Am J Physiol Lung Cell Mol Physiol 286: L546–L553, 2004.
First published November 14, 2003; 10.1152/ajplung.00267.2003.
Human SP-A genetic variants and bleomycin-induced cytokine production by
THP-1 cells: effect of ozone-induced SP-A oxidation
Weixiong Huang,1 Guirong Wang,1 David S. Phelps,2 Hamid Al-Mondhiry,3 and Joanna Floros1,2,4
Departments of 1Cellular and Molecular Physiology, 2Pediatrics, 3Medicine, and 4Obstetrics and
Gynecology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Submitted 31 July 2003; accepted in final form 12 November 2003
Huang, Weixiong, Guirong Wang, David S. Phelps, Hamid
Al-Mondhiry, and Joanna Floros. Human SP-A genetic variants and
bleomycin-induced cytokine production by THP-1 cells: effect of
ozone-induced SP-A oxidation. Am J Physiol Lung Cell Mol Physiol
286: L546–L553, 2004. First published November 14, 2003; 10.1152/
ajplung.00267.2003.—Surfactant protein A (SP-A) plays a role in
innate host defense. Human SP-A is encoded by two functional genes
(SP-A1 and SP-A2), and several alleles have been characterized for
each gene. We assessed the effect of in vitro expressed human SP-A
genetic variants, on TNF-␣ and IL-8 production by THP-1 cells in the
presence of bleomycin, either before or after ozone-induced oxidation
of the variants. The oligomerization of SP-A variants was also
examined. We found 1) cytokine levels induced by SP-A2 (1A, 1A0)
were significantly higher than those by SP-A1 (6A2, 6A4) in the
presence of bleomycin. 2) In the presence of bleomycin, ozoneinduced oxidation significantly decreased the ability of 1A and 1A/
6A4, but not of 6A4, to stimulate TNF-␣ production. 3) The synergistic effect of bleomycin/SP-A, either before or after oxidation, can
be inhibited to the level of bleomycin alone by surfactant lipids. 4)
Differences in oligomerization were also observed between SP-A1
and SP-A2. The results indicate that differences among SP-A variants
may partly explain the individual variability of pulmonary complications observed during bleomycin chemotherapy and/or in an environment that may promote protein oxidation.
which is produced by alveolar type II
cells and consists of surfactant lipids and several associated
proteins, is essential for normal lung function because it
prevents alveolar collapse during end expiration (7). Pulmonary surfactant-associated protein A (SP-A), in addition to
surfactant-related function (14), plays a role in innate host
defense and regulation of inflammatory processes in the lung
(8). It has been previously reported that natural human SP-A
stimulates cytokine production (17, 28), nitric oxide production
(2), alveolar macrophage phagocytosis (52), immune cell proliferation (27), NF-␬B activation (25), and matrix metalloproteinase-9 production (53). In vivo studies suggest a role for
SP-A in neutrophil recruitment in the lung of preterm lambs
(26). However, in other systems, an anti-inflammatory role has
been attributed to SP-A (1, 3, 4, 32). Some in vivo and in vitro
studies show SP-A inhibition of LPS-induced cytokine production, nitric oxide, and lymphocyte proliferation. The reasons
for these apparent discrepancies are not clear, but differences
in the experimental systems are likely to contribute. Because
surfactant lipids can moderate the proinflammatory effect of
SP-A in vitro (25, 27, 28), it has been proposed that surfactant
lipids and SP-A are counterregulatory. Therefore, changes in
the relative amounts of surfactant lipids to SP-A may be
important in determining the immune status of the lung. It is
also possible that the role of SP-A changes at different stages
of the inflammatory response and is modulated by different
activating molecules (15, 47), as has been proposed recently
for NF-␬B (30).
Human SP-A is encoded by two functional genes (SP-A1
and SP-A2) and several alleles have been characterized for
each gene based on nucleotide differences within the coding
region. The most commonly observed alleles are 6A, 6A2, 6A3,
and 6A4 for SP-A1 and 1A, 1A0 1A1, 1A2, 1A3, and 1A5 for
SP-A2 (10, 24). Several studies have shown differences among
human SP-A genetic variants expressed in vitro in their abilities to stimulate cytokine production (55, 56) to bind carbohydrate (35) and lipids (16) and to enhance phagocytosis by
alveolar macrophages (our unpublished observations). Moreover, a number of studies have revealed quantitative associations between SP-A alleles or genotype and mRNA levels, and
different ratios of SP-A1 to SP-A2 mRNA have been observed
among individuals (23). Associations between certain SP-A
alleles and patient groups indicate that functional and/or structural differences of SP-A may play a role in the pathogenesis
of certain diseases (13, 42).
