, and the majority of patients
were ex-smokers. Vital capacity (VC) and/or forced expiratory
volume in one second (FEV1) were ⬍ 80% of predicted values
(30) in 21/46 patients with silicosis, a significant reduction when
compared with control subjects. Patients with COPD had re-
duced FEV1 but not VC when compared with control subjects
Figure 1. Histologic and immuno-
histochemical features of lung
samples from patients with silico-
sis. HO-1 expression (red) was
present in bronchial epithelial cells
containing polarizing particles. (A )
Hematoxylin and eosin, (B ) HO-1
immunostaining, (C ) polarizing
microscopy. Magnification: ⫻400.
The figures are representative
of samples from 10 patients with
silicosis.
but not patients with silicosis. The silicosis group included pa-
tients with disease of varying radiologic severity (28 simple silico-
sis and 18 complicated silicosis) (Table 2). Of the 46 patients,
43 had an ILO profusion of ⭓ 1/0 (with or without large opacit-
ies) and four had large opacities only. Some of the patients with
silicosis or COPD were being treated with theophylline (32.6%
and 40.7%, respectively), short-acting ␤-agonists (28.3% and
25.9%, respectively), anticholinergics (26.1% and 37.0%, re-
spectively), and/or inhaled corticosteroids (8.7% and 22.2%,
respectively).
Serum CRP levels were significantly higher in patients with
silicosis than those with COPD or control subjects (Table 1).
Serum HO-1 levels were also significantly higher in patients with
silicosis than the other groups (Table 1). Patients with silicosis
with only mildly impaired respiratory function showed the high-
est serum HO-1 levels. Thus, increased HO-1 levels correlated
positively with FEV1 (r ⫽ ⫹0.390, p ⫽ 0.008) and VC (r ⫽
⫹0.296, p ⫽ 0.046) (Table 3). Other factors examined, including
age, duration of silica exposure, smoking history, or serum CRP,
Figure 2. HO-1 expression in silicotic nodules. Macro-
phages (red) stained with anti–HO-1 mAb were present
in the central and peripheral areas of silicotic nodules. A
high-power photomicrograph is shown in the inset. (A )
Hematoxylin and eosin, (B ) HO-1 immunostaining, (C )
CD68 immunostaining in serial sections, (D ) negative
control (nonspecific mouse IgG). Magnification: ⫻100
(inset ⫻400). The figures are representative of samples
from 10 patients with silicosis.
Sato, Takeno, Honma, et al.: The Role of HO-1 in Silicosis
did not correlate with serum HO-1 levels. Among patients with
silicosis, occupational history did not correlate with serum HO-1
levels. In contrast to patients with silicosis, lung function parame-
ters and HO-1 levels did not correlate in patients with COPD
(data not shown).
909
Figure 3. Immunohistochemical staining for HO-1 in mice
with silicosis. HO-1 immunostaining in the lung sections
from control mice (A ), or mice with silica-induced lung
injury on Day 1 (B ), Day 14 (C ), or Week 12 (D ). Immuno-
histochemistry detected HO-1 expression in bronchial epi-
thelial cells (B, Day 1), and granulomatous lesions (C, Day
14; D, Week 12) (magnification: ⫻100).
Serum 8-OHdG levels were significantly higher in silicosis
patients than those with COPD or healthy control subjects
(Table 1). Furthermore, a significant negative correlation was
found between serum HO-1 and 8-OHdG levels (r ⫽ ⫺0.311,
p ⫽ 0.035) (Table 3).
Figure 4. High-power photomicrographs of HO-1–
positive granulomatous lesions 12 wk after silica instillation
in mice (magnification: ⫻400). (A ) HO-1 immunostaining.
(B ) Polarizing microscopy of the same field as in A indicates
that HO-1–positive macrophages phagocytized silica
particles. (C ) F4/80 immunostaining. (D ) Negative control
(rat IgG).
910 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 174 2006
Localization of HO-1 Protein in Silicosis Lungs
Since elevated serum HO-1 was preferentially found in patients
with silicosis, we examined HO-1 expression in the lung lesions.
