From the Thoracic Diseases Research Unit,
Division of Pulmonary, Critical Care, and Internal Medicine, and the
§ Department of Biochemistry and Molecular Biology, Mayo
Clinic and Foundation, Rochester, Minnesota 55905
Received for publication, September 21, 2002, and in revised form, October 31, 2002
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ABSTRACT |
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Infiltration of the lungs with
neutrophils promotes respiratory failure during severe
Pneumocystis carinii (PC) pneumonia. Recent studies have
shown that alveolar epithelial cells (AECs), in addition to
promoting PC attachment, also participate in lung inflammation by the
release of cytokines and chemokines. Herein, we demonstrate that a PC
Pneumocystis carinii
(PC)1 pneumonia remains a
life-threatening opportunistic infection in immunocompromised
hosts. Despite the availability of effective medications, the mortality
from PC pneumonia ranges between 15 and 40% (1). Neutrophilic lung inflammation promotes diffuse alveolar damage and contributes significantly to the respiratory failure, which is characteristic of
severe PC pneumonia (2). The exact mechanisms by which PC induce this
lung inflammation are not fully understood, although recent studies
indicate that P. carinii has recently been classified as a fungal organism
based on RNA homology and evidence that PC assembles a cell wall rich
in Fungal glucans have been shown to bind several different receptors. In
particular, the Histological studies consistently identify P. carinii
organisms closely associated with the alveolar epithelium in infected human and animal tissues. Furthermore, the binding of P. carinii to alveolar epithelial cells is believed to be essential
for establishment of infection (17). However, accumulating evidence
demonstrates that pulmonary alveolar epithelial cells not only function
as a passive barrier mediating gas exchange but also actively
participate in the host immune response. Alveolar epithelial cells are
capable of processing and presenting antigen to T lymphocytes (18, 19). Furthermore, isolated alveolar epithelial cells produce and secrete various cytokines and chemokines, including TNF MIP-2 is of particular interest in the pathogenesis of P. carinii pneumonia, because it represents the rodent homologue of the human CXC chemokine IL-8 and is thus a potent
stimulant of neutrophil accumulation and activation (24-26). Although
chemokine expression is an important component of the inflammatory
response against PC pneumonia, exuberant expression can cause
deleterious effects on respiratory system function. Bronchoalveolar
lavage studies in humans demonstrate that greater degrees of lung
inflammation, as evidenced by increased neutrophil burdens, are
associated with worse oxygenation and increased mortality during
P. carinii pneumonia (2).
The current study was therefore undertaken to determine the extent to
which P. carinii cell wall Materials--
Unless otherwise noted, general reagents were
from Sigma Chemical Co. (St. Louis, MO). P. carinii f. sp.
(formae specialis) carinii was originally obtained
through the American Type Culture Collection (ATCC) and maintained in
our immunosuppressed rat colony. The following antibodies were used:
anti-CD11b (M-19, Santa Cruz Biotechnology Inc., Santa Cruz, CA), FITC
anti-CD45 (OX-1, BD Pharmingen, San Diego, CA), anti-CDw17 (Huly-M13,
Ancell, Bayport, MN),
anti-asialoGM12 (Matreya,
State College, PA). We recently described the purification and
characterization of a Primary Alveolar Epithelial Cell Isolation--
Rat alveolar
epithelial cells were isolated according to the method of Dobbs
et al. (27). Briefly, Sprague-Dawley rats (~250 g) were
anesthetized by an intraperitoneal injection of pentobarbital (100 mg/kg) and exsanguinated by transfection of the inferior vena cava.
