Am J Physiol Lung Cell Mol Physiol
279: L110–L117, 2000.
Comparison of SP-A and LPS effects
on the THP-1 monocytic cell line
MINGCHEN SONG AND DAVID S. PHELPS
Department of Pediatrics, Pennsylvania State University College of Medicine,
Hershey, Pennsylvania 17033
Received 25 October 1999; accepted in final form 21 February 2000
is a complex mixture of lipids
and proteins that lines the alveolar surface of the lung.
Although the most well-established function of surfactant is the reduction in surface tension at the air-liquid
interface, surfactant proteins and lipids also have been
shown to be involved in the innate or non-antibodymediated host defense system in the lungs (34). Surfactant is ideally suited to have a role in host defense
processes in the lung because it covers the entire alveolar surface. In this position it is the first substance
encountered by pathogens reaching the alveoli in inspired air. Whole surfactant from normal individuals
and some of its individual lipid components have suppressive effects on many aspects of immune cell function (22, 34). However, surfactant protein A (SP-A) and
some other surfactant components may behave quite
differently.
SP-A, the most abundant of the surfactant-associated proteins, is a member of a family of collagenous
C-type lectins (collectins) that includes serum mannose-binding protein, SP-D and conglutinin. Collectins
are involved in many aspects of host defense function,
and SP-A exerts a variety of stimulatory effects on
alveolar macrophages (7, 22, 28, 34, 35). Among its
many actions, SP-A binds to some pathogens by means
of its carbohydrate recognition domain, thus promoting
the binding and phagocytosis of these pathogens by the
macrophage (26). It also has been shown to interact
with bacterial lipopolysaccharide (LPS) (3, 10). SP-A
also stimulates the generation of oxidative activity in
macrophages (30), immune cell proliferation (16), the
production of proinflammatory cytokines (17), and the
increased expression of cell surface proteins (15) in a
monocyte/macrophage cell line and in other cells of
monocytic origin. SP-A also has been shown recently to
stimulate nitric oxide production (3, 6). The most convincing evidence that SP-A has an important role in
innate immunity comes from the finding that the genetically engineered SP-A-deficient mice, which have
essentially normal lung structure and function, show
an increased susceptibility to infection by group B
streptococcus and Pseudomonas aeruginosa (18, 19).
It is well known that LPS, a constituent of the outer
membrane of gram-negative bacteria, activates macrophages strongly and induces production of a number of
molecules including cytokines, eicosanoids, and free
radicals. It thus participates in various events associated with the inflammatory response at the alveolar
level (32). It has been reported that some of the effects
evoked by LPS are also produced by SP-A, particularly
the induction of the inflammatory mediators such as
tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6,
IL-8, and nitrogen intermediates (3, 6, 17, 30). Because
both SP-A and LPS may be present in the lung during
the inflammatory response and both are capable of
modulating the production of cytokines via cellular
responses that include alveolar macrophage, we explored some of the similarities, differences, and inter-
Address for reprint requests and other correspondence: D. S.
Phelps, Dept. of Pediatrics, Rm. C7814, Pennsylvania State Univ.
College of Medicine, 500 University Drive, Hershey, PA 17033
(E-mail: dsp4@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.
surfactant protein A; lipopolysaccharide; cytokines; tolerance; innate immunity
PULMONARY SURFACTANT
L110
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society
http://www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on September 30, 2016
Song, Mingchen, and David S. Phelps. Comparison of
SP-A and LPS effects on the THP-1 monocytic cell line. Am J
Physiol Lung Cell Mol Physiol 279: L110–L117, 2000.—
Surfactant protein A (SP-A) increases production of proinflammatory cytokines by monocytic cells, including THP-1
cells, as does lipopolysaccharide (LPS). Herein we report
differences in responses to these agents. First, polymyxin B
inhibits the LPS response but not the SP-A response. Second,
SP-A-induced increases in tumor necrosis factor-␣ (TNF-␣),
interleukin-1␤ (IL-1␤), and IL-8 are reduced by ⬎60% if SP-A
is preincubated with Survanta (200 ␮g/ml) for 15 min before
addition to THP-1 cells. However, the LPS effects on TNF-␣
and IL-8 are inhibited by ⬍20% and the effect on IL-1␤ by
⬍50%. Third, at Survanta levels of 1 mg/ml, SP-A-induced
responses are reduced by ⬎90%, and although the inhibitory
effects on LPS action increase, they still do not reach those
seen with SP-A. Finally, we tested whether SP-A could induce tolerance as LPS does. Pretreatment of THP-1 cells with
LPS inhibits their response to subsequent LPS treatment
24 h later, including TNF-␣, IL-1␤, and IL-8. Similar treatment with SP-A reduces TNF-␣, but IL-1␤ and IL-8 are
further increased by the second treatment with SP-A rather
than inhibited as with LPS. Thus, whereas both SP-A and
LPS stimulate cytokine production, their mechanisms differ
with respect to inhibition by surfactant lipids and in ability
to induce tolerance.
