Content uploaded by Francis X Mccormack
Author content
All content in this area was uploaded by Francis X Mccormack
Content may be subject to copyright.
Cutting Edge: The Immunostimulatory Activity of
the Lung Surfactant Protein-A Involves Toll-Like
Receptor 4
1
Loı¨c Guillot,* Viviane Balloy,* Francis X. McCormack,
†
Douglas T. Golenbock,
‡
Michel Chignard,* and Mustapha Si-Tahar
2
*
The collectin surfactant protein-A (SP-A) is involved in the
innate host defense and the regulation of inflammatory pro-
cesses in the lung. In this work we investigated the molecular
mechanisms related to the immunostimulatory activity of SP-A
using macrophages from C3H/HeJ mice, which carry an inac-
tivating mutation in the Toll-like receptor (TLR)4 gene, and
TLR4-transfected Chinese hamster ovary cells. We demon-
strate that SP-A-induced activation of the NF-
B signaling
pathway and up-regulation of cytokine synthesis such as
TNF-
␣
and IL-10 are critically dependent on the TLR4 func-
tional complex. These findings support the concept that TLR4
is a pattern recognition receptor that signals in response to
both foreign pathogens and endogenous host mediators. The
Journal of Immunology, 2002, 168: 5989–5992.
Early interest in pulmonary surfactant focused on the bio-
physical properties of the phospholipid components, es-
pecially their ability to lower surface tension at the air-
epithelial interface within the alveoli. However, there are also
proteins associated with the surface-active material. The most
abundant among them, surfactant protein-A (SP-A),
3
is a member
of the collectin family of preimmune opsonins, which also includes
surfactant protein-D, mannose-binding protein, conglutinin, and
collectin-43 (1, 2). Recent studies suggest an integral role for SP-A
in innate host defense and regulation of inflammatory processes in
the lung. Although several studies have demonstrated that SP-A
enhances macrophage chemotaxis, phagocytosis, immune cell pro-
liferation, and expression of cell surface proteins, the role of SP-A
in regulating production of cytokines is controversial (3–9). Re-
gardless, the observation that SP-A can mediate cell-specific func-
tions suggests the existence of specific receptors. Although a num-
ber of SP-A binding proteins have been identified, their
contributions to the biological activities of SP-A remain debatable
(4). In that context, it is of interest to note that the immune re-
sponses induced by SP-A are similar to those observed when cells
are stimulated by bacterial LPS (10). For instance, it has been
shown that SP-A activates the major downstream signaling NF-
B
pathway and that specific NF-
B inhibitors block SP-A-dependent
increases in TNF-
␣
mRNA levels (11). Based on these data, we
hypothesized that SP-A and LPS may share a common functional
receptor.
According to the current model, the specific cellular recognition
of LPS is initiated by the signal-transducing Toll-like receptor
(TLR)4 and the accessory molecules CD14 and MD-2, leading to
the rapid activation of intracellular signaling pathways, which co-
ordinate the induction of multiple genes encoding inflammatory
mediators (12, 13). TLRs appear to represent a conserved family of
innate immune recognition receptors. A variety of bacterial and
fungal products have been identified that serve as TLR ligands, but
TLRs may also regulate homeostasis via interaction with endog-
enous protein ligands (14–17).
In the present study, we demonstrate for the first time that SP-A
requires a functional TLR4 complex to induce leukocyte
activation.