Blood was evacuated from the pulmonary vasculature by perfusion with
normal saline. The trachea was isolated, and the lungs were lavaged
multiple times to remove alveolar macrophages. AECs were separated from
the basement membrane by filling and incubating the lungs with porcine
elastase (1500 units/animal) for 20 min in situ. Next, the
lungs were minced, filtered, and centrifuged (130 × g). The collected cells were resuspended in DMEM and
incubated for one additional hour on Petri dishes coated with rat IgG
to remove macrophages by Fc-mediated adherence. The non-adherent cells
were collected, centrifuged (400 × g for 10 min), and
resuspended in DMEM with 10% bovine calf serum, penicillin (50,000 units/liter) and streptomycin (50 mg/liter) solution. Cells were
counted using a standard hemacytometer. Freshly isolated alveolar
epithelial cells (3 × 105 cells/well) were plated on
96-well tissue culture plates and incubated over 48 h (37 °C,
5% CO2), with the media being changed after the initial
24 h. A sample of cells was also stained with FITC anti-CD45
(OX-1, BD Pharmingen) and viewed under fluorescence, to ensure minimal
contamination by macrophages (<5%). These AEC monolayers derived
after 48 h of culture were used to study epithelial cell MIP-2
release following MIP-2 Protein Quantification--
To characterize AEC responses
to the PC MIP-2 mRNA Determination--
To further characterize AEC
expression of MIP-2, ribonuclease protection assays (RPA) were
performed to evaluate steady-state MIP-2 mRNA levels following
stimulation with the P. carinii In Vivo Determination of MIP-2 mRNA
Levels--
Intratracheal instillation of PC Immunoprecipitation and Immunoblot Analysis for
CD11b--
Because the integrin CD11b/CD18 represents a major
well-characterized Effect of Anti-CD11b, Anti-asialoGM1, and Anti-CDw17 on AEC MIP-2
Release--
Recent studies indicate that lactosylceramide (CDw17) may
function as an alternate
To further confirm the specificity of the antibody, in parallel
experiments, anti-CDw17 was also preincubated with solubilized lactosylceramide (LacCer, 1 mg/ml) for 20 min to neutralize the antibody prior to addition to the cells. Immunohistochemistry was
performed to verify that both the anti-CDw17 and the anti-asialoGM1 antibodies specifically interacted with the cultured AECs.
Immunohistochemistry was also performed on AEC monolayers, which had
been digested with endoglycosidase Hf (New England BioLabs,
1000 units/ml, 24 h) to remove sugar modifications of the cellular
surfaces. Finally, to further exclude steric hindrance effects of the
bulky IgM anti-CDw17 antibody, in additional experiments, the IgM was
digested into IgG and rIgG fragments prior to use (ImmunoPure IgM
fragmentation kit, Pierce Inc., Rockford, IL).
Effect of Lactosylceramide Synthesis Inhibition on Glucan-induced
Release of MIP-2 by AECs--
We further investigated whether
reduction of AEC membrane lactosylceramide would reduce MIP-2
production following Statistical Analysis--
All data are expressed as the
mean ± S.E. Differences between groups were determined using
two-tailed Student's t test. Statistical testing was
performed using SPSS software program, with statistical differences
considered significant if p was <0.05.
P. carinii
To further determine the relative potency of the AEC response to PC
P. carinii
Although the AEC preparations consistently contained >95% epithelial
cells, with few remaining macrophages, we further confirmed that
P. carinii P. carinii Alveolar Epithelial Cells Lack the CD11b/CD18 Antibodies to Lactosylceramide (CDw17) Strongly Inhibit MIP-2
Release by AECs Challenged with P. carinii
To further verify the specificity of suppression induced by anti-CDw17
on MIP-2 release by AECs in response to PCBG, additional experiments
were performed to assess the ability of AECs to release MIP-2 following
stimulation with LPS (1.0 µg/ml) in the presence of this antibody
(Fig. 5B). LPS stimulates cells using receptor systems
distinct from those know to interact with
The ability of the anti-CDw17 antibody was significantly, though
partially, reversed by preincubation of the antibody with solubilized
lactosylceramide before being added to the AECs (Fig. 6). To exclude the possibility that the
inhibitory effects were solely related to steric hindrance imparted by
the bulky IgM anti-CDw17 binding to the membrane, the anti-CDw17 (IgM)
was digested into IgG and rIgG fragments. Again, when AECs were
incubated with fragmented anti-CDw17 (IgG and rIgG), significant
decreases in MIP-2 still occurred following Inhibition of Lactosylceramide Production in AECs Decreased MIP-2
Production following Stimulation with PC
Additional experiments we performed to assess the ability of AECs to
release MIP-2 following stimulation with a potent alternative agent,
namely LPS (1.0 µg/ml), in the presence of the glycosphingolipid synthesis inhibitor (Fig. 7B). Again, AECs were potently
stimulated to release MIP-2 following stimulation with LPS, and this
release was not significantly altered by the presence of NB-DNJ. Thus, inhibition of glycosphingolipid synthesis specifically inhibits AEC
release of MIP-2 in response to PCBG, yet the cells remain fully viable
and are able to respond normally to an alternative stimulant such as
LPS, which acts through other receptor pathways.