Bleomycin is an effective chemotherapeutic agent and is
used in the treatment of squamous cell carcinoma, lymphomas
testicular carcinoma, etc. However, bleomycin-induced pulmonary fibrosis is a serious problem that limits the usefulness of
the drug in some patients (49). Bleomycin induces inflammatory cells to secrete multifunctional cytokines, such as tumor
necrosis factor (TNF)-␣, interleukin (IL)-1␤, IL-8, and transforming growth factor-␤. These cytokines stimulate fibroblast
proliferation and are probably involved in the development of
pulmonary fibrosis (44, 57). It has been observed that bleomycin specifically stimulates macrophage-like THP-1 cells to
produce TNF-␣, IL-8, and IL-1␤ in a time- and dose-dependent
pattern (22). In a clinical study, the TNF-␣ level was shown to
be significantly increased after bleomycin infusion (46).
TNF-␣ is considered to be a central mediator in bleomycininduced pulmonary fibrosis, and TNF-␣ receptor knockout
mice are protected from lung injury after exposure to bleomycin (38). Although the mechanism of bleomycin-induced toxicity has not been fully elucidated, it has been demonstrated
that bleomycin-induced DNA damage is associated with reactive oxygen species. It is postulated that bleomycin exerts its
Address for reprint requests and other correspondence: J. Floros, Depts. of
Cellular and Molecular Physiology, H166, Penn State College of Medicine,
500 Univ. Dr., Hershey, PA 17033 (E-mail: jfloros@psu.edu).
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. Section 1734 solely to indicate this fact.
inflammation; enzyme-linked immunosorbent assay; oligomerization;
ozone
PULMONARY SURFACTANT,
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HUMAN SP-A GENETIC VARIANTS
toxic effects in the lung through the generation of oxygen free
radicals that initiate pulmonary fibrosis (20). Clinical data have
implicated oxygen supplementation during bleomycin chemotherapy with increased bleomycin toxicity (31).
Lung surfactant lines the surface of lung alveoli and is likely
to be the first target of reactive oxygen species generated by
oxygen and ozone (40). The effects of oxidants on the pulmonary surfactant system include the oxidation of surfactant
proteins (including SP-A) and the peroxidation of surfactant
phospholipids (50). Moreover, a number of studies have shown
that oxidation of SP-A in vitro by ozone (36, 37, 56), nitrogen
dioxide (34), and reactive oxygen and nitrogen species (9, 19)
impairs many aspects of SP-A function, including lipid aggregation, mannose binding capacity, stimulation of cytokine
production, and phagocytosis. In vivo oxidation of SP-A in
different systems (34, 37, 48) has also resulted in functional
differences in SP-A.
Our previous studies showed that SP-A enhances bleomycin-induced inflammatory cytokine production and that this
synergistic effect can be attenuated by surfactant lipid (22). In
the present study, we used the surfactant protein genetic
variants as a model to gain insight into the basis of individual
variability to bleomycin-induced pulmonary toxicity and specifically in microenvironments that may promote protein oxidation. In this regard, we assessed 1) differences among human
SP-A genetic variants, expressed in vitro by insect cells either
before or after ozone-induced oxidation, in their ability to
stimulate cytokine production in the presence of bleomycin;
and 2) whether functional differences correlate with differences in the oligomeric patterns of normal and ozone-oxidized
SP-A variants under various electrophoretic conditions. The
rationale for these studies is twofold. First, the functional and
biochemical properties of SP-A change following ozone exposure (36, 37). In this regard, we hypothesized that the functional ability of SP-A variants is differentially affected by
ozone-induced damage/oxidation. This differential alteration is
due to amino acid differences among alleles. The second
reason is that differences in SP-A oligomer size has already
been associated with disease (21). Therefore, it is possible that
ozone-induced alterations, as assessed by changes in the electrophoretic mobility, correlate with functional differences.
MATERIALS AND METHODS
Cell lines and cell culture conditions. The insect cell line Sf9
(GIBCO-BRL, Life Technologies) from Spodoptera frugiperda was
used for protein expression. The cells were cultured in Sf-900 II SFM
medium (GIBCO-BRL) in an incubator at 27°C. Protein expression
proceeded in suspension culture of cells in a 250-ml Erlenmeyer flask
(50 ml culture medium/flask) with shaking at 110 rpm.
The THP-1 cell line was obtained from the American Type Culture
Collection (Manassas, VA). Cells were grown in RPMI 1640 (Sigma,
St. Louis, MO) with 0.05 mM 2-mercaptoethanol and 10% heatinactivated fetal bovine serum (BioWhittaker, Walkersville, MD) at
37°C in an atmosphere of 5% CO2. The cells were split periodically
and used at passages 8–15 in the various experiments. After differentiation with 10⫺8 M vitamin D3 for 72 h, cells were washed with
cold PBS. The cell pellet was then resuspended in medium at a density
of 2 ⫻ 106 cells/ml in 24-well culture plates and exposed to bleomycin and SP-A. We terminated incubations by pelleting the cells.
Supernatants were harvested and stored at ⫺80°C until assayed.