Materials and Methods
Chemicals and Abs
LPS (Escherichia coli 055:B5), PMA, 4
␣
-phorbol didecanoate, and Gey’s
medium were from Sigma-Aldrich (St. Louis, MO). RPMI 1640 medium,
HBSS, antibiotics, and glutamine were from Life Technologies (Paisley,
U.K.). FCS was from HyClone Laboratories (Logan, UT). Non-phospho-
thioate-modified oligodeoxynucleotide (ODN)1668 was custom synthe-
sized by Genset (Paris, France). The immunostimulatory oligonucleotide
sequence was 5⬘-TCCATGACGTTCCTGATGCT-3⬘and the inactive con-
trol was 5⬘-TCCATGAGCTTCCTGATGCT-3⬘. Human SP-A was isolated
from the lung washings of a patient with alveolar proteinosis by a modi-
fication of a protocol of Suwabe et al. (18), which included serial sedi-
mentation of the surfactant pellet in the presence of 1 mM Ca
2⫹
, elution
with EDTA, and adsorption to mannose-Sepharose. For some experiments,
SP-A was further purified by gel filtration under physiologic ionic strength
conditions (150 mM NaCl, 10 mM Tris) by Superose 6 gel filtration chro-
matography using a fast protein liquid chromatography column with a bed
volume of 10 ⫻300 mm (Amersham Pharmacia Biotech, Piscataway, NJ).
The level of LPS associated with the SP-A was 140 pg LPS per microgram
of SP-A. Another SP-A preparation was obtained having no detectable LPS
using the QCL1000 kit (BioWhittaker, Walkersville, MD).
*Unite´deDe´fense Inne´e et Inflammation, Institut Pasteur, Institut National de la
Sante´ et de la Recherche Me´dicale, Unite´ 485, Paris, France;
†
Division of Pulmonary
and Critical Care Medicine, Department of Medicine, University of Cincinnati, Cin-
cinnati, OH 45267; and
‡
Department of Medicine, Division of Infectious Diseases,
University of Massachusetts Medical School, Worcester, MA 01655
Received for publication February 1, 2002. Accepted for publication April 25, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
L.G. was supported by the De´legation Ge´ne´rale pour l’Armement (Paris, France),
M.S.-T. was partly supported by Vaincre la Mucoviscidose (Paris, France), and
D.T.G. was supported by National Institutes of Health Grant GM54060.
2
Address correspondence and reprint requests to Dr. Mustapha Si-Tahar, Unite´de
De´fense Inne´e et Inflammation, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris,
France. E-mail address: sitahar@pasteur.fr
3
Abbreviations used in this paper: SP-A, surfactant protein-A; TLR, Toll-like recep-
tor; BMDM, bone marrow-derived macrophage; ODN, oligodeoxynucleotide; CHO,
Chinese hamster ovary.
The Journal of Immunology
Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00
●
●
Cell preparation and culture of BMDM
Five- to 6-wk-old female C3H/HeOuJ or C3H/HeJ mice provided by Iffa
Credo (L’Arbresle, France) and Institut Pasteur (Paris, France), respec-
tively, were used to prepare bone marrow-derived macrophages (BMDM),
as previously described (19). Briefly, mice were euthanized by CO
2
expo-
sure and femurs were aseptically collected and placed in cell culture dishes
containing sterile HBSS. Bone marrow was collected with HBSS and
RBCs were lysed with Gey’s medium. The cell suspension was then cen-
trifuged and the pellet was resuspended in RPMI 1640 medium supple-
mented with 10% FCS and 10% CSF-1-conditioned medium. Cells were
then cultured for 3 days. Nonadherent cells were removed and centrifuged
for 10 min at 400 ⫻g. The pellet was resuspended in 1 ml of RPMI 1640
supplemented with 10% FCS and 1% antibiotics. Cell suspension was
flushed through 25-, 27-, and 30-gauge needles successively to separate
cell aggregates. Cells were resuspended at 2 ⫻10
6
cells/ml in RPMI 1640
supplemented with 10% FCS, 2.5% CSF-1-conditioned medium, and 1%
antibiotics. A total of 300
l of the cell suspension was dispensed into
48-well tissue culture plates (Costar, Corning, NY). After an overnight
incubation, wells were washed with prewarmed medium and stimulated as
indicated in the figures.
