Departments of 4 Medicine and 3 Biostatistics and 2 Department of Veterans Affairs Medical Center, University of Iowa, Iowa City, Iowa 52242; and 1 Department of Medicine and 5 Department of Veterans Affairs Medical Center, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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For several decades, the mouse strains C3H/HeJ and C57BL/10ScNCr have been known to be hyporesponsive to endotoxin or lipopolysaccharide (LPS). Recently, mutations in Toll-like receptor (TLR) 4 have been shown to underlie this aberrant response to LPS. To further determine the relationship between TLR4 and responsiveness to LPS, we genotyped 18 strains of mice for TLR4 and evaluated the physiological and biological responses of these strains to inhaled LPS. Of the 18 strains tested, 6 were wild type for TLR4 and 12 had mutations in TLR4. Of those strains with TLR4 mutations, nine had mutations in highly conserved residues. Among the strains wild type for TLR4, the inflammatory response in the airway induced by inhalation of LPS showed a phenotype ranging from very sensitive (DBA/2) to hyporesponsive (C57BL/6). A broad spectrum of airway hyperreactivity after inhalation of LPS was also observed among strains wild type for TLR4. Although the TLR4 mutant strains C3H/HeJ and C57BL/10ScNCr were phenotypically distinct from the other strains with mutations in the TLR4 gene, the other strains with mutations for TLR4 demonstrated a broad distribution in their physiological and biological responses to inhaled LPS. The results of our study indicate that although certain TLR4 mutations can be linked to a change in the LPS response phenotype, additional genes are clearly involved in determining the physiological and biological responses to inhaled LPS in mammals.
Toll-like receptor 4; Toll genes; lipopolysaccharide; asthma
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INTRODUCTION |
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ENDOTOXIN OR
LIPOPOLYSACCHARIDE (LPS), the main component of the cell wall of
gram-negative bacteria, has been shown to elicit an inflammatory and
physiological response that is mediated through the innate immune
system (3). As the first step in host defense against
infection, the innate immune response is characterized by the binding
of bacterial pathogens by pathogen-recognition receptors such as CD14
and Toll proteins (10). Subsequent to the initial
recognition, binding, and signaling, nuclear factor (NF)-B and
activator protein-1 promote transcription of proinflammatory cytokines
[tumor necrosis factor (TNF)-
, interleukin (IL)-1, IL-6, and IL-8]
that serve to enhance the inflammatory response by recruiting and
activating leukocytes (4, 5). In septic shock, this
inflammatory response is amplified to a potentially dangerous level
that can in and of itself have detrimental effects. However, failure to
activate innate immunity can have deleterious outcomes as well, such as
overwhelming bacterial infection. Thus the innate immune response
serves as a sentinel balance to alert the host to bacterial invasion
and modulate the inflammatory response. As such, innate immunity
represents a proximal target for potential intervention in bacterial infections.
Recent evidence has identified Toll-like receptor (TLR) 4 as a major LPS receptor in mammals. This evidence is mainly based on work done in mice. In 1965, the first LPS-hyporesponsive strain, C3H/HeJ, was identified (18) and was subsequently shown to have a mutation in TLR4 (14). This mutation changes a conserved proline to a histidine at position 712 of the open reading frame. In addition, strain C57BL/10ScNCr is also hyporesponsive to LPS. This strain has a deletion on chromosome 4, which encompasses the murine TLR4 locus. Two knockout mouse lines also support a role for TLR4 in the LPS response (6, 8). The knockout lines for both TLR4 and MyD88, a downstream mediator of TLR4 signaling, are unresponsive to LPS (6, 8). The combined genetic evidence from these mouse strains is the strongest evidence yet for a role of TLR4 as a regulator of the response to LPS.
In humans, TLR4 has been associated with LPS signaling. Recently, TLR4 mutations in humans have been associated with a reduced response to inhaled LPS (1). Specifically a mutation at residue 299, which changes a conserved Asp to a Gly, is associated with a reduced response to LPS in vitro (1). However, not everyone with the TLR4 mutations was hyporesponsive to inhaled LPS, and not everyone who was hyporesponsive to inhaled LPS had the TLR4 mutations. These findings suggest that there are genes in addition to TLR4 that determine the response to LPS in humans.