Preparation of native human SP-A. Native human SP-A was
purified from bronchoalveolar lavage (BAL) fluid of an alveolar
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proteinosis patient by the butanol extraction method as described in
our previous report (22). The purified protein was examined by
two-dimensional gel electrophoresis followed by Western blotting and
silver staining and was found to be ⬎98% pure. Western blot analysis
was done with a rabbit anti-human SP-A IgG. For silver staining of
gels, a modified version was used, as reported by Rabilloud (41).
Protein concentration was determined with the micro-bicinchoninic
acid method (Pierce, Rockford, IL) with RNase A as a standard. LPS
content was determined with the QCL-1000 Limulus amebocyte
lysate assay (BioWhittaker). This test indicated that human SP-A used
in this study contained ⬍0.01 pg LPS/␮g SP-A. SP-A was stored at
⫺80°C until use.
Expression of human SP-A genetic variants by insect cells. The
SP-A genetic variants were expressed by the baculovirus-mediated
insect cell system as described in our previous study (55). Briefly,
PCR-amplified cDNA fragment was cloned into donor plasmid pFastBac DUAL (GIBCO-BRL). The recombinant plasmid was then transformed into Escherichia coli DH10Bac in which the foreign gene was
transferred into the baculovirus genome (bacmid). After cloning,
selection, and purification, the recombinant bacmid DNA was transfected into insect cell line Sf9. Three days after transfection, SP-A
expression was examined by Western blot analysis, and recombinant
baculovirus particles in the supernatant of the cell culture were
collected. We achieved the expression of single SP-A gene products
by infecting insect cells with the virus particles containing a single
SP-A allele. A 50-ml culture at 2 ⫻ 106 cells/ml was infected with 1
ml of a viral stock at 1 ⫻ 109 pfu/ml. For coexpression of SP-A1/
SP-A2 products, the virus particles containing SP-A1 and SP-A 2
genes were mixed at a 1:1 ratio of virus titers and used for infecting
the insect cells. To confirm expression of the specific gene and/or
allele, we determined the SP-A genotype of the insect cell mRNA by
the PCR-converted restriction fragment length polymorphism
(cRFLP) method (10). The culture supernatants were harvested at 72 h
after infection and purified with mannose-affinity chromatography.
The purified SP-A variants were then dialyzed against 5 mM Tris (pH
7.5) and examined by Western blotting and silver staining. Protein
concentration and LPS content were determined by the methods
described above. The LPS content of SP-A variants used in this study
was ⬍0.2 pg/␮g SP-A.
Bleomycin. Bleomycin (Blenoxane; Bristol-Myers Squibb, Princeton, NJ) solutions were prepared immediately before use with endotoxin-free saline (American Pharmaceutical Partners, Los Angeles,
CA). LPS was not detected in the stock solution of bleomycin at a
bleomycin concentration of 5 U/ml (1 U ⫽ 1 mg) using the method
described above.
Electrophoretic analysis of in vitro expressed SP-A variants and
native human SP-A under various conditions. Human SP-A genetic
variants and human SP-A from BAL fluid were subjected to electrophoresis under reducing, nonreducing, and native conditions. Reducing PAGE analysis was done following the procedure described by
Laemmli (29). Electrophoresis (90 V, 1.5 h) of each protein sample
was performed in a 10% polyacrylamide gel in the presence of SDS
after reduction of protein by dithiothreitol and heating at 95°C for 10
min. The nonreducing PAGE (75 V, 5 h) was done in a 4–15%
gradient polyacrylamide gel under the same conditions as the reducing
PAGE, except there was no reducing agent in the loading buffer. Gel
electrophoresis under native conditions was performed in a 4–15%
gradient polyacrylamide gel with an electrophoretic running buffer
(90 mM Tris, 80 mM boric acid, and Na2EDTA, pH 8.4) and a loading
buffer without reducing agent and SDS. In addition, electrophoresis
was carried out at 4°C at 50 V for 1 h followed by 95 V for 13 h.
Stimulation of THP-1 cells with SP-A and bleomycin. After differentiation with 10⫺8 M vitamin D3 for 72 h, THP-1 cells were pelleted
and washed with cold PBS. Cells were resuspended in RPMI 1640 at
a density of 2 ⫻ 106 cells/ml and incubated in 24-well culture plates
(0.5 ml/well). Cells were stimulated with nonozone or ozone-exposed
SP-A (20 ␮g/ml), bleomycin (5 mU/ml), or SP-A plus bleomycin. In
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some experiments, Infasurf (Forest Pharmaceuticals, St. Louis, MO),
an extract of natural surfactant from calf lung, was used to assess the
inhibitory effect of surfactant lipid on SP-A, either with or without
ozone-induced oxidation, and bleomycin on induced cytokine production. Cells were incubated for 4 h after treatment. TNF-␣ and IL-8
levels in culture medium were then quantified by ELISA.
ELISA assay. The ELISA assays for TNF-␣ and IL-8 (OptEIA
Human ELISA Sets; Pharmingen, San Diego, CA) were performed
according to the instructions recommended by the manufacturer. The
ELISA kits were capable of measuring levels of 7.8–500 pg/ml for
TNF-␣ and 6.2–400 pg/ml for IL-8. We obtained a reference curve for
each of these cytokines by plotting the concentration of several
dilutions of standard protein versus the corresponding absorbance.