Culture of U937 cells
The promonocytic U937 cells were obtained from the American Type Cul-
ture Collection (Manassas, VA) and grown in RPMI 1640 supplemented
with 10% FCS, 1% antibiotics, 1% glutamine, and 10 mM HEPES. Cells
were dispensed into 48-well plates (Costar) at 1.7 ⫻10
6
cells/ml and
differentiated into macrophages in the presence of 15 nM PMA or 15 nM
4
␣
-phorbol didecanoate as a negative control for 48 h at 37°Cina5%CO
2
humidified air atmosphere. Then, adherent cells were washed with medium
and 300
l of prewarmed medium was dispensed into each well. Twenty-
four hours later, cells were washed and stimulated as indicated in Results.
Determination of TNF-
␣
and IL-10 concentrations
Murine TNF-
␣
and IL-10 concentrations in BMDM supernatants were deter-
mined by an ELISA and a solid-phase immunoenzyme assay as previously
described (19), respectively. Human TNF-
␣
concentrations in the supernatant
of U937 cells were determined by ELISA, according to the manufacturer’s
protocol (Pelikine compact; CLB, Amsterdam, The Netherlands).
CD25 expression analysis
The Chinese hamster ovary (CHO)/CD14/TLR4 reporter line is a stably
transfected human CD14- and TLR4-positive CHO cell line that expresses
inducible membrane CD25 under the transcriptional control of the human
E-selectin promoter (13, 20). The promoter fragment chosen contains an
essential NF-
B binding site. Flow cytometry analysis of NF-
B activity
used cells that were plated in 12-well dishes and stimulated in Ham’s F12
medium containing 10% FCS, as indicated in Fig. 2. Subsequently, the
cells were harvested with trypsin-EDTA and labeled with FITC-CD25
mAb. Fluorescence analysis was performed using a FACScan flow cytom-
eter (BD Immunocytometry Systems, Mountain View, CA). Binding of
anti-CD25 Ab to its epitope is expressed as the fold increase in median
fluorescence intensity over basal values measured on nontreated cells.
Statistical analysis
Each point corresponds to the mean ⫾SD of the indicated number of
experiments. Statistical significance between the individual groups was
analyzed using the unpaired Student ttest with a threshold of p⬍0.05.
Results
SP-A stimulates murine and human macrophage cytokine
secretion
A growing number of reports have suggested that SP-A may have
some host defense-related properties (3, 4). We first sought to
characterize the effect of purified SP-A on murine BMDM, in
terms of cytokine production. Supernatants from BMDM cultured
in the absence or the presence of increasing concentrations of
SP-A (2.5–20
g/ml) were harvested after 6 or 24 h and assayed
for TNF-
␣
and IL-10 concentrations, respectively (Fig. 1). For
comparison, BMDM were also activated by an optimal concentra-
tion of LPS (1
g/ml). To rule out any possible effect associated
with contamination of SP-A by LPS, all experiments performed
during this investigation used SP-A supplemented with 20
g/ml
polymyxin B, a well-characterized LPS inhibitor (21) (Fig. 1). Un-
der these experimental conditions, TNF-
␣
and IL-10 levels in-
creased to levels of up to ⬃250 and ⬃3500 pg/ml upon exposure
of BMDM with a concentration of SP-A above 10
g/ml, respec-
tively (p⬍0.001; n⫽4). LPS elicited a stronger immunostimu-
latory effect that was about twice that measured upon SP-A cell
activation (Fig. 1). Experiments were also performed to determine
the effect of SP-A on the secretion of cytokines from human cells.
