Departments of 1 Cell Biology and 2 Pediatrics, Duke University, Durham, North Carolina 27710; and 3 Division of Pulmonary Biology, Children's Hospital Research Foundation, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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The role
of surfactant-associated protein (SP) A in the mediation of pulmonary
responses to bacterial lipopolysaccharide (LPS) was assessed in vivo
with SP-A gene-targeted [SP-deficient;
SP-A(/
)] and wild-type [SP-A(+/+)]
mice. Concentrations of tumor necrosis factor (TNF)-
, macrophage
inflammatory protein-2, and nitric oxide were determined in recovered
bronchoalveolar lavage fluid after intratracheal administration of LPS.
SP-A(
/
) mice produced significantly more TNF-
and
nitric oxide than SP-A(+/+) mice after LPS treatment. Intratracheal
administration of human SP-A (1 mg/kg) to SP-A(
/
) mice
restored regulation of TNF-
, macrophage inflammatory protein-2, and
nitric oxide production to that of SP-A(+/+) mice. Other markers of
lung injury including bronchoalveolar fluid protein, phospholipid
content, and neutrophil numbers were not influenced by SP-A. Data from
experiments designed to test possible mechanisms of SP-A-mediated
suppression suggest that neither binding of LPS by SP-A nor enhanced
LPS clearance are the primary means of inhibition. Our data and others
suggest that SP-A acts directly on immune cells to suppress LPS-induced
inflammation. These results demonstrate that endogenous or exogenous
SP-A inhibits pulmonary LPS-induced cytokine and nitric oxide
production in vivo.
lipopolysaccharide; lung inflammation
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INTRODUCTION |
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PULMONARY SURFACTANT is a mixture of phospholipids, neutral lipids, and proteins that coats the alveolar surface, the site of gas exchange. This mixture reduces the surface tension at the air-liquid interface of the alveoli, preventing collapse at end expiration (20, 21). Surfactant-associated protein (SP) A is an abundant phospholipid-associated protein that plays a role in the formation of tubular myelin and in host defense. SP-A is a member of a family of calcium-dependent lectins (collectins) that includes serum mannose binding protein, SP-D, and conglutinin (29, 38, 44). Collectins, including SP-A, have a collagen-like amino-terminal domain and a globular COOH-terminal carbohydrate recognition domain that binds various carbohydrates including D-mannose, L-fucose, D-galactose, and D-glucose (1, 2, 16, 46). SP-A binds to a variety of bacterial and viral pathogens as well as to lipopolysaccharide (LPS) from some serotypes of bacteria (10).
Collectins have multiple host defense functions. For example,
collectins bind to immune cells, opsonize and enhance bacterial clearance, and affect oxygen radical production (36, 37, 39, 40,
49-52, 54). In addition, SP-A acts as an anti-inflammatory agent
in vitro, suppressing LPS-induced production of tumor necrosis factor
(TNF)- by alveolar macrophages and inhibiting mitogen-induced T-cell
proliferation and interleukin (IL)-2 production in vitro (6, 31).
Contradictory results to these findings have been reported with
different culture systems and methods of SP-A purification (23, 25).
Recent studies (17, 22, 26, 27) with SP-A-deficient
[SP-A(/
)] mice showed that the absence of
SP-A has a minimal effect on surfactant homeostasis but a major impact
on bacterial clearance. LeVine et al. (26) showed that
clearance of intratracheal group B streptococcus and Pseudomonas
aeruginosa from the lungs of SP-A(
/
) mice was
impaired compared with clearance from the lungs of wild-type
[SP-A(+/+)] mice. Decreased clearance of bacteria was
associated with decreased binding and uptake by alveolar macrophages. Systemic spread of group B streptococcus infection was also increased in SP-A(
/
) mice (26, 27). Intratracheal administration of exogenous human SP-A restored bacterial killing in
SP-A(
/
) mice at 6.67 and 4.44 mg/kg but not at 2.22 mg/kg
(27). Concentrations of TNF-
and macrophage inflammatory protein-2
(MIP-2; the murine homolog for IL-8) (8) in lung homogenates were
increased in SP-A(
/
) mice compared with those in
SP-A(+/+) mice after infection with P. aeruginosa (26).