Exposure of SP-A to ozone and detection of protein oxidation.
SP-A protein at a concentration of ⬃0.5 mg/ml was exposed to ozone
in 24-well tissue culture plates as described previously (51). Briefly,
each well contained 100 ␮l of the protein solution and was exposed to
ozone (1 ppm for 4 h) for protein oxidation. Protein oxidation was
detected with the OxyBlot oxidized protein detection kit (Intergen,
Purchase, NY). The ozone-exposed proteins were derivatized with
2,4-dinitrophenylhydrazine (DNPH), 200 ng of DNPH-derivatized
protein were blotted onto nitrocellulose, and immunodetection of
oxidized proteins was done with anti-DNPH and goat anti-rabbit IgG
(horseradish peroxidase-conjugated) antibodies. Blots were exposed
to XAR film following enhanced chemiluminescent detection.
Statistics. Values are presented as means ⫾ SE. Data were analyzed using SigmaStat statistical software (SPSS, Chicago, IL). For
comparison among SP-A variants, statistical treatment included a
one-way analysis of variance followed by a Student-Newman-Keuls
test for pairwise comparison. A paired t-test was used for comparing
the effect of SP-A before and after ozone-induced oxidation exposure.
A value at P ⬍ 0.05 was judged to be significantly different.
RESULTS
Differentiated THP-1 cells were stimulated with SP-A variant (20 ␮g/ml), bleomycin (5 mU/ml), or SP-A variant plus
bleomycin. In this study, in vitro expressed genetic variants of
two SP-A1 alleles (6A2, 6A4), two SP-A2 alleles (1A, 1A0)
that are commonly observed in the general population (12), as
well as two combinations of SP-A1 and SP-A2 alleles (1A0/
6A2, 1A/6A4) were used. For the SP-A1/SP-A2 combinations
we used a ratio of 1:1 viral SP-A1 and SP-A2 titers as noted in
MATERIALS AND METHODS. Although it has been proposed that
SP-A consists of a 2:1 ratio of SP-A1 to SP-A2 (54), the ratio
of SP-A1 to SP-A2 at the mRNA level among unrelated
individuals varies considerably (23). Therefore, it remains to
be determined whether there is a unique ratio of SP-A1 to
SP-A2 that applies to all individuals. A dose of 20 ␮g/ml of
SP-A was chosen rather than the dose of 50 ␮g/ml we have
used in previous experiments (55, 56), because the lower dose
more effectively shows the combined effects of bleomycin and
SP-A before or after ozone-induced oxidation. The doses of
bleomycin and ozone used in this study were chosen based on
findings of our previous studies (22, 56) and on other published
reports (36, 44).
Combined effect of in vitro expressed SP-A variants and
bleomycin on cytokine production by THP-1 cells. As depicted
in Fig. 1A, TNF-␣ levels induced by SP-A1 variants (6A2, 6A4)
are significantly lower than those by SP-A2 variants (1A, 1A0)
and by coexpressed variants (1A0/6A2, 1A/6A4). When these
variants were combined with bleomycin, a synergistic effect on
TNF-␣ level was observed for all SP-A variants. In the presence of bleomycin, 6A2 and 6A4 stimulated production of
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Fig. 1. Combined effect of surfactant protein (SP)-A variants and bleomycin
on TNF-␣ and IL-8 levels by THP-1 cells. Differentiated THP-1 cells were
stimulated with the indicated SP-A variant (20 ␮g/ml) plus bleomycin (BLM,
5 mU/ml) for 4 h. TNF-␣ (A) and IL-8 (B) levels in culture medium were
quantified by ELISA. Data are derived from 6 separate experiments, and the
results are given as means ⫾ SE. *SP-A1 single allele product (6A2, 6A4)
values are significantly different (P ⬍ 0.05) from the corresponding SP-A2
single allele values or the coexpressed variants. SP-A1 variants in the presence
of BLM differ from SP-A2 variants plus BLM (P ⬍ 0.05). Native human SP-A
purified from bronchoalveolar lavage (BAL) of an alveolar proteinosis patient
was used as a positive control. #Human (h) SP-A synergistic effect is
significantly higher than that of the coexpressed (6A2/1A0, 6A4/1A) (P ⬍
0.05), or SP-A2 (1A0, 1A) (P ⬍ 0.05), or SP-A1 (6A2, 6A4) (P ⬍ 0.01).
TNF-␣ by THP-1 cells to levels of 441 ⫾ 34.9 pg/ml and
432 ⫾ 64.9 pg/ml, respectively, which are significantly lower
than those induced by 1A (635 ⫾ 91.5 pg/ml) and 1A0
(603.8 ⫾ 49.5 pg/ml). When IL-8 production was studied after
the cells were treated with SP-A variants and bleomycin, the
response pattern of IL-8 was very similar to that observed in
TNF-␣ assay (Fig. 1B). No significant difference was found
between SP-A2 and coexpressed variants in their ability to
stimulate cytokine production in the presence of bleomycin.