COMPARISON OF SP-A AND LPS IN THP-1 CELLS
actions between these two proinflammatory stimuli
regarding their effects on immune cells.
In the present study, using the surfactant replacement preparation Survanta, we investigated the effects of surfactant lipids on proinflammatory functions
in THP-1 cells stimulated by either SP-A or LPS, and
we did similar studies with polymyxin B. We also
investigated whether tolerance, a well-characterized
consequence of repeated exposure to LPS, could be
induced by SP-A in THP-1 cells.
MATERIALS AND METHODS
Cell Culture
Preparation of SP-A
SP-A was purified by preparative isoelectric focusing
(Rotofor, Bio-Rad; Hercules, CA) from the alveolar lavage
fluid of alveolar proteinosis patients as described elsewhere
(13). The purified protein was examined by two-dimensional
gel electrophoresis and silver staining and was found to be
greater than 95% pure. Endotoxin content was determined
with the QCL-1000 Limulus amebocyte lysate (LAL) assay
(BioWhittaker; Walkersville, MD). This test indicated an
average endotoxin level in our samples of ⬍3 pg LPS/mg
SP-A.
Polymyxin B (Sigma) was added to the cells at various concentrations (1 or 10 ␮g/ml) in the presence of SP-A (50 ␮g/ml)
or LPS (0.1 ng/ml). The incubation was continued for 2 h, and
cells were harvested for RNA isolation.
Survanta Inhibition Experiments
Survanta (Ross Laboratories; Columbus, OH), a lipid extract of bovine lung homogenate that is supplemented with
specific lipids and contains SP-B and SP-C (but no SP-A), was
used as a source of surfactant lipid. THP-1 cells were plated
at a density of 2 ⫻ 106 cells/ml in 2 ml (4 ⫻ 106 cells/
treatment) in 24-well culture plates. SP-A or LPS from Escherichia coli 055:B5 (Sigma) at final concentrations of 50
␮g/ml or 0.1 ng/ml, respectively, were preincubated with
200–1,000 ␮g/ml of Survanta for 15 min before addition to
the THP-1 cells. Then the cells were incubated for an additional 2 h.
Tolerance Experiments
THP-1 cells were plated at a density of 2 ⫻ 106 cells/ml
(4 ⫻ 106 cells/treatment) in 24-well culture plates. THP-1
cells were pretreated for 24 h with SP-A (0 or 50 ␮g/ml) or
LPS (0 or 0.1 ng/ml). These pretreatment exposures to SP-A
or LPS were designated as SP-A1 or LPS1. After this incubation, the cells were carefully washed with cold PBS and then
exposed to a second SP-A (0 or 50 ␮g/ml) or LPS (0 or 0.1
ng/ml) treatment, designated as SP-A2 or LPS2, for an additional 2-h period after which the cells were harvested for
RNA analysis (Fig. 1).
THP-1 Cell RNA Analysis
Total RNA was prepared using RNAzol B (Tel-Test;
Friendswood, TX) according to the recommendations of the
manufacturer. RNA concentrations of samples were determined spectrophotometrically by absorbance at 260 nm. Total RNA (4 ␮g) was heat denatured and applied to Immobilon-S transfer membranes (Millipore; Bedford, MA) using a
Bio-Dot apparatus (Bio-Rad; Richmond, CA). The membranes were then baked for 30 min at 50°C, and the RNA was
cross-linked by exposure to ultraviolet light to the membrane. Specific mRNAs were analyzed by hybridization of the
blots with the appropriate oligonucleotide probes.