These studies showed that the bacterially infected
SP-A(/
) mice had higher levels of cytokines and
chemokines than the SP-A(+/+) mice. However, it was unclear whether the
increases were a direct effect of SP-A on pulmonary cytokine production
or related to the decreased capacity of SP-A(
/
) mice to
clear bacteria. In the present study, the role of SP-A in the
regulation of inflammatory responses to LPS was assessed in vivo.
Inflammatory cytokines, nitric oxide (NO) production, and other markers
of lung injury were measured in SP-A(+/+) mice, SP-A(
/
)
mice, and SP-A(
/
) mice treated with exogenous SP-A after
intratracheal administration of LPS.
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MATERIALS AND METHODS |
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Animal care and treatment. The gene encoding murine SP-A was disrupted by homologous recombination to generate an inbred strain of SP-A-deficient mice. It was previously shown that neither SP-A mRNA nor protein can be detected in these mice (22). Mice of either sex, 20-24 wk of age, and ranging in weight from ~25 to 32 g were housed under pathogen-free conditions until they were used for experiments. SP-A(+/+) mice were of the same genetic background (129J) and were age matched. The animals were anesthetized by exposure to gauze soaked in Metaphane (methoxyflurane; Mallinckrodt Veterinary) in a closed jar until the mice were no longer conscious and breathing had slowed. Mice were then restrained on a Plexiglas board and continuously exposed to Metaphane from a soaked gauze pad placed inside a 15-ml conical tube. After the skin was swabbed with 70% ethanol, an incision of ~1.5 cm in length was made 2 cm above the sternum to expose the trachea. A 27-gauge needle attached to a 1-ml syringe (Becton Dickinson) containing 100 µl of either 70 µg/kg (2 µg/animal) of Escherichia coli LPS 026:B6 (Sigma) or 70 µg/kg of LPS and 1 mg/kg (30 µg/animal) of human SP-A was inserted into the trachea. The fluid was injected slowly, and the incision was closed with 35 R Reflex One (Richard-Allen) surgical staples.
Animals were killed 3 h after injection. Previous studies (2, 18, 33,
48, 56) in rats and mice demonstrated that peak concentrations of
TNF- were present after intratracheal administration of LPS doses
ranging from 10 ng/kg to 0.5 mg/kg 3-6 h after instillation.
Animals were given an intraperitoneal injection of 200 µl of Nembutal
(pentobarbital sodium; Abbott Laboratories). After loss of both
consciousness and the ability to respond to tail or foot pad squeezes,
the surgical staples were removed, the trachea was cannulated, and the
mice were killed by exsanguination. The lungs were exposed by removal
of the front portion of the rib cage and lavaged three times with 0.8 ml of lavage buffer. Lavage buffer consisted of phosphate-buffered
saline-2 mM EDTA (pH 7.4) prepared with endotoxin-free water (Picopure Water, Hydro Water Management Systems, Research Triangle Park, NC). BAL
fluid was chilled on ice in 15-ml conical tubes before centrifugation
at 400 g for 10 min. The volume of cell-free lavage fluid
recovered was measured for each animal. The samples were stored in
aliquots in sterile 1.5-ml microfuge tubes at
80°C. The
number of mice and method of experimentation were approved by the Duke
University (Durham, NC) Medical Center Institutional Animal Care and
Use Committee.
Isolation of human SP-A. SP-A was isolated as previously
reported (55). BAL fluid from patients undergoing therapeutic lavage for treatment of alveolar proteinosis was obtained with approval of the
Duke University Medical Center Institutional Review Board. The large
aggregates of surfactant were allowed to settle for at least 24 h under
unit gravity. Removal of lipid and hydrophobic proteins from the pellet
was achieved by butanol extraction with subsequent high-speed
centrifugation of the butanol-lavage mixture (105 g
for 60 min). On evaporation of butanol, the insoluble pellet was
resuspended in 100 mM octylglucopyranoside, 150 mM NaCl, and 5 mM Tris,
pH 7.4, and mixed with polymyxin-agarose (1:5 vol/vol; Sigma) for 30 min at room temperature. To remove octylglucopyranoside, the mixture
was dialyzed (14,000 mol wt cutoff) for a minimum of four complete
changes of buffered endotoxin-free water (5 mM Tris, pH 7.4) followed
by another high-speed centrifugation to remove insoluble proteins. The
supernatant was then collected and stored at 20°C. Protein
concentration was determined with a micro-bicinchoninic acid (BCA)
protein assay kit (Pierce, Rockford, IL) and bovine serum albumin as a
standard. Identity of the isolated protein was confirmed by SDS-PAGE
and Western blot analysis with a rabbit polyclonal anti-human SP-A
antiserum as previously reported (32). The SP-A used for the study was
assayed for endotoxin content, which was found to be 0.013 pg
endotoxin/µg protein.