Effect of ozone-induced oxidation on SP-A/ bleomycin-induced TNF-a production. Functional changes in SP-A following ozone-induced oxidation are shown in Fig. 2. TNF-␣
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Fig. 2. Effect of ozone-induced oxidation on SP-A variants plus BLM to
enhance TNF-␣ levels by THP-1 cells. SP-A variants were exposed to 1 ppm
ozone for 4 h. Differentiated THP-1 cells were stimulated with normal or
oxidized SP-A variant (20 ␮g/ml) plus BLM (5 mU/ml) for 4 h. TNF-␣ levels
in culture medium were quantified by ELISA. Data are derived from 5 separate
experiments, and the results are given as means ⫾ SE. Values are significantly
different (P ⬍ 0.05) (*) compared with the same experimental points but in the
absence of ozone and (ˆ) compared with the corresponding points for 1A and
1A/6A4 variants. Native hSP-A purified from BAL of an alveolar proteinosis
patient was used as a positive control (C).
production was significantly reduced after ozone-induced oxidation of SP-A (99.0 ⫾ 16.2 vs. 48.5 ⫾ 5.8 pg/ml for 1A,
45.8 ⫾ 2.8 vs. 33.0 ⫾ 2.1 pg/ml for 6A4, 93.3 ⫾ 7.1 vs. 47.1 ⫾
2.7 pg/ml for 1A/6A4). The synergistic effect of SP-A plus
bleomycin on TNF-␣ production was also significantly decreased with oxidized 1A and 1A/6A4 variants but not with the
6A4 variant. When differences among SP-A variants in their
ability to stimulate cytokine production before and after SP-A
oxidation were compared, it was found that the TNF-␣ levels
induced by 6A4 were significantly lower than those induced by
1A or 1A/6A4 in all circumstances except in the presence of
bleomycin. Enhancement of TNF-␣ production by native human SP-A was also reduced after in vitro oxidation both in the
presence and absence of bleomycin.
Effect of surfactant lipids (Infasurf) on oxidized SP-A-induced TNF-␣ production by THP-1 cells in the presence of
bleomycin. The inhibitory effect of surfactant lipids (Infasurf)
on native human SP-A plus bleomycin-induced TNF-␣ production was examined, both before and after ozone-induced
oxidation of SP-A. Significant findings were observed in both
the presence and absence of bleomycin (Fig. 3) either before or
after SP-A oxidation.
Patterns of oligomerization of in vitro expressed SP-A variants and human SP-A under various electrophoretic conditions. To study whether the functional differences observed
above were related to structural differences among SP-A variants, we analyzed in vitro expressed SP-A variants by PAGE
under reducing, nonreducing, and native conditions. Under
reducing conditions, the SP-A variants showed a monomeric
form with a lower molecular mass than native human SP-A by
both Western blot analysis (Fig. 4A) and silver staining (Fig.
4B) due to the absence of certain posttranslational modifications in the baculovirus-mediated expression system. A lowintensity dimeric band (⬃60 kDa) of SP-A variants was observed sometimes, especially by Western blot. The SP-A1
(6A2, 6A4) presented with a larger molecular mass than the
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Fig. 3. Effect of surfactant lipids (Infasurf) on the ability of oxidized SP-A to
enhance TNF-␣ production by THP-1 cells in the presence of BLM. Native
hSP-A was exposed to 1 ppm ozone for 4 h. Differentiated THP-1 cells were
stimulated with normal or ozone-exposed SP-A variant (20 ␮g/ml) and/or
BLM (5 mU/ml) in the presence or absence of Infasurf (200 ␮g/ml) for 4 h.
TNF-␣ levels in culture medium were quantified by ELISA. Data are derived
from 4 separate experiments, and the results are given as means ⫾ SE.
*Significantly different (P ⬍ 0.05) compared with the same experimental
points in the absence of Infasurf.
SP-A2 (1A, 1A0) variants. The native human SP-A presented
with both monomeric and dimeric forms under the reducing
condition.
Under nonreducing conditions, the inter- and intramolecular
disulfide bonds remain intact because no reducing agents, such
as dithiothreitol and 2-mercaptoethanol, are used. The majority
of oligomers of the in vitro expressed SP-A variants under this
condition (Fig. 5A) are resolved into dimers (2X) and trimers
Fig. 4. Electrophoretic analysis of in vitro expressed SP-A variants and native
hSP-A under reducing condition. hSP-A genetic variants (1A, 1A0, 6A2, 6A4,
1A0/6A2, 1A/6A4) were expressed from baculovirus-mediated insect cell
system, and native hSP-A was purified from BAL of an alveolar proteinosis
patient. Electrophoresis of 0.5 ␮g of each protein sample was performed in a
10% polyacrylamide gel in the presence of SDS after reduction of protein in 50
mM dithiothreitol at 95°C for 10 min. Protein was detected by silver staining
(B) and Western blot analysis with rabbit anti-hSP-A IgG is shown in A.