Synthesis and labeling of oligonucleotide probes. Oligonucleotides were terminally labeled with [␣-32P]dATP (5,000
Ci/mmol; DuPont-NEN; Boston, MA) using recombinant terminal deoxynucleotidyltransferase (Tdt; GIBCO BRL) with
Dose-Response Studies With SP-A and LPS
THP-1 cells were plated at a density of 2 ⫻ 106 cells/ml in
2 ml (4 ⫻ 106 cells/treatment) in 24-well culture plates. SP-A
(5–100 ␮g/ml) or LPS (10 pg/ml to 10 ng/ml) was added to the
cells. The incubation was continued for 2 h, and the cells were
harvested for TNF-␣ mRNA analysis. We chose to use cytokine mRNA as an end point rather than protein because the
kinetics of production and secretion of different cytokines by
THP-1 cells vary widely (17) and multiple time points would
have been required to adequately measure effects on cytokine
levels.
Polymyxin B Inhibition Experiments
THP-1 cells were plated at a density of 2 ⫻ 106 cells/ml in
2 ml (4 ⫻ 106 cells/treatment) in 24-well culture plates.
Fig. 1. Schematic representation of the experimental design. After
differentiation with 1,25-dihydroxycholecalciferol (VD3), THP-1 cells
were given their first treatment with surfactant protein A (SP-A1) or
lipopolysaccharide (LPS1). Incubation was continued for 24 h, the
cells were washed with PBS, and then the second treatment was
given (SP-A2 or LPS2). The cells were incubated for 2 more hours and
then harvested for RNA preparation and analysis. SP-A1, pretreatment with 0 or 50 ␮g/ml; LPS1, pretreatment with 0 or 0.1 ng/ml;
SP-A2, treatment with 0 or 50 ␮g/ml; LPS2, treatment with 0 or 0.1
ng/ml.
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The THP-1 cell line was obtained from the American Type
Culture Collection (Manassas, VA). Cells were grown in suspension in complete RPMI 1640 (Sigma; St. Louis, MO) culture
medium with 0.05 mM ␤-mercaptoethanol and 10% fetal calf
serum (FCS; Summit Biotechnology; Ft. Collins, CO) at 37°C in
a humidified incubator with a 5% CO2 atmosphere. Because the
properties of the THP-1 cell can change dramatically after
prolonged periods in culture, cells were discarded and replaced
by early frozen stocks after ⬃25 passages. THP-1 cells were
differentiated for 72 h with 10⫺8 M 1,25-dihydroxycholecalciferol (Biomol Research Laboratories; Plymouth Meeting, PA) at
a starting density of 5 ⫻ 105 cells/ml. After differentiation, cells
were pelleted at 250 g for 10 min at 4°C, and the cell pellet was
washed once with 50 ml cold PBS and pelleted as before. The
pellet was then resuspended in 2 ml of complete RPMI 1640
medium with 10% FCS at 2 ⫻ 106 cells/ml (4 ⫻ 106 cells/
treatment) in 24-well culture plates (Fisher Scientific; Pittsburgh, PA). After each incubation period, cells were counted
and viability was assessed by trypan blue exclusion. Under the
conditions employed in this study, SP-A, LPS, and Survanta did
not appear to have any effect on the viability of THP-1 cells. All
experiments were conducted in culture medium containing 10%
FCS.
L111
L112
COMPARISON OF SP-A AND LPS IN THP-1 CELLS
Statistical Analysis
RNA values given are the means of triplicate determinations. Data were analyzed using SigmaStat statistical software (Jandel Scientific; San Rafael, CA) and were judged to
be significantly different at P ⬍ 0.05.
RESULTS
Dose-Response Studies and TNF-␣ mRNA Levels
1,25-Dihydroxycholecalciferol-differentiated THP-1
cells were treated with either SP-A or LPS at the
indicated concentrations (Fig. 2). The range of concentrations of SP-A we chose to use is likely to be within
the physiological range for SP-A in the alveolar lining
layer. SP-A concentrations as low as 5 ␮g/ml increased
TNF-␣ mRNA levels. Cytokine mRNA production continued to increase as the SP-A dose went up to 100
Fig. 2. Dose-response studies with SP-A and LPS. SP-A or LPS at
the indicated concentrations were added to THP-1 cells for 2 h. Cells
were then harvested and processed for tumor necrosis factor-␣
(TNF-␣) mRNA analysis by dot blotting, and relative mRNA levels
were quantified by densitometry. OD, optical density in arbitrary
units.