Analysis of cytokines. Both the L929 bioassay (30) and ELISA
were used to determine TNF- concentrations in BAL fluid so that a
comparison between immunoreactive TNF-
and bioactive TNF-
could
be made. For the bioassay, serial dilutions of lavage sample (37.5 µl/well) were tested in duplicate alongside buffer controls containing no TNF-
. These samples were added to L929 cells (American Type Culture Collection, Manassas, VA) that had been
plated in 96-well plates at a concentration of 3 × 105 cells in 75 µl tissue culture medium/well. MEM (with
glutamine and Earle's salts; GIBCO BRL) tissue culture medium was
supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin,
and 10% horse serum (GIBCO BRL). After 24 h in culture, the medium was replaced with serum-free MEM with 0.2% bovine serum albumin (fraction V, low endotoxin; Sigma), actinomycin D (2 µg/ml; Sigma), and the
diluted lavage sample. L929 cells were incubated for a further 24 h
with sample, standard, or buffer before being fixed with 5%
formaldehyde for 5 min, stained with 1% crystal violet for 5 min, and
dried. TNF-
concentrations were calculated by comparing the decrease
in the absorbance at 540 nm to decreases obtained with serial dilutions
of recombinant mouse TNF-
(R&D Systems).
ELISA for inflammatory cytokines. TNF- or MIP-2
concentrations in BAL fluid were determined with assay kits from
Genzyme Diagnostics and R&D Systems, respectively, in conjunction with a Bio-Rad model 550 microplate reader with accompanying software (Bio-Rad, Hercules, CA) as directed by the manufacturer. BAL samples were diluted 1:10 and 1:25 in the diluent provided with the assay. TNF-
and MIP-2 standards were individually added to BAL samples to
ensure that BAL fluid constituents did not interfere with the assay.
The range of detection for the TNF-
and MIP-2 ELISA kits were
35-2,240 and 7.8-500 pg/ml, respectively.
Determination of nitrite recovered from lung lavage. Nitrite, which is a by-product of NO production, was assayed in the BAL fluid with the method of Schmidt et al. (45). Griess reagent (1 mM sulfanilamide, 1 mM napthylethylenediamine, and 0.1 M HCl) was added to each sample (1:1 vol/vol) in a 96-well tissue culture plate and incubated for 10 min at room temperature. The color change in the samples was quantified with a spectrophotometric plate reader (540 nm) and compared with dilutions of a known standard (NaNO2).
Cellular content of BAL fluid. Cells from BAL samples were isolated by centrifugation at 400 g for 10 min. The cells were resuspended in 1 ml of RPMI 1640 tissue culture medium and 10% (by volume) fetal bovine serum (GIBCO BRL). The cells were diluted 1:1 with Turk's solution and counted with a hemocytometer. Approximately 1-2 × 105 cells from each sample were used to prepare cytospins in a Cytospin 2 (Shandon, PA) centrifuge at a speed of 450 rpm for 5 min. These slides were then air-dried, stained with the Hemacolor stain set (EM Diagnostic Systems), and permanently mounted with a glass coverslip. A minimum of 200 cells/sample were counted and evaluated.
BAL fluid lipid and protein. The method of Bligh and Dyer (5) was used to extract lipids from the mouse lavage fluid before assay of these extracted samples for inorganic phosphorus by the method of Bartlett (1). Protein concentrations were determined with a micro-BCA protein assay kit and bovine serum albumin as a standard.