To further evaluate the relationship between TLR4 and LPS responsiveness, we genotyped 18 genetically diverse strains of mice for TLR4 and measured the physiological and biological responses of these strains to inhaled LPS. Mouse strains were chosen from a variety of backgrounds to ensure genetic diversity (2). Twelve of the eighteen strains of mice were found to have mutations in TLR4. Although two mutant strains (C3H/HeJ and C57BL/10ScNCr) were clearly hyporesponsive to inhaled LPS, there was a broad physiological and biological response to inhaled LPS among the remaining mutant and wild-type strains. These findings provide evidence that other genes apart from the TLR4 receptor gene are important determinants of the physiological and biological responses to inhaled LPS.
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MATERIALS AND METHODS |
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In this paper, we present the physiological, biological, and
genotypic results used to determine the role of TLR4 in the response to
inhaled endotoxin in mice. We used a total of 18 mouse strains in our
analysis. These mouse strains were chosen to represent various
evolutionary backgrounds to ensure genetic diversity (2) with respect to potential polymorphisms in the genes, such as TNF-
(7), that affect airway responsiveness and innate
immunity. With regard to the known hyporesponsive strains C3H/HeJ and
C57BL/10ScNCr, the respective parental strains were included as well
(C3H/HeN and C57BL/10ScSn) to ensure that any sign of
hyporesponsiveness to inhaled LPS was not a result of the strain
background. In addition, all strains were genotyped for TLR4 (open
reading frame and splice sites), the major LPS response receptor in
mammals, to avoid any observed LPS hyporesponsiveness that may be due
to a stop codon or deletion in the TLR4 gene. Phenotyping was performed
by measuring airway reactivity to methacholine pre- and postinhalation
of LPS and by evaluating the inflammatory response with ELISA assays performed on lavage fluid. A minimum of four mice were used for the
phenotypic analysis, with the exception of strains SEA/GNJ, A/J, SM/J,
and KK/HiJ in which five mice were used and strain SOD/Ei in which six
mice were used.
TLR4 genotyping. Genomic DNA for each mouse strain was either purchased from Jackson Laboratory or extracted from the kidney according to the standard protocol with the PUREGENE method (Gentra Systems, Minneapolis, MN). Overlapping primers (17) covering the entire TLR4 open reading frame as well as the splice sites were used to amplify the TLR4 genomic DNA. PCRs were done with the PE Taq polymerase kit, including nucleotides and buffer. All PCR products were sequenced in both directions, and the sequences were compared with the mouse TLR4 cDNA in the GenBank database (accession no. AF177767).
Aerosol exposure protocol and monitoring. LPS for aerosolization was purchased as lyophilized purified Escherichia coli 0111:B4 (Sigma, St. Louis, MO). LPS was solubilized in Hanks' balanced salt solution (HBSS) to a concentration of 1 mg/ml, stored at 4°C, and diluted further in HBSS to the appropriate concentration on the day of the experiment. LPS aerosol was generated and directed into a 20-liter glass exposure chamber with a Collison nebulizer (BGI, Waltham, MA). High-efficiency particle apparatus-filtered air was supplied to the nebulizer at a constant pressure of 20 psi. The chamber atmosphere was exchanged at a rate of 0.25-1.0 changes/min, and LPS concentrations were determined by sampling the total chamber outflow. During the exposure, filter samples were taken from the outflow aerosol at regular intervals and assayed to ensure even endotoxin concentrations. Assays for endotoxin content are described in Endotoxin exposures.
Endotoxin exposures. Aerosol exposures were performed in a 20-liter exposure chamber with a Collison nebulizer delivering the endotoxin. The endotoxin concentrations generated by the aerosols during the exposure period were assayed with the chromogenic Limulus amebocyte lysate (LAL) assay (BioWhittaker, Walkersville, MD) with sterile pyrogen-free labware and a temperature-controlled microplate block and microplate reader (405 nm), as previously described (9). Briefly, four separate samples were taken during each 4-h exposure period by drawing air from the exposure chamber through 47-mm binder-free glass microfiber filters (EPM-2000, Whatman, Maidstone, UK) held within a 47-mm stainless in-line air-sampling filter holder (Gelman Sciences, Ann Arbor, MI). Endotoxin was extracted from the filters with pyrogen-free water at room temperature with gentle shaking. The extracts were then serially diluted and assayed for endotoxin with a LAL assay. For this series of experiments, the intended endotoxin concentration used in the exposures was 6 µg/m3. LAL assays on the filters used showed that the endotoxin concentration was in the range of 6-6.9 µg/m3.