Surgical specimens from 2 patients with silicosis and with lung
cancer, 8 autopsied subjects with silicosis, and 17 control subjects
were studied (Table 4). HO-1 protein was detected by immuno-
histochemical staining in bronchial epithelial cells (Figure 1) and
macrophages (Figure 2) of granulomatous tissue from areas free
of tumor. Silica particles were consistently associated with lesions
containing HO-1–expressing cells (Figure 1). The number of
HO-1–expressing cells was significantly higher in patients with
silicosis than in control subjects (HO-1–positive cell number/
cm2; 409 ⫾ 72 versus 39 ⫾ 12, p ⬍ 0.05) (Table 4).
HO-1 Expression in Murine Silicosis
A murine model of silicosis was studied to clarify the role of
HO-1. Disease was induced by administering crystalline silica
intratracheally to BALB/c mice. This resulted in acute leukocyte
infiltration followed by granuloma formation similar to that ob-
served in human silicosis (Figure E1 in the online supplement).
Immunohistochemical analysis showed that HO-1 expression
had increased in bronchial epithelial cells within 1 d of silica
exposure (Figure 3). HO-1–expressing cells were also found in
granulomatous tissues during the chronic phase of disease
(Figure 4). Consistent with results from the human lung biopsies,
HO-1–expressing cells were found close to silica particles depos-
ited in the lungs throughout the clinical course of disease in
mice (Figure 4). Semiquantitative analysis of HO-1 protein by
immunoblotting indicated that HO-1 expression in the lungs
gradually increased as disease progressed (Figures 5A and 5B).
Plasma 8-OHdG concentrations gradually increased through
12 wk, and then decreased to the baseline by 24 wk (Figure 5C).
This contrasts with the persistent elevation in HO-1 expression
late in the disease process (Figures 5A and 5B).
Super-induction of HO-1 Attenuates Acute Silicosis in Mice
Consistent with the acute inflammatory responses caused by
silica in the lower respiratory tract (31), neutrophils were the domi-
nant cells in BALF during the first 3 d after silica exposure; later
macrophages and lymphocytes became dominant (Figure 6).
To evaluate the impact of HO-1 on the development of silico-
sis, mice were treated with hemin, a potent HO-1 inducer, or
Figure 5. Time course of HO-1 expression in the lung and
plasma 8-OHdG level in silicosis model mouse. (A ) Immu-
noblotting analysis of HO-1 in the microsomal fraction of
mouse silicosis lungs. (B ) Semiquantitative analysis
of immunoblots with anti–HO-1 mAb showed a gradual
increase in HO-1 protein in the lung after silica exposure.
Four mice were examined at each time point (*p ⬍
0.05 versus control). (C ) Plasma 8-OHdG levels were de-
termined by ELISA. The level was increased until 12 wk
after silica instillation and then returned to baseline (*p ⬍
0.05, **p ⬍ 0.01 versus control). Data are shown as
mean ⫾ SEM. Numbers inside the bars indicate the number
of mice investigated in each group.
Sato, Takeno, Honma, et al.: The Role of HO-1 in Silicosis
ZnPP, an inhibitor of HO. In normal mice, a single administra-
tion with hemin induced sustained HO-1 expression for at least
7 d, whereas ZnPP had no effect (Figure E2). In mice with
silicosis, HO-1 expression in the lung was enhanced by hemin
and suppressed by ZnPP (Figures 7A and 7B). Infiltrating cell
numbers were decreased by hemin but increased by ZnPP (Fig-
ure 6A). Hemin pretreatment reduced neutrophil infiltration and
the subsequent accumulation of macrophages, whereas ZnPP
pretreatment enhanced the influx of neutrophils and the subse-
quent accumulation of lymphocytes (Figures 6B–6D). Histopath-
ologic analysis showed that cellular infiltration into alveolar
spaces was suppressed by hemin but augmented by ZnPP (Figure
E1A). Thus, hemin suppressed acute inflammation after silica
exposure, whereas lesion development was accelerated by ZnPP.