Measurement of LPS-SP-A interactions. FITC-labeled E. coli LPS 026:B6 (20 µg) was added to 300 µg of SP-A in Dulbecco's PBS (GIBCO BRL) plus 1 mM Ca2+ and Mg2+ (total volume 1 ml) and incubated for 3 h at 37°C in the dark. The LPS-SP-A sample was dialyzed for 24 h (in the dark) at room temperature against three 1-liter changes of Dulbecco's PBS (plus Ca2+ and Mg2+) with Slide-A-Lyzer dialysis cassettes (mol wt cutoff of 10,000). Simultaneously, a FITC-LPS control (no protein) was used in the same procedure. The resulting dialysates were analyzed for FITC-LPS with a spectrofluorimeter. The amounts of FITC-LPS contained in the control (no SP-A included) and experimental samples were calculated by generating a standard curve of relative fluorescent units versus known amounts of FITC-LPS. The FITC-LPS used as a standard was obtained from the same stock solution added to the binding assay. Furthermore, the SP-A used for the binding assay was from the same preparation of SP-A used for in vivo experiments. The protein content of the SP-A-LPS sample was measured with the micro-BCA assay kit after the 24 h of dialysis.
Statistics. Statistical analysis was performed with the Primer
for Biostatistics computer program and manual (13). Analysis of
variance was used to determine differences among experimental groups. A
multiple comparison procedure, the Student-Newman-Keuls test, was used.
A P value of 0.05 was considered to be significant.
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RESULTS |
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SP-A inhibits TNF- after LPS treatment. Significantly more
bioactive (Fig. 1) and
immunoreactive (Fig. 2) TNF-
was
recovered in the BAL fluid from SP-A(
/
) mice compared
with that from SP-A(+/+) mice 3 h after intratracheal administration of
70 µg/kg of LPS. Coadministration of human SP-A and LPS to
SP-A(
/
) mice decreased TNF-
concentrations in BAL
fluid to 84% of values measured in SP-A(+/+) mice (control). Amounts
of immunoreactive TNF-
in the BAL fluid from untreated SP-A(+/+) or
SP-A(
/
) mice were similar and were approximately fivefold
lower than those of LPS-treated SP-A(+/+) mice.
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SP-A inhibits MIP-2 after LPS challenge. MIP-2 in BAL fluid
increased after LPS treatment (Fig. 3) in
both SP-A(/
) and SP-A(+/+) mice. Coadministration of SP-A
and LPS to SP-A(
/
) mice decreased MIP-2 concentrations in
BAL fluid below amounts detected in either SP-A(
/
) or
SP-A(+/+) mice challenged with LPS. MIP-2 was not detectable in the BAL
fluid from SP-A(+/+) or SP-A(
/
) mice that did not receive
LPS (data not shown).
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SP-A inhibits BAL fluid nitrite after LPS treatment. NO
production was analyzed in the BAL fluid from mice from all three experimental groups and control animals by quantifying nitrite, a
product created when NO reacts with O2 in solution (12).
The amount of nitrite was greater in BAL fluid from
SP-A(/
) mice compared with that from SP-A(+/+) mice.
Coadministration of LPS and human SP-A rescued the
SP-A(
/
) phenotype by significantly reducing the amount of
nitrite in the BAL fluid from SP-A(
/
) mice compared with
amounts detected in the BAL fluid from SP-A(
/
) mice (Fig.
4). There was detectable
nitrite in the BAL fluid from untreated SP-A(+/+) (n = 3) or
SP-A(
/
) (n = 3) mice that was 80.3 ± 33.7 and
122 ± 60.4%, respectively, of that from LPS-treated SP-A(+/+) mice.
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SP-A did not influence other indexes of pulmonary inflammation.
LPS treatment increased BAL fluid protein in both
SP-A(/
) and SP-A(+/+) mice. No significant differences
were detected in the concentration of protein or phospholipid recovered
from LPS-treated SP-A(
/
) or SP-A(+/+) mice (Table
1). Coadministration of SP-A and LPS did
not alter the concentration of BAL fluid phospholipid or
protein from SP-A(
/
) mice compared with that from
SP-A(+/+) mice (Table 1).
|
LPS treatment increased the number and altered the types of cells in
the BAL fluid from SP-A(+/+) and SP-A(/
) mice (Table 2). Greater than 90% of the
BAL fluid cells were alveolar macrophages in untreated animals. The
percentage of macrophages dropped to ~50% and neutrophils increased
to ~50% in all three of the LPS treatment groups (Table
3).