Assessment of pulmonary function.
A methacholine challenge test was performed with a whole body
plethysmograph 24 h before and immediately after the inhalation of
LPS. The exposure protocol used in these experiments allowed us to
determine how pulmonary function changes in response to LPS exposure.
Mice were placed in an 80-ml whole body plethysmograph (Buxco
Electronics, Troy, NY) ventilated by bias airflow at 0.2 l/min. This
unit was interfaced with differential pressure transducers, analog-to-digital converters, and computers. The breathing patterns and
pulmonary function of each individual mouse were monitored over time,
and direct measurement was made of respiratory rate, pressure changes
within the plethysmograph and "box flow" (the difference between
the nasal airflow of the animal and the flow induced by thoracic
movement, which varies in the presence of airflow obstruction because
of pulmonary compression). The Buxco system measured both the magnitude
of the box pressure variations and the slope of the box pressure.
Estimates of airway resistance are expressed as enhanced pause
(Penh). Penh = (expiratory time/40% of
relaxation time 1) × peak expiratory flow/peak
inspiratory flow × 0.67. The validity of Penh as a
measure of airway hyperreactivity was examined in a recent publication
(15). Lung function was measured at baseline and after
stimulation with inhaled methacholine (5, 10, and 20 µg/ml) according
to a standard protocol (15).
Whole lung lavage.
At specific time points after the completion of the inhalation exposure
and second pulmonary function test, mice were killed, the chest was
opened, and the lungs were lavaged in situ via PE-90 tubing inserted
into the exposed trachea. A pressure of 25 cmH2O was used
to wash the lungs with 6.0 ml of sterile pyrogen-free saline, 1 ml at a
time. The volume was noted, and the cells were pelleted by
centrifugation for 5 min at 200 g. The supernatant fluid was
frozen at 70°C for subsequent use. The residual pellet of cells was
resuspended and washed twice in HBSS (without Ca or Mg). After the
second wash, a small aliquot of the sample was taken for cell count.
The cells were washed once more and resuspended in RPMI medium to a
final concentration of 1 × 106 cells/ml. The cells
that were present in 10-12 µl of the 1 × 106
ml cell suspension were spun for 5 min onto a glass slide with a
cytocentrifuge (Cytospin-2; Shandon Southern, Sewickley, PA). Standard
staining was carried out with a Diff-Quik stain set (HARLECO, Gibbstown, NY).
Lung lavage fluid.
Murine intercellular adhesion molecule (ICAM)-1 was measured in lung
lavage fluid with an ELISA prepared with a hamster monoclonal antibody
specific for murine ICAM-1 (Endogen, Woburn, MA). The detection limit
of this assay was determined to be 5 ng/ml, and it is specific for
mouse ICAM-1. Commercial murine ELISA kits were used for the
measurement of TNF- and macrophage inflammatory protein (MIP)-2 in
the lavage fluid (R&D Systems, Minneapolis, MN), according to the
manufacturer's instructions. The detection limits for the ELISA assays
were 5 ng/ml for ICAM-1, 23.4 ng/ml for TNF-
, and 7.8 pg/ml for
MIP-2.
Statistical analysis.
Statistical analyses were performed on the inflammatory variables and
pulmonary function variables. The inflammatory variables are based on
cell counts and ELISA measurements of TNF-, MIP-2, and ICAM-1 in the
lavage fluid. The pulmonary function variables measured included
Penh at the methacholine concentrations pre- and post-LPS
exposure indicated in Assessment of pulmonary
function. Previously, (12) the
Penh reading at 20 mg/ml of methacholine has given the best
measure for the effect of LPS exposure on pulmonary function. All
statistical analysis in this paper is therefore based on readings at
the concentration of 20 mg/ml of methacholine and compared with the
baseline (0 mg/ml of methacholine).
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RESULTS |
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Inbred mice differ with respect to their TLR4 genotype.