␮g/ml. TNF-␣ mRNA levels were increased by LPS
concentrations as low as 10 pg/ml and continued to
increase as the LPS went up, approaching a plateau at
LPS concentrations between 1 and 10 ng/ml. We found
that the effects caused by SP-A at 50 ␮g/ml or by LPS
at 0.1 ng/ml were roughly equal with regard to the
production of TNF-␣ mRNA. Therefore, most of our
experiments were undertaken with these two fixed
doses.
Effect of Polymyxin B on SP-A- or LPS-Induced
Cytokine mRNA Levels
Production of the proinflammatory cytokines by
THP-1 cells and other cells of monocytic lineage is
markedly enhanced by LPS. A number of experiments
were conducted to eliminate the possibility that contaminating LPS in our SP-A preparation was responsible for the stimulatory effects of proinflammatory
cytokines by THP-1 cells. All of the SP-A preparations
contained ⬍3 pg LPS/mg SP-A as measured by the
QCL-1000 LAL assay. THP-1 cells exposed to doses of
LPS in this range did not exhibit any detectable response. Moreover, when polymyxin B (1 or 10 ␮g/ml), a
known inhibitor of LPS, was added in some experiments, it was found that it nearly completely blocked
production of TNF-␣, IL-1␤, and IL-8 after stimulation
of THP-1 cells by LPS (0.1 ng/ml). However, addition of
polymyxin B had little or no effect on SP-A-induced
changes in the production of TNF-␣, IL-1␤, and IL-8
(Fig. 3).
Survanta Decreases SP-A- or LPS-Induced Cytokine
mRNA Levels
Dot blot analysis of total RNA from THP-1 cells was
performed to assess the effect of Survanta on SP-A- or
LPS-stimulated cytokine mRNA expression. When
Survanta was preincubated with SP-A (50 ␮g/ml) 15
min before addition to the cells, significant Survanta
dose-dependent decreases in the SP-A-induced in-
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supplied Tdt buffer. Labeling was done for 1 h at 37°C, and
the reaction was stopped by rapid cooling to 4°C. Unincorporated material was removed using STE Midi Select-D, G-25
Microcentrifuge Spin Columns (5⬘ 3 3⬘, Boulder, CO). Oligonucleotides were routinely labeled to a specific activity of
1–5 ⫻ 106 counts per min (cpm)/ng DNA. The antisense
sequences for TNF-␣, IL-1␤, IL-8, and ␤-actin (37) were as
follows: TNF-␣, 5⬘-GCAATGATCCCAAAGTAGACCTGCCC-3⬘;
IL-1␤, 5⬘-ACACAAATTGCATGGTGAAGTCAGTT-3⬘; IL-8, 5⬘TCTCAGCCCTCTTCAAAAACTTCTC-3⬘; ␤-actin, 5⬘-CTAGAAGCATTTGGGGTGGACGATGGAGGGGCC-3⬘.
Hybridization of blots. The optimal temperatures for hybridization with TNF-␣, IL-1␤, and IL-8 probes were 56°C,
51°C, and 52°C, respectively. Prehybridization was done
overnight at the optimal temperature in hybridization bottles
containing 10 ml of solution [1⫻ saline-sodium phosphateEDTA buffer (SSPE), 2⫻ Denhardt’s solution, 10% dextran
sulfate, 1% milk, 2% sodium dodecyl sulfate (SDS), 200 ␮g/ml
fish sperm DNA, 200 ␮g/ml polyadenylic acid, and 200 ␮g/ml
yeast tRNA]. All of the above reagents were purchased from
Sigma except milk (Carnation), which was treated with diethyl pyrocarbonate to inhibit ribonuclease activity, fish
sperm DNA (US Biochemicals; Cleveland, OH), and yeast
tRNA (Boehringer Mannheim; Indianapolis, IN). Fish sperm
DNA was boiled and added to the solution immediately
before use. After prehybridization, all of the solution was
removed and replaced with 10 ml of hybridization solution
with the addition of 5 ⫻ 106 cpm/ml of labeled probe. Hybridization was done overnight at the optimal temperature. After
hybridization, the blots were briefly rinsed and then washed
twice for 30 min at room temperature with 100 ml of 1⫻
SSPE, 0.5% SDS, and 0.1% milk. The blots were then washed
for 30 min at room temperature with 100 ml of 0.2⫻ SSPE
and 1% SDS. The blots were given a final wash with 100 ml
of 0.1⫻ SSPE and 0.5% SDS for 30 min at the optimal
temperatures (42°C, 35°C, and 42°C for TNF-␣, IL-1␤, and
IL-8, respectively). After hybridization with the probes for
these three cytokines, the membranes were stripped and
reprobed with ␤-actin; the optimal temperatures for ␤-actin
hybridization and the final wash were 58°C and 41°C, respectively.