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SP-A binds minimal amounts of E. coli LPS 026:B6 in solution. After 3 h of incubation at 37°C, only 5.8 ng FITC-labeled LPS 026:B6/µg SP-A were bound. We calculated that the maximum amount of LPS 026:B6 that could have bound SP-A after 3 h in vitro was 8.65%.
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DISCUSSION |
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Summary. We have shown here that SP-A-deficient mice are more
sensitive to intratracheal endotoxin than wild-type mice. For example,
production of biologically active and immunoreactive TNF- in the
airways of SP-A(
/
) mice was greater than that in SP-A(+/+) mice challenged with intratracheal LPS. In addition, the
amount of nitrite measured in the BAL fluid from SP-A(
/
) mice was greater than the amount in SP-A(+/+) mice. Cotreatment of
SP-A(
/
) mice with human SP-A and LPS rescued the knockout phenotype. Specifically, the amounts of TNF-
, MIP-2, and nitrite were similar in SP-A(+/+) mice and SP-A(
/
) mice treated
with SP-A. However, the amounts of protein and phospholipid and the number of infiltrating neutrophils did not vary among the three experimental groups tested. Thus SP-A appears to act as an
anti-inflammatory protein because it inhibits LPS-induced cytokine and
NO production in vivo. However, SP-A does not totally protect the lung
from LPS-induced inflammation in the experimental model tested.
Effects of SP-A on cytokine and nitrite production. Inhibition
of cytokine production by SP-A has been reported by several groups. For
example, McIntosh et al. (31) previously reported that
SP-A inhibits LPS-induced (E. coli 026:B6 and 0111:B4) TNF- production by alveolar macrophages and that SP-A treatment of a mixed
culture of lung fibroblasts and LPS-activated alveolar macrophages
protected fibroblast growth. It was also found that SP-A inhibits IL-2
production by human peripheral blood mononuclear cells stimulated in
vitro with mitogen (7). Similarly, Sano et al. (43) reproduced
the finding that SP-A inhibits LPS-induced TNF-
production by a
macrophage-like tumor cell line stimulated with different serotypes of
LPS. Recently, Cheng et al. (9) showed that SP-A decreased
ionomycin-induced IL-8 production and release by human eosinophils.
Hickling et al. (14) demonstrated that SP-A reduced production of
TNF-
by buffy coat cells stimulated with P. aeruginosa LPS.
Rosseau et al. (41) examined the effect of SP-A on cytokine production
by monocytes and macrophages treated with Candida albicans and
showed that SP-A inhibited TNF-
production by both cell types.
Further analysis of cytokine production by alveolar macrophages showed
that the amount of several proinflammatory mediators (IL-1, IL-8, MIP-1
and monocyte chemoattractant protein-1) was reduced by SP-A, and it was
suggested that SP-A acted directly on monocytes and macrophages to
suppress cytokine production (41). These in vitro reports are
consistent with our conclusion that SP-A exerts an anti-inflammatory
effect in vivo, perhaps by several different mechanisms.
Our results also show that SP-A reduces the amount of nitrite in lavage
fluid from LPS-challenged mice. These results are consistent with those
from a study by LeVine et al. (26), which demonstrated that levels of
nitrite were greater in SP-A(/
) mice than in SP-A(+/+)
mice after intratracheal infection with P. aeruginosa.
In contrast to our results and the studies summarized above, SP-A has
been shown to stimulate cytokine and NO production (4, 23, 25). For
example, Kremlev and Phelps (23) reported that SP-A
stimulates the production of TNF-, IL-1
, IL-1
, and IL-6 by rat
splenocytes. SP-A also enhanced the production of immunoglobulins A, G,
and M by rat splenocytes as well as their proliferation (23, 24). Blau
et al. (4) reported that SP-A, LPS, and combinations of the two
stimulated production of nitrite by alveolar macrophages.