The genotype analysis showed that a total of six mouse strains had a
common allele for TLR4, whereas the remaining 12 strains had a variety
of point mutations throughout the open reading frame of the
TLR4 gene (Table 1). Of these 12 strains
with TLR4 mutations, 9 had mutations in highly conserved residues. The
results in Table 1 show that Cast/Ei, which has a different
evolutionary origin than the other inbred strains used in this study,
has a large number of unique mutations in the TLR4 coding region,
particularly in the NH2 terminus of the protein. For the
remaining 11 mutant strains, most of the mutations occur in exon 3, the
largest exon of mouse TLR4, and are clustered at the COOH terminus of
the protein. In addition to the mutations in the open reading frame of
the TLR4 gene, we found several mutations in noncoding regions of the
genes such as the 5'-untranslated region and introns. These mutations
are summarized in Table 2. At this point,
it is not known whether any of the strains except C3H/HeJ and
C57BL/10ScNCr express TLR4 protein with altered activity, with either
reduced or increased signaling activity in response to LPS.
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Inbred strains show very different physiological responses to inhaled LPS. We determined the effect of methacholine and LPS inhalation on lung function in 18 different inbred strains of mice. The two known LPS-hyporesponsive strains, C3H/HeJ and C57BL/10ScNCr, when compared with the other strains, were not markedly different in their estimates of airway resistance either before exposure to LPS (Fig. 1A) or after inhaling LPS for 4 h (Fig. 1B). In fact, the pre-LPS estimates of airway resistance for C3H/HeJ and C57BL/10ScNCr were similar to those for other mouse strains such as C57BL/10ScSn and FVB/NJ, which express the common (or wild-type) TLR4 allele. Even after LPS inhalation, the estimates of airway resistance did not reveal any differences between the hyporesponsive strains C3H/HeJ and C57BL/10ScNCr, and several of the remaining 16 strains used in the study (Fig. 1B), such as BALB/c, LP/J, SM/J, A/J, and KK/HiJ, also had similar baseline estimates of airway resistance. Interestingly, most of the strains expressing wild-type TLR4 protein, with the exception of FVB/NJ, had higher airway resistance levels than the hyporesponsive C3H/HeJ strain.
Methacholine-induced airway hyperreactivity revealed interesting strain-dependent variation. Strain C57BL/10ScSn, the wild-type progenitor of the TLR4 deletion mutant C57BL/10ScNCr, was very sensitive to the effect of LPS exposure. Both at baseline and at 20 mg/ml of methacholine, strain C57BL/10ScSn was almost 10 times more sensitive as measured by pulmonary function assay compared with preexposure levels of airway responsiveness. On the other hand, the TLR4 mutant derivative of C57BL/10ScNCr had almost no change in airway responsiveness as a result of either methacholine or LPS inhalation. Strain C3H/BFeJ, in contrast, showed only slightly more sensitivity to both LPS and methacholine compared with the TLR4 mutant C3H/HeJ strain. This potentially implicates strain background and additional polymorphisms in other genes besides TLR4 as the basis for the lack of hyporesponsiveness. In general, the variation in airway responsiveness with respect to inhaled LPS seems to be slightly masked by the addition of methacholine. Ratios for saline baseline exposures were significantly higher than the ratios at 20 mg/ml of methacholine. In addition, the airway hyperreactivity at 20 mg/ml of methacholine showed much less variation both in the TLR4 wild-type strains and in the strains expressing mutated forms of the TLR4 protein (Fig. 2, A and B).The concentration of LPS-induced polymorphonuclear leukocytes in
the airspace is influenced by TLR4 as well as by other genes.
The concentration of neutrophils in the lavage fluid after
inhalation of LPS has previously been used as an indicator of the inflammatory response to LPS (4). As expected, of all 18 strains tested, the hyporesponsive strains C3H/HeJ and C57BL/ 10ScNCr had by far the lowest concentration of polymorphonuclear leukocytes (PMNs) in the lavage fluid (Fig. 3). All
other strains tested had at least two orders of magnitude more
neutrophils in the lavage fluid. The remaining 10 strains with
mutations in TLR4 demonstrated a wide range of LPS-induced airway
inflammation with PMNs, ranging from ~100,000 (BALB/c) to >400,000
(LP/J) cells/ml lavage fluid. There did not appear to be a clear
correlation between the TLR4 genotype of these strains and the
concentration of neutrophils in the airspace because strains with a
shared TLR4 genotype (such as BALB/c and A/J) showed a significant
difference in PMNs per milliliter of lavage fluid (Fig. 3). On the
other hand, the marked difference between the results for C3H/HeJ and
C57BL/10ScNCr mice suggests that the mutations in TLR4 encountered in
strains C3H/HeJ and C57BL/ 10ScNCr, compared with those mutations
found in the other TLR4 mutant strains, had a much more profound effect
on TLR4 function.