The undifferentiated human U937 monocytic cell line failed to
up-regulate TNF-
␣
release in response to 10
g/ml SP-A or 1
g/ml LPS. However, when U937 cells were cultured with PMA,
which promotes differentiation to a more macrophage-like pheno-
type (22), this cell line became responsive to both stimuli. Thus,
TNF-
␣
levels were 482 ⫾70 and 1259 ⫾26 pg/ml upon SP-A and
LPS treatment, respectively (p⬍0.001 when compared with rest-
ing cells; n⫽3). Denaturation of SP-A by boiling reduces this
activity by ⬃95% (data not shown). The heat-sensitive nature of
SP-A as well as the failure of polymyxin B to inhibit its stimula-
tory effect indicates that SP-A-induced cytokine secretion from
macrophages is LPS independent. Because purity of the natural
SP-A is critical to the interpretation of these experiments, we also
assessed the immunostimulatory activity of SP-A that was further
purified by Superose 6 gel filtration chromatography (6 million
m.w. cut off), a method that excludes lower and high molecular
components such as surfactant protein-D. Upon incubation of
BMDM with 10
g/ml of the purified SP-A in the presence of 20
FIGURE 1. SP-A stimulates murine mac-
rophage cytokine secretion. BMDM from
C3H/HeOuJ mice were stimulated by LPS (1
g/ml) or increasing doses of SP-A (2.5–20
g/ml), in the presence (⫹) or absence (⫺)
of 20
g/ml polymyxin B sulfate. After 6 or
24 h, BMDM supernatants were collected
and TNF-
␣
(A) and IL-10 (B) concentrations
were determined by ELISA, respectively. ⴱ,
Values are significantly different from non-
treated cells (pis ⬍0.05). Results are ex-
pressed as the mean ⫾SD and are represen-
tative of four distinct experiments performed
in triplicate.
5990 CUTTING EDGE: SP-A STIMULATORY ACTIVITY REQUIRES TLR4
g/ml polymyxin B, TNF-
␣
levels increased up to 264 ⫾49 pg/ml
(n⫽3). This value is consistent with the TNF-
␣
release (263 ⫾51
pg/ml; Fig. 1A) observed in supernatants of BMDM treated with
SP-A purified using the modified protocol of Suwabe et al. (18)
alone.
SP-A activates the NF-
B signaling pathway through TLR4
Based on the common ability of SP-A and LPS to induce NF-
B-
dependent signals leading to similar immune cell responses, we
hypothesized that SP-A and LPS may share a common stimulatory
pathway. In that context, we evaluated whether SP-A could be an
endogenous ligand of the TLR4 complex. Thus, we determined
whether SP-A could activate TLR4 in a CHO-K1 cell line trans-
fected with expression plasmids for TLR4 and CD14, and the re-
porter construct NF-
B-dependent pELAM1-CD25. This cell line
was challenged with 10
g/ml LPS-free SP-A supplemented with
polymyxin B or with 1
g/ml LPS as a positive control. SP-A
induced a significant CD25 expression (p⬍0.01, n⫽3) that was
⬃70% of the LPS response (Fig. 2). In comparison, 10
g/ml
SP-A did not induce any NF-
B-dependent CD25 expression in a
control cell line that was not transfected with TLR4 and CD14
expression plasmids. By contrast, this control cell line was
strongly responsive to 50 ng/ml murine IL-1

, under the same
experimental conditions (CD25 expression was increased 6.3 ⫾
0.5-fold over basal values measured on resting cells; n⫽3; p⬍
0.01; data not shown).
Cytokine production by SP-A-stimulated macrophages is TLR4
dependent
SP-A-induced cytokine secretion was analyzed using BMDM iso-
lated from the LPS nonresponsive C3H/HeJ mice, carrying an in-
activating mutation in the tlr4 gene (23). We compared this effect
to that observed using BMDM isolated from C3H/HeOuJ mice, a
genetically related but TLR4-sufficient murine strain. Strikingly,
SP-A-induced IL-10 secretion was markedly reduced in TLR4-
deficient BMDM, in comparison with control BMDM (270 ⫾164
vs 2079 ⫾131 pg/ml; n⫽3; p⬍0.001; Fig. 3). A similar pattern
was observed when TNF-
␣
secretion was examined (data not
shown). The BMDM from C3H/HeJ did not appear to be globally
refractory to inflammatory stimuli, because cell activation was not
reduced when BMDM were challenged with a specific TLR9 li-
gand, i.e., ODN1668, a CpG ODN derived from a mycobacterial
sequence (24). As expected, the corresponding nonactive ODN did
not trigger any cytokine secretion (n⫽3, Fig. 3).