There are several possible explanations for these conflicting observations. For example, SP-A is purified by several different methods, and its activity may be dependent on this variable (6, 7). The means by which cells are activated to elaborate cytokines is also important. The in vitro studies have investigated responses of a variety of cell types including cultured splenocytes and differentiated THP-1 cells, and it is possible that SP-A may exert cell-specific effects. In addition, the effects of SP-A appear to vary with the type of pathogen stimulus. For example, Hickman-Davis et al. (15) demonstrated that SP-A increased the nitrite production of alveolar macrophages stimulated with Mycoplasma pneumoniae. In contrast, Pasula et al. (35) reported that SP-A suppresses reactive nitrogen intermediates by murine alveolar macrophages exposed to Mycobacterium tuberculosis. In any case, our studies show that endogenous SP-A in the alveolar milieu can suppress some proinflammatory responses that are induced by LPS.
Not all indexes of inflammation were altered by SP-A. Exogenous
or endogenous SP-A did not alter the amount of LPS-induced protein
leak, phospholipid, or cellular profile in BAL fluid. These results
suggest that the decreases in the detectable amounts of cytokines were
not an artifactual result of differences in total recoverable protein.
Our results are consistent with the finding that pretreatment of rats
with dexamethasone before LPS challenge decreased TNF- accumulation
in BAL fluid but not the total amount of protein in BAL fluid (28, 34).
Inhibition of some but not all indexes of inflammation suggests that
that SP-A has selective actions in vivo or that its action as an
anti-inflammatory compound is not potent enough to attenuate more
potentially sensitive markers of lung injury.
There was no significant difference in the total amount of MIP-2
recovered from the LPS-challenged lungs of SP-A(+/+) or
SP-A(/
) mice despite the fact that TNF-
concentrations
were significantly different in the same samples. Data from the
referenced study by Xing et al. (56) showed that LPS-induced MIP-2 and
TNF-
mRNA expression in the whole rat lung as well as in alveolar
macrophages differed in several ways. For example, the amount of
LPS-induced MIP-2 mRNA appeared greater than TNF-
mRNA in the whole
lung and alveolar macrophages at the earliest tested time points (30 min and 1 h, respectively). MIP-2 mRNA expression was also much higher
than TNF-
at much later time points (12 and 24 h, respectively). These data suggest that MIP-2 production, like protein leak and cellular influx, is a more sensitive index of inflammation and one on
which SP-A does not exert an effect unless present in high concentrations.
Two lines of evidence suggest that SP-A can inhibit MIP-2 production
under certain circumstances. LeVine et al. (26) showed that MIP-2
production was greater in lung tissue homogenates from SP-A(/
) mice than in those from SP-A(+/+) mice after
infection with P. aeruginosa. Our results with exogenous human
SP-A (rescue group) show that coadministration of SP-A and LPS to
SP-A(
/
) mice does decrease the amount of MIP-2 measured
in BAL fluid relative to that in BAL fluid from SP-A(
/
)
mice or SP-A(+/+) mice challenged with LPS.
We do not know definitively why exogenously administered SP-A was a
more effective inhibitor of MIP-2 production than endogenous SP-A. One
explanation is that when the exogenous SP-A was administered concurrently with LPS to ensure codistribution of LPS and SP-A, an
acute inflammatory response occurred in the context of a larger than
normal concentration of SP-A. It is also possible that exogenous SP-A
exerts a more potent inhibitory response before associating with lipid.
However, it has been previously shown (31) that the anti-inflammatory
effect of SP-A on LPS (026:B6)-stimulated alveolar macrophages was
maintained in the presence of surfactant-like lipids and that the
magnitude of this inhibition was comparable to SP-A added in the
absence of lipid. It is important to note that the dose of exogenous
SP-A administered to SP-A(/
) mice is estimated to be
within a physiological range. For example, Ryan et al. (42) estimated
that the lungs from rats weighing 250-300 g contain 600-720
µg recoverable SP-A/kg body weight.
Possible mechanisms by which SP-A inhibits LPS-stimulated inflammation. SP-A may inhibit LPS-induced cytokine production in vivo by several different mechanisms. It is possible that SP-A blocks LPS interactions with immune cells, possibly by inhibiting the interaction of LPS with cells by binding CD14 (43) or conversely binding to LPS and preventing its interaction with the cell surface (19). In vitro data also raise the possibility that SP-A increases the clearance of LPS from the lung (47).