Despite effective antimicrobial agents, P. carinii
pneumonia continues to inflict a high rate of respiratory failure and
death. Severe PC pneumonia is characterized by intense neutrophilic
lung inflammation leading to impaired gas exchange, diffuse alveolar damage, and respiratory failure. In fact, neutrophil infiltration in
the lungs of patients with PC pneumonia is a stronger predictor of
respiratory failure and death than the organism burden itself (2).
Additional investigations indicate that P. carinii cell wall
It is well established that alveolar macrophages produce and secrete
cytokines and chemokines such as TNF It is important to note that strict precautions were taken to minimize
alveolar macrophage contamination in the AEC isolates used in these
studies. Anti-CD45 immunofluorescence consistently demonstrated fewer
than 5% macrophages in these preparations, and negligible CD11b, a
prominent macrophage marker, was detected by sequential
immunoprecipitation and immunoblotting. Our data further demonstrate
that, on a cell-by-cell basis, AECs secrete greater amounts of MIP-2
than alveolar macrophages. In addition, assuming a 5% contamination of
the AEC with macrophages, that number of macrophages would have only
generated minimal MIP-2 (89 pg/ml) in these cultures (3, 11). Thus, it
is highly unlikely that the substantial levels of MIP-2 produced by
P. carinii Although MIP-2 is an important chemokine involved in lung inflammation,
it is only one of a number of neutrophil-directed cytokines. Our RPA
analysis indicates that AECs also increase expression of TNF Several potential The glycosphingolipid, lactosylceramide (LacCer), is widely distributed
in the plasma membranes of many different cell types, including
alveolar epithelial cells. LacCer has been shown to bind several
different bacteria and yeast (41). Recently, LacCer has been
demonstrated to bind
poly-(1,6)-beta-D-glucopyranosyl-(1,3)-beta-D-glucopyranose glucan, a purified How CDw17 transmits its signal has yet to be elucidated. Accumulating
evidence indicates that glycosphingolipids are concentrated in
microdomains on the plasma membrane surface (44, 45). These microdomains or "rafts" are thought to represent platforms where signaling molecules are concentrated. Interestingly, antibodies to
asialoGM1, another glycosphingolipid on AECs and a known
Pseudomonas pilin receptor, did not result in any
attenuation of MIP-2 production. It might be postulated that ligation
of In summary, our study demonstrates that -glucan rich cell wall isolate (PCBG) stimulates the release of
macrophage inflammatory protein-2 (MIP-2) from isolated AECs through a
lactosylceramide-dependent mechanism. The results
demonstrate that MIP-2 mRNA and protein production is significantly
increased at both early and late time points after PCBG challenge.
Although CD11b/CD18 (Mac-1, CR3) is the most widely studied
-glucan
receptor, we demonstrate that CD11b/CD18 is not present on AECs. This
study instead demonstrates that preincubation of AECs with an antibody
directed against the membrane glycosphingolipid lactosylceramide
(CDw17) results in a significant decrease in MIP-2 secretion.
Preincubation of the anti-CDw17 antibody with solubilized
lactosylceramide reverses this effect. Furthermore, incubation of AECs
with inhibitors of glycosphingolipid biosynthesis, including
N-butyldeoxyno jirimycin and
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-HCl, also results in a significant decrease in AEC MIP-2 production following challenge with PCBG. These data demonstrate that PC
-glucan induces significant production of MIP-2 from AECs and that
CDw17 participates in the glucan-induced inflammatory signaling in lung
epithelial cells during PC infection.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucan components of the organism cell wall drive
neutrophil accumulation during this pneumonia (3).
-glucans (4, 5). Fungal
-glucans consisting of 1,3-linked
-D-glucopyranosyl residues with varying amounts of 1,6-linked
-D-pyranosyl side chains represent essential
structural components of many fungal cell walls (6, 7). Purified
-glucans from various fungal species strongly induce the production
of proinflammatory cytokines and chemokines from inflammatory cells (8-10). Previous studies demonstrate that the PC cyst cell wall contains complex carbohydrates rich in
-glucans, chitin, and a
mannose-rich major surface glycoprotein complex termed gpA (5, 7). We
have recently purified particulate
-glucan from P. carinii and have demonstrated that alveolar macrophages produce tumor necrosis factor-
(TNF
) and macrophage inflammatory
protein-2 (MIP-2) in response to this PC cell wall component (3,
11).