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Inbred strains of mice demonstrate clear differences in the release
of specific proteins after inhalation of LPS.
A possible cause for the difference in neutrophil migration could be
the secretion of cellular proteins such as cytokines, chemokines, and
adhesion molecules. All three types of proteins have been implicated in
mediating neutrophil migration in response to inhaled LPS. We therefore
measured the concentration of TNF-, MIP-2, and ICAM-1 in the lavage
fluid after inhalation of LPS. The hyporesponsive strains C3H/HeJ and
C57BL/10ScNCr had the lowest concentrations of TNF-
and MIP-2 of all
the strains tested in this study (P < 0.0001 for both
MIP-2 and TNF-
; Fig. 4, A
and B), suggesting that the lack of TNF-
and MIP-2
secretion may be responsible for the absence of LPS-induced neutrophil
infiltration into the airspace. However, the concentration of ICAM-1 in
the lavage fluid for both of these strains (C3H/HeJ and C57BL/10ScNCr) was not significantly different from that of the other 16 strains tested (P = 0.7), suggesting that the hyporesponsive
phenotype of C3H/HeJ and C57BL/10ScNCr cannot be explained by ICAM-1
(Fig. 4C).
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DISCUSSION |
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Our results suggest that TLR4 is important in the response
to inhaled endotoxin in mice but that additional genes are likely involved in modulation of the complex physiological and biological phenotype. For the 18 mouse strains tested, 6 had a common TLR4 allele
and 12 had mutations throughout the open reading frame of TLR4. The
LPS-hyporesponsive strains (C3H/HeJ and C57BL/10ScNCr) demonstrated a
blunted response to inhaled LPS; however, this response was most
clearly observed in the concentrations of neutrophils, TNF-, and
MIP-2 in the lavage fluid. Surprisingly, mice with the common allele
for TLR4 had substantial variability in their physiological and
biological responses to inhaled endotoxin.
As a major LPS receptor in mammals, TLR4 influences a variety of physiological responses that are an important component of the host defense against bacterial pathogens. Although our data indicate that TLR4 influences the physiological response to inhaled LPS, our findings in mice also suggest that other genes may be important in regulating this response. Mutant strains of mice are less responsive physiologically than strains with the common TLR4 allele. Importantly, these findings parallel results in humans in which our laboratory (1) found that mutations in the TLR4 gene were associated with a blunted response to inhaled LPS in some test subjects. Thus both the results presented in this paper and the findings of our laboratory in humans indicate that TLR4 is important in determining the physiological response to inhaled LPS but that other genes are undoubtedly involved.
The ICAM-1 results in this paper suggest that TLR4 is not involved in
cell adhesion and that changes in cell adhesion may not be part of the
cellular response to inhaled endotoxin. Previous evidence had suggested
a potential involvement of adhesion molecules in mediating the LPS
response in mice. ICAM-1-deficient mice have shown an increased
resistance to inhaled LPS and lack the airway hyperreactivity of
wild-type mice exposed to high doses of LPS (21). In
contrast, TNF- and, to a lesser degree, MIP-2 show a correlation of
increased expression with increased responsiveness to inhaled endotoxin
and suggest a role for both proteins in mediating the LPS response in
mice. This correlates with previous evidence (4) that has
shown both cytokines to be regulated by the transcription factor NF-
B, a protein downstream of TLR4 in the LPS response pathway. TNF-
levels have frequently been used as a measure of LPS
reactivity because both C3H/HeJ and C57BL/10ScNCr mice lack the
increase in TNF-
levels that follows LPS exposure. Our results indicate that TNF-
expression is a reliable indicator for measuring LPS responsiveness in mice.
Besides TLR4, potential genes that would affect cytokine expression and
NF-B translocation include other Toll receptors such as TLR2 that
also signal through NF-
B, as well as downstream molecules in the
LPS-induced signaling cascade such as MyD88 and MD-2, a cofactor
required for TLR4-dependent LPS response (16, 19). The
action of other genes that modulate the LPS response in mammals is less
well characterized. Ran, a G protein (22), and class II
myosin heavy chain molecules (13) have also been indicated
as mediators of the mammalian LPS response.