To address the possibility that SP-A-mediated decreases in cytokine
production exclusively through LPS binding and clearance from the
airways, SP-A(+/+) and SP-A(/
) mice and a rescue group received an intratracheal injection of FITC-LPS (E. coli
026:B6, 2 µg/animal). Animals were lavaged 1 h after instillation
rather than 3 h due to limitations of detecting fluorescence above
background. The amounts of fluorescence detected in the cell-free
lavage fluid and the 150-g pellet were similar in the three
groups [SP-A(+/+), SP-A(
/
), and rescue; 7 animals/group]. These findings are particularly important because
it has been shown that optimum LPS-induced stimulation of cultured
monocytes occurs in a matter of minutes (11). These data suggest that
SP-A is not exclusively exerting its anti-inflammatory effect through
enhanced LPS clearance.
Blau et al. (3) and Kalina et al. (19) have previously reported that interactions of SP-A with LPS can affect their functions. For example, they showed that conditioned medium from SP-A-treated alveolar macrophages stimulates the in vitro formation of myeloid progenitor cell colonies to a similar magnitude as conditioned medium from LPS- or IL-1-treated alveolar macrophages (3). A subsequent report found that coculturing SP-A and LPS with alveolar macrophages reduced the colony-forming activity in the medium compared with macrophages treated with SP-A or LPS alone. The observed mechanism for the inhibition of both LPS and SP-A stimulatory activity was correlated with LPS-SP-A binding (E. coli strain 346) (19).
For our study, we used a serotype of LPS that was reported not bind to
SP-A. This serotype of LPS was chosen to minimize the possibility that
the mechanism of immune suppression of SP-A is through the binding of
LPS. A previous study (43) showed that SP-A does not bind to E. coli LPS 026:B6. These results were confirmed in another binding
assay in which SP-A did not bind LPS 026:B6 but did bind E. coli LPS LCD25 (C. Stamme and J. R. Wright, unpublished observations). Additionally, we designed a binding assay to determine whether SP-A bound LPS 026:B6 in solution because adherence of LPS to a
microtiter plate may alter its binding properties. The soluble binding
assay showed that when 100 µg of SP-A were incubated with 20 µg of
LPS 026:B6, it bound only 0.00577 µg LPS/µg SP-A after a 3-h
incubation (total SP-A bound 8.65% of total LPS). Although we cannot
exclude the possibility that this very low amount of LPS binding is
sufficient to inhibit LPS-stimulated cytokine production, these data as
well as the previous finding that SP-A can suppress TNF- production
by alveolar macrophages when SP-A is added several hours after LPS
activation (31) suggest that SP-A inhibition of LPS-induced
inflammation is by means of a mechanism that does not require SP-A to
physically interact with LPS.
Physiological relevance of SP-A with respect to the pulmonary immune system. Use of SP-A-deficient mice has established that SP-A functions in the resolution of pulmonary bacterial infections and regulation of the resulting pulmonary inflammation. These findings are consistent with other in vitro observations that SP-A inhibits a variety of potentially proinflammatory responses including T-lymphocyte proliferation, IL-2 production (7, 53), and IL-8 production (9) as well as nitrite production (35). The in vivo data presented here and the growing number of reports confirming our initial in vitro observation that SP-A also acts as an anti-inflammatory molecule suggest that a fundamental role of SP-A within the airways is immunologic homeostasis.
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ACKNOWLEDGEMENTS |
---|
This research was supported by National Heart, Lung, and Blood Institute Grants HL-51134 (to J. R. Wright), HL-58795 (to T. Korfhagen and J. R. Wright), and HL-28623 (to J. A. Whitsett); by a Clinical Research Grant from the March of Dimes (to J. C. McIntosh); and by fellowships from the Canadian Lung Association and the Parker B. Francis Foundation (to P. Borron).
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: J. R. Wright, Dept. of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710 (E-mail: j.wright{at}cellbio.duke.edu).
Received 23 March 1999; accepted in final form 8 November 1999.
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