After the final wash, blots were partially dried and exposures were done at ⫺85°C with Kodak X-Omat XAR film
(Rochester, NY) and two intensifying screens (DuPont-NEN).
Levels of mRNAs for specific cytokines were quantitated on
the X-ray films by laser densitometry and normalized to
␤-actin mRNA.
COMPARISON OF SP-A AND LPS IN THP-1 CELLS
L113
responses were nearly completely inhibited. When the
cells were treated with Survanta combined with LPS
(0.1 ng/ml) following a similar schedule, Survantainduced dose-dependent decreases in cytokine levels
were seen, although the inhibition of the LPS effect by
Survanta was far less than the inhibition of the SP-A
effect. The LPS-induced production of TNF-␣ and IL-8
were inhibited by only ⬃20% and IL-1␤ by ⬍50% when
200 ␮g/ml Survanta were added. When Survanta doses
were increased to 1 mg/ml, although the inhibitory
effects on LPS stimulation increased (the percent inhibition for TNF-␣, IL-1␤, and IL-8 was ⬃88%, ⬃91%,
and ⬃45%, respectively), they still did not reach the
degree of inhibition seen on the SP-A effect (Fig. 4).
The phenomenon of LPS-induced tolerance is well
described. In the present studies, THP-1 cells were
preincubated with LPS or medium alone for 24 h. The
medium was then changed, and cytokine mRNA levels
were analyzed 2 h after a second incubation with LPS
or medium alone (Fig. 1). All three cytokine mRNAs
were assayed using aliquots of total RNA from the
same set of THP-1 cells. Pretreatment with 0.1 ng/ml
LPS resulted in a profound inhibition (⬃67% inhibition) of TNF-␣ in response to a subsequent LPS stimulus (0.1 ng/ml) compared with pretreatment with medium alone followed by LPS treatment (Fig. 5). Similar
inhibition occurred with regard to IL-1␤ (Fig. 6) and
IL-8 (Fig. 7) (⬃41% and ⬃45% inhibition, respectively).
However, when SP-A was introduced using a similar
experimental schedule, pretreatment with SP-A resulted in a slight reduction in TNF-␣ production due to
a subsequent SP-A stimulus (⬃22% inhibition) (Fig. 5).
However, the production of IL-1␤ (Fig. 6) and IL-8 (Fig.
7) by the same cells was further increased by the
second treatment with SP-A (⬃28% and ⬃54% increase, respectively) rather than inhibited as was the
case with LPS.
DISCUSSION
Fig. 3. Inhibitory effect of polymyxin B on SP-A- or LPS-induced cytokine mRNA levels. Polymyxin B at concentrations of 0, 1.0, or 10 ␮g/ml
was added to THP-1 cells with SP-A (50 ␮g/ml, solid bars) or LPS (0.1
ng/ml, open bars) for 2 h. Cells were then harvested and processed for
TNF-␣ (A), interleukin-1␤ (IL-1␤; B), and IL-8 (C) mRNA analysis by
dot blotting. Relative mRNA levels were quantified by densitometry.
Data are from 3 identical experiments. Cytokine mRNA values from
cells not treated with polymyxin B, the controls for these experiments,
are set equal to 100%, and polymyxin B-treated cell values are expressed as percent of control. *Values from cells treated with both
polymyxin B and SP-A that are significantly different from control (P ⬍
0.05). **Values from cells treated with both LPS and polymyxin B that
are significantly different from control values.
creases in cytokine levels were seen. Production of
TNF-␣, IL-1␤, and IL-8 was inhibited by more than
60% by Survanta at 200 ␮g/ml in SP-A-stimulated
THP-1 cells. At 1 mg/ml of Survanta, the SP-A-induced
A growing number of reports have suggested that
surfactant may have some host defense-related functions. SP-A has a stimulatory effect on numerous aspects of immune cell function, including phagocytosis,
chemotaxis, generation of reactive oxidant species, expression of cell surface markers, and production of
proinflammatory cytokines (8, 15, 17, 26, 30). In most
cases, surfactant lipids have been found to inhibit
various functions, including the stimulation of alveolar
macrophages by SP-A (14, 15, 17, 24). Thus surfactant
lipids and SP-A may be counterregulatory, and changes
in the relative amounts of surfactant lipid to SP-A may be
important in regulating the immune status of the lung.