Discussion
The lung is protected by innate and adaptive immune mechanisms
as well as by a unique local immunoregulatory system, i.e., pul-
monary surfactant. Surfactant modulates several inflammatory pro-
cesses including cell proliferation and the release of inflammatory
mediators. The lipid components of surfactant appear to have pre-
dominantly immunosuppressive effects, but the hydrophilic surfac-
tant protein, SP-A, has been reported to exhibit both immuno-
stimulatory and immunosuppressive activity (25). Thus, in vitro
studies by McIntosh et al. (5) and Sano et al. (6) suggest that SP-A
can inhibit LPS-induced TNF-
␣
production by alveolar macro-
phages. These results, which are contradictory with previous data,
may be explained by the primary activation state of these leuko-
cytes (7) and/or the method of SP-A purification. A role of SP-A
in the down-regulation of pulmonary inflammation has also been
suggested using mice deficient in this protein, which have in-
creased inflammatory cytokine in response to an infectious insult
compared with wild-type mice (8, 9). However, it is of note that
this anti-inflammatory effect of SP-A is not observed in a murine
model of sepsis-induced lung injury (26).
In this work we investigated the effect of human SP-A on the
production of cytokines by two well-defined populations of human
and murine macrophages. We show that SP-A, at a concentration
likely to be found in the alveolar spaces (11), induces the secretion
of immunoregulatory molecules such as TNF-
␣
and IL-10. These
observations suggest that, under normal in vivo conditions, the
inhibitory effects of the lipids prevail. However, in some lung dis-
eases or after certain insults, the balance between the inhibitory
and stimulatory influences may be disrupted and result in inflam-
matory injury (27). In that regard, an increased level of SP-A has
been found in pulmonary lavages from patients with pneumonitis,
asbestosis, or exposure to hyperoxic conditions. Interestingly,
these pathological conditions are characterized by elevated levels
of cytokines and the presence of an inflammatory state (28). One
could speculate that these changes are the result of an increased
level of SP-A.
FIGURE 2. SP-A activates the NF-
B signaling pathway through
TLR4. CHO-K1 cells transfected with expression plasmids for human
TLR4 and human CD14, and the reporter construct pELAM1-CD25 were
stimulated by LPS (1
g/ml) or SP-A (10
g/ml). After 24 h, CD25 ex-
pression was determined by flow cytometry. Results are expressed as the
fold increase in median fluorescence intensity over basal value measured
on nontreated cells. ⴱ, Values are significantly different from nontreated
cells (pis ⱕ0.05). Results are mean ⫾SD of three distinct experiments.
FIGURE 3. Cytokine production by SP-A-stimulated macrophages is
TLR4 dependent. BMDM from C3H/HeOuJ and C3H/HeJ mice were stim-
ulated by SP-A (10
g/ml) and the CpG ODN1668 and the corresponding
control ODN (ODN Ctrl, 1
M). ⴱ, Value is significantly different from
wild-type BMDM (p⬍0.001). Results are expressed as the mean ⫾SD
and are representative of three to four distinct experiments performed in
triplicate.
5991The Journal of Immunology
During the course of our study, we compared SP-A- and LPS-
induced immunostimulatory activity and found that both mediators
stimulate the expression of the same cytokines. LPS contamination
could not account for SP-A effects because the neutralizing mol-
ecule polymyxin B was systematically added to all SP-A samples
and the protein denaturing heat treatment suppressed SP-A activ-
ity. In addition, LPS-free SP-A was efficient in activating the
NF-
B signaling pathway through TLR4.
The finding that SP-A induces cell-specific functions led to the
search for its functional receptor. Kuroki and colleagues (29, 30)
recently reported that SP-A binds to both CD14 and TLR2 and that
this interaction likely contributes to the ability of SP-A to affect
LPS- and peptidoglycan-mediated cell responses, respectively.