The silica-induced increases in plasma 8-OHdG levels were
markedly increased by pretreatment with ZnPP and suppressed
by hemin (Figure 7C). There was an inverse correlation between
lung HO-1 and plasma 8-OHdG levels (Figures 7A–7C). This
observation is consistent with the finding that serum HO-1 levels
were not elevated in patients with silicosis with high serum levels
of 8-OHdG.
DISCUSSION
More than 10,000 patients with silicosis are currently followed
up in Japan, although the incidence has been gradually decreas-
ing due to efforts to prevent further silica exposure (32). Unfortu-
nately, silicosis continues to occur in many developing countries
(due to a paucity of preventive medicine) and in some advanced
countries (especially in the fields of chemical and civil engi-
neering, where workers are exposed to silica-containing dusts)
(33). Although this disease can be prevented by avoiding silica
exposure, equally important is the development of therapy for
those with pre-existing disease.
This work is the first to demonstrate that HO-1 is synthesized
in the lungs of patients with silicosis, thus contributing to a
significant elevation of serum HO-1 levels in patients. Our stud-
ies suggest that the lung is a major source of circulating HO-1,
since silicotic nodules contain abundant HO-1–expressing cells.
911
Figure 6. Effects of pretreatment with hemin or ZnPP in
the murine silicosis model. (A ) Total cell number in the
BALF 1–14 d after silica exposure. The cell number was
significantly reduced by pre-treatment with hemin (*p ⬍
0.05 versus silica alone, n ⫽ 4–5 mice each), and signifi-
cantly increased by ZnPP (*p ⬍ 0.05 versus silica alone,
n ⫽ 4–5 mice each). (B ) Neutrophils (PMN) were signifi-
cantly reduced or elevated after Day 1 in mice pretreated
with hemin or ZnPP, respectively (*p ⬍ 0.05 versus silica
alone, n ⫽ 4–5 mice each). (C ) Macrophages were signifi-
cantly reduced after Day 7 in hemin-pretreated mice
(*p ⬍ 0.05 versus silica alone, n ⫽ 4–5 mice each). (D )
Lymphocytes were significantly enhanced after Day 7 in
ZnPP-pretreated mice (*p ⬍ 0.05 versus silica alone, n ⫽
4–5 mice each). Data are shown as mean ⫾ SEM of four
or five experiments.
In contrast, peripheral blood mononuclear cells (PBMC) are
unlikely to be the source of serum HO-1, both because HO-1
expression was not elevated in PBMC from patients with silicosis
and HO-1 protein levels in serum did not correlate with protein
production by PBMC (r ⫽ ⫹0.185, p ⫽ 0.301) (unpublished
observations). If serum HO-1 derives primarily from lung le-
sions, it could provide a novel biomarker for evaluating the
severity of silicosis.
Although HO-1 expression in the lung is increased in diseases
characterized by increased oxidative stress (34, 35), changes in
serum HO-1 levels have not been reported in pulmonary disor-
ders. We previously demonstrated that serum HO-1 levels were
elevated in patients with adult-onset Still’s disease and hemopha-
gocytic syndrome, two systemic inflammatory disorders charac-
terized by hyper-ferritinemia and macrophage activation, and
that serum HO-1 levels correlate closely with disease activity
(36).
Our present results indicate that serum HO-1 levels correlate
significantly with FEV1 and VC in patients with silicosis. A previ-
ous necropsy study revealed that pathologic changes in the lung
parenchyma and large airways were associated with impairment
of lung function in patients with silicosis (37). Although involve-
ment of ROS and increased circulating carbon monoxide levels
are present in patients with COPD (38, 39), serum HO-1 levels
are not elevated in those patients (whose lung function is compa-
rable to those with silicosis). Rather, data from human lung
samples and from experimental silicosis in mice indicates that
silica exposure directly and persistently induces HO-1 expres-
sion in granulomatous lung tissue.
The present study examined HO-1 expression in human lung
samples. Considerable care was taken to ensure that studies
involving these samples were free from artifacts (potentially
introduced due to the terminal stage of illness, postmortem phe-
nomena, and/or extrapulmonary disease). Several groups have
shown that HO-1 can be accurately detected in autopsy material
(40–42). Moreover, there were no differences between surgically
resected versus autopsied lung tissue with respect to levels of
HO-1 expression (HO-1–positive cell number/cm2; 414 ⫾ 204
versus 407 ⫾ 88), suggesting that the terminal stage of disease
912 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 174 2006
did not affect expression of this protein. Finally, lungs from
patients with various extrapulmonary diseases expressed HO-1
at levels far below those found in silicotic lungs.