Notation on the right (1X, 2X) indicates oligomers.
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ozone exposure at a concentration of 1 ppm for 4 h (Fig. 6A).
Although oxidation of native human SP-A was detected before
ozone exposure (Fig. 6A), a higher level of oxidation was
observed after ozone exposure as assessed by a shorter film
exposure (Fig. 6B). Densitometric analysis of two independent
experiments performed in duplicate indicates that SP-A oxidation before ozone exposure is approximately (42 ⫾ 4%) that
following ozone exposure. Examination of native SP-A indicated that SP-A from the BAL fluid of patients with alveolar
proteinosis is partially oxidized in vivo in the human body, and
this has been shown previously (51).
Under reducing conditions, SP-A variants, but not native
human SP-A, demonstrated bands of higher apparent molecular weight after ozone exposure (Fig. 7A). Although no significant loss of SP-A was detected by protein quantification after
oxidation, bands of lower intensity in oxidized SP-A variants
were observed in gel electrophoresis under nonreducing conditions (Fig. 7B). A major electrophoretic change in 6A4 was
seen, compared with other SP-A variants after oxidation (Fig.
7, A and B). Increased smearing in the spaces between the
oligomeric bands was observed in ozone-oxidized SP-A variants under nonreducing conditions (Fig. 7B). These observations point to the possibility that higher-size oligomers are
formed and some of these may or may not enter the gel,
explaining perhaps both the lighter band intensity and the
smearing.
Fig. 5. Patterns of oligomerization of in vitro expressed SP-A variants and
native hSP-A under nonreducing (A) and native (B) conditions. hSP-A genetic
variants (1A, 1A0, 6A2, 6A4, 1A0/6A2, 1A/6A4) were expressed from baculovirus-mediated insect cell system and native hSP-A was purified from BAL of
an alveolar proteinosis patient. Electrophoresis of 5 ␮g of each protein sample
was performed in a 4–15% gradient polyacrylamide gel in the presence of SDS
after 95°C for 10 min without dithiothreitol where inter- and intramolecular
disulfide bonds remain intact (nonreducing condition, A) and in the absence of
heating, SDS, and dithiothreitol where SP-A molecules should maintain their
native conformation (native condition, B). Protein was detected by silver
staining. Notation on the right (2X, 3X, etc.) indicates oligomers.
(3X). In contrast, the oligomers of native human SP-A are
highly oligomerized and most of them are ⬎6X. Differences
between SP-A1 and SP-A2 variants in the pattern of oligomerization under the nonreducing condition were observed. These
include the presence of bands of higher intensity corresponding
to the trimer (3X) and an additional band between the dimer
(2X) and the trimer (3X) for SP-A1. A band corresponding to
a higher-order oligomer (⬎6X) is present in SP-A2 and absent
in SP-A1.
Differences among SP-A variants were also observed under
the native condition (Fig. 5B), where proteins are not denatured
by chemicals and heating and both covalent and noncovalent
interactions are maintained. Differences between SP-A1 and
SP-A2 variants include the presence in SP-A1 (and absence in
SP-A2) of a high-intensity band, approximately a 9X oligomer.
Conversely, a high-intensity band at ⬃12X is present in SP-A2
and absent in SP-A1. Subtle differences between alleles of a
given gene and/or of coexpressed SP-A1/SP-A2 gene products
are also observed.
Electrophoretic characteristics of oxidized of SP-A variants
and native human SP-A. No significant oxidation was detected
for each of the SP-A variants (1A, 6A4, 1A/6A4) before ozone
exposure, but all of the SP-A variants were oxidized after
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DISCUSSION
SP-A, a major surfactant-associated protein, plays important
roles in surfactant-related activities and in the innate host
defense of the lung. Due to its high level of heterogeneity and
polymorphism, SP-A may contribute to the individual variability of susceptibility to pulmonary disease under certain conditions. The effect of bleomycin therapy on pulmonary toxicity
may present an opportunity where the impact of SP-A variants
could be studied. In the present study, we assessed the functional and structural differences among human SP-A genetic
variants either before or after ozone-induced oxidation on
Fig. 6. Detection of ozone-induced oxidation to SP-A variants and native
hSP-A. In vitro expressed hSP-A genetic variants (1A, 6A4, 1A/6A4) and
native hSP-A purified from BAL of an alveolar proteinosis patient were
exposed to ozone at a concentration of 1 ppm for 4 h. Then 200 ng of each
sample were blotted onto a membrane in duplicate (A) and the oxidized
proteins were detected using the OxyBlot oxidized protein detection method.
A short exposure of the film was developed to demonstrate the oxidation of
native hSP-A by ozone exposure (B).