CD14 lacks an intracellular domain necessary for signal transduc-
tion and is believed to present various pathogen-associated molec-
ular patterns to signal-transducing molecules of the TLR family
(15). Although SP-A may directly bind to the extracellular domain
of TLR2, it does not induce activation of the major downstream
signaling NF-
B pathway (29). Our data indicate that SP-A-in-
duced activation of the NF-
B pathway and up-regulation of cy-
tokine synthesis are strongly dependent on a TLR4 functional
complex.
Although the detailed mechanism by which SP-A interacts with
this receptor remains to be determined, the results presented in this
work argue that TLR4 is a pattern recognition receptor that signals
in response to both foreign pathogens and host endogenous medi-
ators. This finding is consistent with recent studies showing that
fibrinogen and heat shock protein 60 are putative ligands of the
TLR4 complex (16, 17).
In summary, the data presented in this report demonstrate that
cell activation by the lung SP-A is mediated by TLR4 and support
the concept that SP-A may be an important mediator of
inflammation.
References
1. Khubchandani, K. R., and J. M. Snyder. 2001. Surfactant protein A (SP-A): the
alveolus and beyond. FASEB J. 15:59.
2. Mason, R. J., K. Greene, and D. R. Voelker. 1998. Surfactant protein A and
surfactant protein D in health and disease. Am. J. Physiol. 275:L1.
3. Shepherd, V. L., and J. P. Lopez. 2001. The role of surfactant-associated protein
A in pulmonary host defense. Immunol. Res. 23:111.
4. Crouch, E., and J. R. Wright. 2001. Surfactant proteins A and D and pulmonary
host defense. Annu. Rev. Physiol. 63:521.
5. McIntosh, J. C., S. Mervin-Blake, E. Conner, and J. R. Wright. 1996. Surfactant
protein A protects growing cells and reduces TNF-
␣
activity from LPS-stimu-
lated macrophages. Am. J. Physiol. 271:L310.
6. Sano, H., H. Sohma, T. Muta, S. Nomura, D. R. Voelker, and Y. Kuroki. 1999.
Pulmonary surfactant protein A modulates the cellular response to smooth and
rough lipopolysaccharides by interaction with CD14. J. Immunol. 163:387.
7. Stamme, C., E. Walsh, and J. R. Wright. 2000. Surfactant protein A differentially
regulates IFN-
␥
- and LPS-induced nitrite production by rat alveolar macro-
phages. Am. J. Respir. Cell Mol. Biol. 23:772.
8. Harrod, K. S., B. C. Trapnell, K. Otake, T. R. Korfhagen, and J. A. Whitsett.
1999. SP-A enhances viral clearance and inhibits inflammation after pulmonary
adenoviral infection. Am. J. Physiol. 277:L580.
9. Borron, P., J. C. McIntosh, T. R. Korfhagen, J. A. Whitsett, J. Taylor, and
J. R. Wright. 2000. Surfactant-associated protein A inhibits LPS-induced cyto-
kine and nitric oxide production in vivo. Am. J. Physiol. 278:L840.
10. Song, M., and D. S. Phelps. 2000. Comparison of SP-A and LPS effects on the
THP-1 monocytic cell line. Am. J. Physiol. 279:L110.
11. Koptides, M., T. M. Umstead, J. Floros, and D. S. Phelps. 1997. Surfactant
protein A activates NF-
B in the THP-1 monocytic cell line. Am. J. Physiol.
273:L382.
12. Golenbock, D. T., and M. J. Fenton. 2001. Extolling the diversity of bacterial
endotoxins. Nat. Immunol. 2:286.
13. Lien, E., T. K. Means, H. Heine, A. Yoshimura, S. Kusumoto, K. Fukase,
M. J. Fenton, M. Oikawa, N. Qureshi, B. Monks, et al. 2000. Toll-like receptor
4 imparts ligand-specific recognition of bacterial lipopolysaccharide. J. Clin. In-
vest. 105:497.
14. Means, T. K., D. T. Golenbock, and M. J. Fenton. 2000. Structure and function
of Toll-like receptor proteins. Life Sci. 68:241.