The distinct phenotype of strains C3H/HeJ and C57BL/10ScNCr, both of which have mutations or deletions in the TLR4 gene, suggests that these particular mutations or deletions in TLR4 may specifically affect neutrophil migration. This would explain the near normal levels of neutrophils in the lavage fluid of the Cast/Ei strain, which has numerous mutations spread throughout the TLR4 open reading frame. Further experiments are necessary to determine whether the hyporesponsive phenotype in C3H/HeJ and C57BL/10ScNCr mice is a result of the specific mutation in TLR4 or additional mutations in other genes. There is evidence that mutations other than those in TLR4 contribute to the lack of LPS response in strain C57BL/10ScNCr (11).
The phenotypic variability for endpoints such as TNF- secretion or
neutrophil infiltration underscores the involvement of such additional
genes in mediating the immune response to LPS. This scenario
would put all such genetic effects downstream of TLR4 and secondary to
the initial recognition of LPS by the TLR4 receptor. On the other hand,
inherent genetic variability in the TNF-
gene could be the
underlying cause for the variability in TNF-
secretion. Naturally
occurring mutations in genes involved in neutrophil migration, such as
CD11 and CD18, could cause a seemingly hypo- or hyperresponsive
phenotype unrelated to the actual TLR4-mediated LPS response. Clearly,
more work is necessary to determine the genetic variations in different
inbred mouse strains. The ongoing mouse genomic sequencing project will
aid in identifying some of the genetic variations in the most commonly used inbred strains.
However, assuming that TLR4 is a key gene in the response to inhaled
LPS, the specific mutation in TLR4 in C3H/HeJ mice should be considered
further. It is worth noting that the TLR4 protein from mouse strain
C3H/HeJ has been found to exert a dominant loss-of-function effect in
vitro (20), suggesting an absolute requirement for the
presence of the proline residue at position 712 of the open reading
frame for protein function. In the same context, mutations in genes
other than TLR4 in strains such as DBA/2J and 129/Svlm could result in
hyperresponsiveness to inhaled LPS, whereas any of the mutations in
TLR4 in hyperresponsive strains such as P/J and NZW/LacJ could
constitute gain-of-function mutations. It nevertheless is surprising to
see the large number of polymorphisms within the TLR4 gene, given that
it is central to the host response to a variety of pathogenic
organisms. Smirnova et al. (17) have reported similar
findings with respect to the highly polymorphic nature of TLR4 in
additional mouse strains as well as in other species. The C3H/HeJ
mutation seems to be crucial in reducing the activity of TLR4 because
the proximal intracellular domain of TLR4 is highly conserved, whereas
the extracellular domain as well as the COOH terminus of the
protein varies significantly between mouse strains and species. The
recent resolution of the Toll/IL-1 cytoplasmic domain crystal structure
(23) seems to support the requirement for a highly
organized protein and would explain the varying degrees of polymorphic
sites in the different protein domains. It is important to note as well
that known polymorphisms in genes such as TNF- (7) have
not been fully characterized for their functional effects, nor have
such genes been sequenced in all of the 18 strains tested in this
study. Only a full understanding of the functional effects of such
polymorphisms in relation to inhaled LPS and to existing polymorphisms
in other genes such as TLR4 will yield a complete genetic explanation
for the varied response to inhaled LPS. Clearly, more research is
necessary to understand the specific role of TLR4 in response to
inhaled LPS in mice and potential involvement of these additional genes
in this complex phenotype. The results of this study present a first attempt to identify potentially useful strains for further study of
this phenotype.
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ACKNOWLEDGEMENTS |
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This study was supported by a Department of Veterans Affairs Merit Review Grant; National Institute of Environmental Health Sciences Grants ES-07498, ES-09607, and ES-011375; and National Heart Lung, and Blood Institute Grants HL-62628, HL-66611, and HL-66604.
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FOOTNOTES |
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Present address of E. Lorenz: Wake Forest Univ. School of Medicine, Sect. of Infectious Diseases, Medical Ctr. Blvd., Winston-Salem, NC 27157-1042.
Address for reprint requests and other correspondence: D. A. Schwartz, Pulmonary and Critical Care Medicine, Duke Univ. Medical Center, Research Drive, Room 275 MSRB, DUMC Box 2629, Durham, NC 27710 (E-mail: david.schwartz{at}duke.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.
Received 13 February 2001; accepted in final form 7 June 2001.
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