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HUMAN SP-A GENETIC VARIANTS
Fig. 7. Electrophoretic analysis of in vitro expressed SP-A variants and native
hSP-A after ozone exposure under reducing and nonreducing conditions. In
vitro expressed hSP-A genetic variants (1A, 6A4, 1A/6A4) and native hSP-A
purified from BAL of an alveolar proteinosis patient were exposed to ozone at
a concentration of 1 ppm for 4 h. A: 1 ␮g protein was subjected to 10%
polyacrylamide gel electrophoresis in the presence of SDS after reduction of
protein in 50 mM dithiothreitol at 95°C for 10 min. B: 5 ␮g of each protein
sample were subjected to a 4–15% gradient polyacrylamide gel electrophoresis
in the presence of SDS after 95°C for 10 min without dithiothreitol where
inter- and intramolecular disulfide bonds remain intact (nonreducing condition). Protein bands were detected by silver staining. Notation on the right (2X,
3X, etc) indicates oligomers.
cytokine production by THP-1 cells in the presence of bleomycin, as well as the electrophoretic pattern of SP-A variants.
The rationale for the latter is based on findings whereby SP-A
oligomers of different sizes have been shown to associate with
pulmonary disease (21). We found that the levels of cytokines
induced by SP-A2 (1A, 1A0) were significantly higher than
those by SP-A1 (6A2, 6A4) in the presence of bleomycin.
Ozone-induced oxidation significantly decreased the ability of
1A and 1A/6A4, but not of 6A4, to stimulate TNF-␣ production
in the presence of bleomycin. Moreover, the synergistic effect
of bleomycin/SP-A either before or after oxidation was inhibited by surfactant lipids to the level of bleomycin alone.
Structural differences in oligomerization were also observed
between SP-A1 and SP-A2 variants, as assessed by gel analysis
where changes were observed in both the molecular weight and
the band intensity of SP-A variants following ozone-induced
oxidation. These findings show that functional and structural
differences exist among SP-A variants in the presence of
bleomycin either before or after ozone-induced oxidation and
indicate that structural differences may explain functional differences. The present findings also show that the ability of
surfactant lipids to attenuate cytokine production is not influenced by the oxidation state of SP-A.
The high level of polymorphism of the SP-A genes may lead
to quantitative and/or qualitative differences of the SP-A proAJP-Lung Cell Mol Physiol • VOL
L551
teins among individuals, and this may contribute to the individual variability of susceptibility to pulmonary disease and
bleomycin-induced pulmonary fibrosis. In the present study,
we found that the ability of SP-A variants to enhance bleomycin-induced cytokine production is similar to that of native
SP-A. However, differences with regards to the level of cytokine enhancement and the electrophoretic pattern between
SP-A1 and SP-A2 variants were observed. Published data have
shown the level of SP-A mRNA, as well as the ratio of SP-A1
to SP-A2 mRNA, to vary among individuals, indicating that, in
some individuals, single SP-A gene products are in excess (23).
An overabundance of one SP-A gene product over the other
(23) may be reflected in functional differences among individuals, and such differences under certain compromised conditions may contribute to disease pathogenesis (11, 13, 42).
Support for such a possibility is provided by two groups of
experiments. First, functional differences between SP-A1 and
SP-A2 have been observed not only by the findings in the
present study but by several in vitro studies. These include
differences between the two SP-A gene products in cytokine
production (55, 56), carbohydrate binding (16, 35), lipid aggregation (16, 35), structural stability (16), and phagocytosis
(our unpublished observation). Second, association between
certain SP-A alleles and patient groups has been observed (13,
42), suggesting that SP-A functional differences play a role in
the pathogenesis of certain diseases. The present data, along
with previously published reports, suggest that the SP-A1 and
SP-A2 single gene products are not entirely equivalent in their
functional capabilities at the protein concentrations studied.
A remarkable difference in the oligomeric pattern between
SP-A1 (6A2, 6A4) and SP-A2 (1A, 1A0) was observed under
various electrophoretic conditions. This may be of potential
clinical interest in view of previous findings where patients
with birch pollen allergy were identified with a larger fraction
of smaller-size oligomers of SP-A (21). The difference in
oligomeric patterns between SP-A1 and SP-A2 may relate to
differences, among SP-As, in posttranslational modification
(39), assembly (18), and thermal stability (16). For example,
biochemical studies have shown: 1) the SP-A2 structure to be
more stable than the SP-A1 structure and 2) differences in the
ability of SP-A to undergo self-aggregation and to induce lipid
and LPS aggregation, in order of native SP-A ⬎ SP-A2 ⬎
SP-A1/SP-A2 ⬎ SP-A1 (16). The higher structural stability of
SP-A2 and/or of other structural differences between SP-A1
and SP-A2 may explain its higher capacity in cytokine production. In the present study, we did not observe a difference
between the coexpressed variants and the single SP-A2 gene
product, either in the function or in the oligomerization pattern,
as we observed previously (16, 55). This is probably due to the
variable ratio of SP-A1 to SP-A2 in the baculovirus-mediated
expression system. In this system, the SP-A1/SP-A2 gene
product was coexpressed by inoculating baculovirus containing
both genes at a ratio of 1:1 (55). Although mRNA expression
of both genes was confirmed by genotyping using PCR-cRFLP
analysis (10), the ratio of SP-A1 to SP-A2 expression in each
preparation may vary, and this variation may result in subtle
differences in function and/or structure.
