Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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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 µg/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.
surfactant protein A; lipopolysaccharide; cytokines; tolerance; innate immunity
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INTRODUCTION |
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PULMONARY SURFACTANT 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-antibody-mediated 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 interactions 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.
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MATERIALS AND METHODS |
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Cell Culture
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 mMPreparation 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.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 µg/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-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. Polymyxin B (Sigma) was added to the cells at various concentrations (1 or 10 µg/ml) in the presence of SP-A (50 µg/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 µg/ml or 0.1 ng/ml, respectively, were preincubated with 200-1,000 µg/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 µg/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 µg/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).
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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 µg) 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 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', 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
phosphate-EDTA buffer (SSPE), 2× Denhardt's solution, 10% dextran
sulfate, 1% milk, 2% sodium dodecyl sulfate (SDS), 200 µg/ml fish
sperm DNA, 200 µg/ml polyadenylic acid, and 200 µg/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.
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 |
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Dose-Response Studies and TNF-
mRNA Levels
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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 µg/ml), a known inhibitor of LPS, was added in some experiments, it was found that it nearly completely blocked production of TNF-
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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 µg/ml) 15 min before addition to the cells, significant Survanta dose-dependent decreases in the SP-A-induced increases in cytokine levels were seen. Production of TNF-
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Effect of Pretreatment With SP-A or LPS on Cytokine mRNA Levels
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-
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DISCUSSION |
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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 both SP-A- and LPS-stimulated THP-1
cells. The LPS inhibitor polymyxin B completely inhibits the
LPS-induced 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 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-A-stimulated 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 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 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
surfactant lipids may in part involve transcriptional regulation
through inhibition of nuclear factor-B (NF-
B) activation
(1, 13).
We have found other differences in the responses of THP-1 cells to LPS
and SP-A. Cells exposed to LPS become refractory to further challenge
with this agent. This phenomenon, referred to as tolerance, is
characterized by decreased cytokine production after a second dose of
LPS compared with those levels induced by an initial dose. Tolerance
has been demonstrated in a variety of primary cells of monocytic
lineage and in monocyte/macrophage cell lines (2,
5, 11, 12, 20,
23). In the present experiments, THP-1 cells pretreated
with LPS showed decreased cytokine production following a second LPS
stimulus. Thus LPS pretreatment in our system induced the development
of tolerance, which was characterized by a marked attenuation of the
monocyte/macrophage response to subsequent LPS stimulation. In
contrast, pretreatment with SP-A resulted in unique changes in cytokine
release after a subsequent SP-A stimulation. Specifically, TNF- was
markedly suppressed, whereas IL-1
and IL-8 were significantly
augmented rather than inhibited. This clearly shows a dichotomy among
the three cytokine responses to SP-A. The differential release of these
inflammatory cytokines suggests that different regulatory pathways
control the responses to LPS and SP-A in tolerant THP-1 cells.
When monocytes are stimulated with LPS repeatedly, the initially strong
response is modified so that only low amounts of the proinflammatory
cytokines, in particular TNF-, are produced. Decreased responses of
tolerant monocytes have been documented not only for TNF-
but also
for IL-1
, IL-6 and other cytokines, for arachidonic acid
metabolites, for responses like fever, and for LPS-induced death rate
(38). In the present study, we have confirmed that LPS tolerance in
THP-1 cells resulted in decreased levels of TNF-
, IL-1
, and IL-8.
Tolerance is not strictly LPS specific because it can also be induced
by some cytokines, including TGF-
(4, 31). Our results
show that SP-A induces tolerance in THP-1 cells, although the pattern
of cytokine changes is different from that resulting from LPS treatment.
The molecular mechanisms leading to LPS tolerance have been studied
extensively but are still not completely delineated. Studies by
Ulevitch and Tobias (27) have shown that CD14, a component of the LPS receptor, is not downregulated when cells are rendered tolerant. Blackwell et al. (2) have found that in the
LPS-desensitized rat alveolar macrophage cell line NR8383 the initial
steps of signal transduction are activated up to the stage of the
NF-B transcription factor. Several other studies also suggest the
involvement of this factor in tolerance. For example, it has been shown
that in tolerant macrophages there is a predominance of the p50
homodimer of NF-
B, which lacks transactivation activity
(11, 38). It also has been demonstrated that
in tolerant THP-1 cells, the endotoxin-tolerant phenotype may be
partially due to an enhanced rate of synthesis of the inhibitor
I
B-
in these cells (12).
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.
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ACKNOWLEDGEMENTS |
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The authors thank Todd M. Umstead and Jill Hayden for technical assistance.
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FOOTNOTES |
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The study was supported by National Heart, Lung, and Blood Institute Grant HL-54683.
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{at}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. §1734 solely to indicate this fact.
Received 25 October 1999; accepted in final form 21 February 2000.
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