There is accumulating evidence that HO-1 helps protect the
host from progression of certain inflammatory diseases (43). In
the current study, super-induction of HO-1 by administration of
hemin suppressed silica-induced leukocyte infiltration in the
lung, whereas treatment with ZnPP augmented it. When hemin
was administered to normal mice (without silica exposure), HO-1
expression was induced in the lung, and persisted for at least
7 d (Figure E2). ZnPP did not alter HO-1 expression level in
the absence of silica exposure, but did suppress HO-1 expression,
leukocyte infiltration, and subsequent pathologic changes in mice
with silicosis (Figure 7). This discrepancy suggests that ZnPP
does not directly act on HO-1 expression but instead suppresses
a mediator induced by silica that stimulates HO-1 expression.
ROS play a critical role in the development of silicosis (2).
In addition to causing direct tissue toxicity, ROS damage nuclear
and mitochondrial DNA, leading to increased generation of
Figure 7. Effects of hemin and ZnPP on HO-1
expression and plasma 8-OHdG levels in mu-
rine silicosis. (A ) Lung immunoblots demon-
strated that HO-1 expression was up- or
down-regulated by pretreatment with hemin
or ZnPP, respectively (n ⫽ 4 at each time
point of each group). (B ) Semiquantitative
analysis of HO-1/actin expression by densi-
tometry (n ⫽ 4 at each time point of each
group; *p ⬍ 0.05 versus silica alone). (C )
Plasma 8-OHdG determined by ELISA was
significantly reduced 1 d after hemin pre-
treatment of mice and was elevated 2 d after
ZnPP pretreatment of mice (n ⫽ 4–7 at each
time point of each group). Data are shown as
mean ⫾ SEM (*p ⬍ 0.05 versus silica alone).
8-OHdG (44). In the murine silicosis model, plasma 8-OHdG
levels were elevated from 1–12 wk after particle exposure and
then returned to baseline by 24 wk. In contrast, HO-1 expression
was persistently increased in lungs of mice with silicosis. The
dissociation between lung HO-1 expression and plasma levels
of 8-OHdG may have several explanations. First, because ROS
activity reflects the balance between oxidative stress and antioxi-
dant systems (45), it is likely that antioxidant activity exceeds
silica-induced ROS after 12 wk in this model. Second, by 12
wk, some of the silica particles may have left the lung via the
lymphatics (accumulation of silica in the draining lymph nodes
was confirmed histologically). The negative relationship between
8-OHdG and HO-1 was particularly evident in mice treated with
hemin or ZnPP. Hemin enhanced the expression of HO-1 while
reducing 8-OHdG levels compared with controls, whereas ZnPP
treatment had the opposite effects. Indeed, serum 8-OHdG lev-
els were increased in patients with silicosis having normal serum
HO-1 levels. Previous studies suggest that ROS are generated
via the Fenton mechanism after silica exposure (2), and that
Sato, Takeno, Honma, et al.: The Role of HO-1 in Silicosis
HO-1 acts as an important scavenger of ROS (46, 47), probably
via the heme degradation products bilirubin and CO (43).
In summary, the present study shows that HO-1 is persistently
expressed in the lung lesions of patients with silicosis. This ap-
pears to reflect the induction of ROS by silica, leading to an
elevation in serum HO-1 levels. The increased HO-1 can protect
the host by suppressing silica-induced ROS activity. Thus, upreg-
ulation of HO-1 may represent a novel strategy for the treatment
of silicosis.
Conflict of Interest Statement : None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
Acknowledgment : The authors thank Dennis M. Klinman (Center for Biologics
Evaluation and Research, FDA) for helpful discussions and Toshihiro Shibata, CT
(Division of Pathology, Rosai Hospital for Silicosis) for technical help. The authors
also thank the nursing and laboratory staff of Rosai Hospital for Silicosis.
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