Bleomycin, which is used in several protocols of chemotherapy, is an exogenous lung oxidant and produces reactive
oxygen species (20). In addition, patients who receive bleomycin therapy may need oxygen supplementation, and this hyper286 • MARCH 2004 •
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L552
HUMAN SP-A GENETIC VARIANTS
oxia in turn may increase the oxidant burden in the lung via
oxygen free radical production (31). A microenvironment with
increased oxidative stress may promote oxidation of SP-A that
in turn may alter its structure and/or its function. Oxidants such
as ozone and nitrogen dioxide from air pollution contribute to
surfactant phospholipid oxidation (40) and function, and thus
these agents may also contribute to SP-A oxidation. In the
present study, ozone-induced oxidation of SP-A resulted in a
significant decrease in its synergistic effect with bleomycin to
stimulate TNF-␣ production. The functional reduction of SP-A
after ozone-induced oxidation is consistent with other in vitro
and in vivo reports, where various oxidants including ozone
were used to study aspects of SP-A function (9, 34, 36, 37).
Müller and coworkers (34) compared SP-A functions following in vivo and in vitro exposure of SP-A to nitrogen dioxide
and found that the in vivo exposure was less effective in
altering SP-A function with regard to its mannose binding
capacity, protein-lipid aggregation, and lipid secretion. It is
possible that under relatively normal conditions protective
mechanisms, such as the presence of adequate levels of antioxidants, ions, and lipids, exist to prevent SP-A from being
fully oxidized. Moreover, animal studies showed SP-A, but not
SP-B and SP-C, to be increased following bleomycin treatment
(43) and nitrogen dioxide treatment (34). We speculate that the
increased amount of free SP-A in the presence of oxidantproducing molecules overloads the antioxidant system and
further contributes to the bleomycin-induced proinflammatory
effect. Alternatively, the increased amount of SP-A in bleomycin-treated animals may help alleviate bleomycin-induced surfactant dysfunction due to derangement of other surfactant
components. For example, SP-A is capable of reversing the
biophysical properties of oxidized surfactant lipids (6) and can
inhibit lipid peroxidation (5). It is also possible that the ability
of oxidized SP-A to protect surfactant lipids from peroxidation
is diminished and the resulting oxidized surfactant lipids no
longer attenuate the SP-A/bleomycin synergism. In this scenario a prolonged inflammatory response ensues that may lead
to lung injury and fibrosis.
There was no significant change of the 6A4/bleomycin
synergistic effect on TNF-␣ level after ozone-induced oxidation of 6A4 in the presence of bleomycin. This indicates that
the response of 6A4 may differ from that of 1A and 1A/6A4
under the present experimental conditions. A tryptophan instead of an arginine at position 219 within the carbohydrate
recognition domain that distinguishes the 6A4 from other
frequently found SP-A1 alleles may contribute to this observation, although the finding is rather paradoxical because a
tryptophan is more susceptible to ozone oxidation than an
arginine (33). Of interest, the 6A4 allele has been shown to
exhibit inferior capacity to induce LPS aggregation (16), enhance cytokine production (56), and be a risk factor in the
pathogenesis of idiopathic pulmonary fibrosis (45). However,
the mechanism as to how the 6A4 may contribute to these
different responses and specifically whether the distinguishing
amino acid (Trp) plays a role is unknown. It is possible that
differences in structure and stability play a role in these
processes. The electrophoretic data (Fig. 7) following ozoneinduced oxidation show a more pronounced change in 6A4
compared with 1A and 1A/6A4, with a loss of higher-size
oligomers.
AJP-Lung Cell Mol Physiol • VOL
In summary, with regard to cytokine production by macrophage-like cells in response to SP-A, we found 1) differences
between the SP-A1 and SP-A2 single gene products in the
presence of bleomycin, 2) a difference between the SP-A 6A4
variant and the other SP-A variants after ozone-induced oxidation in the presence of bleomycin, and 3) electrophoretic
differences in the oligomeric band pattern between SP-A variants either before or after ozone-induced oxidation. We speculate that differences observed by gel analysis have an impact
on the function of SP-A variants under unperturbed conditions
and conditions that promote protein oxidation. We further
speculate that a better understanding of the mechanisms that
underlie the differences among SP-A variants may contribute
to our better understanding of individual variability of pulmonary complications observed during bleomycin chemotherapy
and/or in an environment with increased oxidative burden.
GRANTS
This work was supported by National Institutes of Health Grants 1R01
ES-09882–01 and R37 HL-34788, American Heart Association Grant
0160354U (G. Wang), and the Julia Cotler Hematology Research Fund.
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