2 integrin CD11b/CD18 (CR3,
Mac-1) has been extensively studied as a major leukocyte membrane
receptor for
-glucans (12, 13). CD11b/CD18 possesses one or multiple lectin binding sites on the C-terminal domain of the CD11b/CD18
subunit capable of interacting with complex carbohydrates (14). Additional studies using leukocytes have further demonstrated that the
cell membrane glycosphingolipid, lactosylceramide (CDw17), also
interacts with
-glucans (15). Interaction of glucans with CDw17 has
also been reported to promote enhancement of the oxidative burst
response and NF-kB activation of neutrophils (16).
and MIP-2, when stimulated with bacterial components like lipopolysaccharide (LPS) or
whole organisms such as Mycobacterium tuberculosis
(20-23).
-glucans, induce the
expression of MIP-2 from primary AECs. We further sought to determine
whether the mechanisms of AEC activation by PC
-glucans was mediated through CD11b/CD18 or through the alternate lactosylceramide
(CDw17)-associated
-glucan receptor. Enhanced local production of
MIP-2 by AECs may significantly promote neutrophilic lung inflammation
that characterizes severe P. carinii pneumonia and
contributes to respiratory failure in this infection.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucan-rich cell wall isolate from P. carinii (3). This isolate was found to contain 95.7% carbohydrate and 4.3% protein by weight using the orcinol-sulfuric acid method and
BCA protein determinations, respectively. A sample of the P. carinii cell wall isolate was largely, though not completely, hydrolyzed with 2 M HCl thereby releasing 82% of its
content as D-glucose measured by the glucose oxidase
method. Thus, the majority of this material represents P. carinii-derived glucose polymer, namely glucans (3). This material
was previously reported to be active in stimulating alveolar
macrophages to release TNF
and was markedly degraded by
-1,3-glucanases, losing most of its stimulatory activity following
digestion (3). The
-glucan-rich P. carinii cell wall
isolate was exhaustively washed with SDS (1%) and polymyxin (10 µg/ml) to remove any contaminating endotoxin. The final wash was
assayed for endotoxin using a modified Limulus amebocyte lysate assay
(Whittaker M.A. Bioproducts, Walkersville, MD) and consistently found
to contain <0.125 unit/ml endotoxin (3).
-glucan stimulation.
-glucan-rich cell wall fraction, varying concentrations
(1-10 × 106 particles/ml) of the PCBG isolate were
used to stimulate the alveolar epithelial cell monolayers. In
preliminary studies, a concentration of (5 × 106
particles/ml) was found to cause optimal cell stimulation. AECs were
then stimulated, and supernatants were collected at 0, 2, 6, 12, 16, or
24 h. A MIP-2 sandwich ELISA (BIOSOURCE,
Camarrilo, CA) was used to determine MIP-2 protein concentrations
released into the media at the different time points. To further
determine the relative potency of MIP-2 release from AECs compared with alveolar macrophages (AMS), and to exclude the possibility that residual macrophages in the AEC preparations significantly contributed to the observed MIP-2 release, identical numbers of AECs and alveolar macrophages (3 × 105 cells/well), as well as the
maximal number of residual alveolar macrophages in the AEC isolates
(5% AMS, equal to 1.5 × 104 cells) were stimulated
with PCBG (5 × 106 particles/ml) for 24 h. MIP-2
release into the medium was again determined by ELISA.