That these amounts change in disease states is clear
(7), but it is not known whether the surfactant alterations are a cause of the disease or an effect of it. The
results presented here show enhanced production of
proinflammatory cytokines, TNF-␣, IL-1␤, and IL-8, in
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Effect of Pretreatment With SP-A or LPS
on Cytokine mRNA Levels
L114
COMPARISON OF SP-A AND LPS IN THP-1 CELLS
surfactant lipids can cause an overall suppression of
cytokine release. When we compared the effects of
Survanta on the production of cytokines in SP-A- or
LPS-stimulated THP-1 cells, we found that Survanta
exerted more pronounced inhibitory effects on SP-Astimulated cells than on LPS-stimulated cells, although Survanta-induced dose-dependent decreases
occurred in both groups. This suggests that the inhibitory effects of Survanta on SP-A and LPS may occur
through different mechanisms. Similar inhibitory effects have been reported with human alveolar macrophages (24, 25). In the normal lung, the inhibitory
both SP-A- and LPS-stimulated THP-1 cells. The LPS
inhibitor polymyxin B completely inhibits the LPSinduced increases in cytokines but has no effect on the
increases resulting from SP-A treatment. However,
Survanta, when combined with either SP-A or LPS,
significantly suppressed the production of these cytokines in a dose-dependent manner. This suggests that
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Fig. 4. Effect of Survanta on SP-A- or LPS-induced cytokine mRNA
levels. Survanta at the indicated concentration was preincubated
with SP-A (50 ␮g/ml; solid bars) or LPS (0.1 ng/ml; open bars) 15 min
before addition to THP-1 cells. After an additional 2 h, cells were
harvested and processed for TNF-␣ (A), IL-1␤ (B), and IL-8 (C)
mRNA analysis by dot blotting, and relative mRNA levels were
quantified by densitometry. Values presented are a percentage of
values obtained from cells treated with SP-A or LPS without Survanta. * P ⬍ 0.05 vs. cells treated with SP-A without Survanta.
** P ⬍ 0.05 vs. cells treated with LPS without Survanta. The data
shown are from a single experiment but are representative of the 3
or 4 experiments performed.
Fig. 5. Effect of pretreatment with SP-A or LPS on TNF-␣ mRNA
levels. THP-1 cells were pretreated for 24 h with SP-A (SP-A1, 0 or 50
␮g/ml; B) or LPS (LPS1, 0 or 0.1 ng/ml; A), washed, and then
stimulated with SP-A (SP-A2, 0 or 50 ␮g/ml; B) or LPS (LPS2, 0 or 0.1
ng/ml; A) for an additional 2 h. Cells were harvested and processed
for TNF-␣ mRNA analysis by dot blotting, and relative mRNA levels
were quantified by densitometry. * P ⬍ 0. 05 vs. cells pretreated with
SP-A (0 ␮g/ml) or LPS (0 ng/ml). Data shown are from 1 experiment
but are representative of the 3 experiments performed. RNA from
the same cells was also used for the determinations shown in Figs. 6
and 7.