15. Akira, S., K. Takeda, and T. Kaisho. 2001. Toll-like receptors: critical proteins
linking innate and acquired immunity. Nat. Immunol. 2:675.
16. Ohashi, K., V. Burkart, S. Flohe, and H. Kolb. 2000. Cutting edge: heat shock
protein 60 is a putative endogenous ligand of the Toll-like receptor-4 complex.
J. Immunol. 164:558.
17. Smiley, S. T., J. A. King, and W. W. Hancock. 2001. Fibrinogen stimulates
macrophage chemokine secretion through Toll-like receptor 4. J. Immunol. 167:
2887.
18. Suwabe, A., R. J. Mason, and D. R. Voelker. 1996. Calcium dependent associ-
ation of surfactant protein A with pulmonary surfactant: application to simple
surfactant protein A purification. Arch. Biochem. Biophys. 327:285.
19. Salez, L., V. Balloy, N. van Rooijen, M. Lebastard, L. Touqui, F. X. McCormack,
and M. Chignard. 2001. Surfactant protein A suppresses lipopolysaccharide-in-
duced IL-10 production by murine macrophages. J. Immunol. 166:6376.
20. Delude, R. L., A. Yoshimura, R. R. Ingalls, and D. T. Golenbock. 1998. Con-
struction of a lipopolysaccharide reporter cell line and its use in identifying mu-
tants defective in endotoxin, but not TNF-
␣
, signal transduction. J. Immunol.
161:3001.
21. Cooperstock, M. S. 1974. Inactivation of endotoxin by polymyxin B. Antimicrob.
Agents Chemother. 6:422.
22. Ways, D. K., W. Qin, T. O. Garris, J. Chen, E. Hao, D. R. Cooper, S. J. Usala,
P. J. Parker, and P. P. Cook. 1994. Effects of chronic phorbol ester treatment on
protein kinase C activity, content, and gene expression in the human monoblas-
toid U937 cell. Cell Growth Differ. 5:161.
23. Beutler, B., and A. Poltorak. 2001. The sole gateway to endotoxin response: how
LPS was identified as Tlr4, and its role in innate immunity. Drug Metab. Dispos.
29:474.
24. Bauer, S., C. J. Kirschning, H. Hacker, V. Redecke, S. Hausmann, S. Akira,
H. Wagner, and G. B. Lipford. 2001. Human TLR9 confers responsiveness to
bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci.
USA 98:9237.
25. Arias-Diaz, J., I. Garcia-Verdugo, C. Casals, N. Sanchez-Rico, E. Vara, and
J. L. Balibrea. 2000. Effect of surfactant protein A (SP-A) on the production of
cytokines by human pulmonary macrophages. Shock 14:300.
26. Malloy, J. L., R. A. Veldhuizen, F. X. McCormack, T. R. Korfhagen,
J. A. Whitsett, and J. F. Lewis. 2002. Pulmonary surfactant and inflammation in
septic adult mice: role of surfactant protein A. J. Appl. Physiol. 92:809.
27. Phelps, D. S. 2001. Surfactant regulation of host defense function in the lung: a
question of balance. Pediatr. Pathol. Mol. Med. 20:269.
28. Griese, M. 1999. Pulmonary surfactant in health and human lung diseases: state
of the art. Eur. Respir. J. 13:1455.
29. Murakami, S., D. Iwaki, H. Mitsuzawa, H. Sano, H. Takahashi, D. R. Voelker,
T. Akino, and Y. Kuroki. 2002. Surfactant protein A inhibits peptidoglycan-
induced tumor necrosis factor-
␣
secretion in U937 cells and alveolar macro-
phages by direct interaction with Toll-like receptor 2. J. Biol. Chem. 277:6830.
30. Sano, H., H. Chiba, D. Iwaki, H. Sohma, D. R. Voelker, and Y. Kuroki. 2000.
Surfactant proteins A and D bind CD14 by different mechanisms. J. Biol. Chem.
275:22442.
5992 CUTTING EDGE: SP-A STIMULATORY ACTIVITY REQUIRES TLR4