COMPARISON OF SP-A AND LPS IN THP-1 CELLS
L115
with Survanta for 15 min before exposing the cells to
either SP-A or LPS. We found that production of cytokines was reduced, although the degree of inhibition
was less than that achieved by preincubation of Survanta with SP-A or LPS before treatment (data not
shown). These results suggest that the lipids may exert
at least part of their influence by interacting directly
with SP-A or LPS and changing the way in which these
agents interact with the cells. However, several other
lines of evidence suggest that the suppressive effects of
lipids on LPS are not due to extracellular binding or
inactivation of LPS (24). These include some recent
studies proposing that the suppressive effects of surDownloaded from http://ajplung.physiology.org/ by 10.220.33.3 on September 30, 2016
Fig. 6. Effect of pretreatment with SP-A or LPS on IL-1␤ mRNA
levels. THP-1 cells were pretreated for 24 h with SP-A (SP-A1, 0 or 50
␮g/ml; B) or LPS (LPS1, 0 or 0.1 ng/ml; A), washed, and then
stimulated with SP-A (SP-A2, 0 or 50 ␮g/ml; B) or LPS (LPS2, 0 or 0.1
ng/ml; A) for an additional 2 h. Cells were harvested, processed for
IL-1␤ mRNA analysis by dot blotting, and relative mRNA levels were
quantified by densitometry. * P ⬍ 0. 05 vs. cells pretreated with SP-A
(0 ␮g/ml) or LPS (0 ng/ml). Data shown are from 1 experiment but
are representative of the 3 experiments performed. RNA from the
same cells was also used for the determinations shown in Figs. 5
and 7.
actions of the surfactant lipids appear to control the
actions of immune cells in the alveolar spaces. However, if the lipids are reduced in amount or SP-A is
increased, the stimulatory influence of SP-A could be
enhanced.
The precise mechanism by which surfactant lipids
mediate these suppressive effects remains elusive.
Surfactant lipids may exert their effects by causing
changes in membrane fluidity (33). To address the
possibility that surfactant lipids interact with cells and
alter some of their characteristics (e.g., membrane fluidity or receptor affinity), we incubated THP-1 cells
Fig. 7. Effect of pretreatment with SP-A or LPS on IL-8 mRNA
levels. THP-1 cells were pretreated for 24 h with SP-A (SP-A1, 0 or 50
␮g/ml; B) or LPS (LPS1, 0 or 0.1 ng/ml; A), washed, and then
stimulated with SP-A (SP-A2, 0 or 50 ␮g/ml; B) or LPS (LPS2, 0 or 0.1
ng/ml; A) for an additional 2 h. Cells were harvested, processed for
IL-8 mRNA analysis by dot blotting, and relative mRNA levels were
quantified by densitometry. * P ⬍ 0. 05 vs. cells pretreated with SP-A
(0 ␮g/ml) or LPS (0 ng/ml). Data shown are from 1 experiment but
are representative of the 3 experiments performed. RNA from the same
cells was also used for the determinations shown in Figs. 5 and 6.
L116
COMPARISON OF SP-A AND LPS IN THP-1 CELLS
The potential physiological relevance of SP-A-induced tolerance is not clear at present. Deranged levels
of SP-A have been associated with a variety of lung
disease states. Increased SP-A has been found in lavage from patients with acquired immunodeficiency
syndrome-related pneumonia, sarcoidosis, hypersensitivity pneumonitis, acute farmer’s lung disease, and
asbestosis (7, 9). We speculate the SP-A tolerance may
be a protective mechanism that prevents damage to the
lung by avoiding excessive inflammation as has been
postulated for LPS tolerance.
Currently, there remains some controversy about
whether native SP-A is pro- or anti-inflammatory. For
instance, McIntosh et al. (21) have reported that SP-A
inhibited the production of TNF by alveolar macrophages stimulated by LPS. Our laboratory, on the
other hand, has reported that SP-A stimulated production of several cytokines, including TNF-␣ (17). The
reasons for these conflicting observations on the effects
of SP-A on TNF-␣ release are not known but may be
due to differences in SP-A purification methods, the
oligomeric state of the SP-A, the cell types studied, the
assays used, or other subtle technical differences.
In summary, although many of the effects caused by
SP-A resemble those produced by LPS, there are some
marked differences between these two agents. SP-A
effects are readily inhibited by surfactant lipids,
whereas the effects on LPS are seen only at the higher
doses of lipids. Regarding tolerance, both SP-A and
LPS exhibit a much reduced secondary response with
respect to TNF-␣ production. However, whereas similarly reduced LPS responses are seen for IL-1␤ and
IL-8, a second dose of SP-A further increases levels of
both cytokines. These differences suggest that while
SP-A and LPS may act in part through a common
mechanism, it is likely that these two agents also act
through unique mechanisms as well. Further studies
will be needed to determine whether SP-A has an effect
on the response to LPS and vice versa and the mechanisms responsible for this effect.
The authors thank Todd M. Umstead and Jill Hayden for technical assistance.
The study was supported by National Heart, Lung, and Blood
Institute Grant HL-54683.
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