-glucan. Rat alveolar
epithelial cell monolayers (5 × 106 cells/well) were
stimulated with P. carinii
-glucan (5 × 106 particles/well) for 0, 2, 6, and 24 h and MIP-2
RNA was measured by ribonuclease protection assay. To confirm our
findings in primary AEC, and to further exclude the possibility that
MIP-2 was derived from residual alveolar macrophages in the AEC
preparations, MLE-12 cells (ATCC, Rockville, MD), a transformed
alveolar type II cell line, were similarly cultured with identical
concentrations of P. carinii
-glucan at parallel time
points. Total RNA was isolated at the end of each time point using the
RNeasy Mini kit (Qiagen Inc., Valencia, CA). Ribonuclease protection
assays were then performed according to the manufacturer's
instructions (BD Pharmingen Inc., San Diego, CA). Briefly, 15 µg of
RNA was precipitated using ammonium acetate and ethanol. A custom
cytokine template set and the T7 polymerase were used to transcribe
32P-radiolabeled, antisense probes for rat MIP-2, MCP-1,
IL-1
, TNF
, TNF
, IL-3, IL-4, IL-5, Il-10, IL-2, IFN
, L32,
and glyceraldehyde-3-phosphate dehydrogenase. The radioprobes were
hybridized in excess of the target RNA. At the end of the
hybridization, free probe and other single-stranded RNA were digested
with RNase. The RNase-protected probes were purified using
chloroform/isoamyl alcohol/phenol and precipitated with ethanol. The
probes were then resuspended in Laemmli loading buffer, separated on
6% polyacrylamide gels, dried, and exposed to x-ray film.
-glucan was used to
evaluate the in vivo expression of MIP-2 by AECs.
Sprague-Dawley rats were briefly anesthetized, the trachea was isolated
through a neck incision, and 0.5 ml of P. carinii
-glucan-rich cell wall isolate (2 × 107particles/ml) was injected into the lungs. After 24 h, alveolar epithelial cells were isolated from the inoculated rats as
described above. A sample of cells was also stained with FITC anti-CD45 (OX-1, BD Pharmingen) and viewed under fluorescence, to ensure minimal
contamination by macrophages (<5%). RNA was immediately extracted
from the freshly isolated AECs, and RPA were performed to determine
MIP-2 mRNA levels.
-glucan receptor, we evaluated whether it was
present on cultured AECs. AECs (5 × 106) were
cultured over 48 h. Rat macrophages (2 × 106)
were also isolated from the same animals to serve as a reference source
for the CD11b integrin. The cells were extracted with
octyl-thioglucoside (100 mM) containing a protease
inhibitor mixture (Complete Mini, Roche Molecular Biochemicals,
Mannheim, Germany). Lysates were precleared with 1 µg of goat IgG and
20 µl of Protein G-Sepharose for 30 min. Two micrograms of anti-CD11b
(M-19, Santa Cruz Biotechnology) or goat IgG was added to lysates
containing 200 µg of cellular protein and incubated for 2 h. 20 µl of Protein-G Sepharose was then added to the lysates, and the
mixture was incubated an additional hour. Immunoprecipitates were
collected by centrifugation (1000 × g for 5 min),
washed, and resuspended in Laemmli buffer. The immunoprecipitated
products were separated over a 10% Tris-HCl SDS-PAGE gel, and CD11b
was detected by immunoblotting with anti-CD11b using
streptavidin-biotinylated alkaline phosphatase complex detection.
-glucan receptor mediating cellular
activation (16). To test this, freshly isolated AECs (3 × 105) were plated on 96-well plates and cultured for 48 h. Cells were next preincubated for 20 min with anti-CD11b, anti-CDw17,
anti-asialoGM1, or isotype control mouse IgM (Sigma Chemical Co., St.
Louis, MO), and stimulated with the PCBG (5 × 106
particles/ml) for 16 h in the presence of these antibodies at the
indicated concentrations. In an identical fashion, AECs were also
cultured with LPS (1.0 µg/ml), an independent potent stimulant of
epithelial cells. Supernatants were collected after 16 h, and MIP-2 concentrations were determined by ELISA. Cell viability was
confirmed using the Cell Proliferation Kit II (Roche Molecular Biochemicals, Mannheim, Germany). This assay measures the conversion of
sodium-3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT) to a formazan dye through electron coupling in
metabolically active mitochondria using the coupling reagent N-methyldibenzopyrazine methyl sulfate. Only metabolically
active cells are capable of mediating this reaction, which is detected by absorbance of the dye at 450-500 nm.
-glucan challenge. AECs (3 × 105) were cultured in DMEM/F-12 medium containing lipid
free fetal calf serum (Intracel, Issaquah, WA), in the presence of the
glycosphingolipid biosynthesis inhibitors,
N-butyldeoxynojirimycin (NB-DNJ, 200 and 400 µM) or
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-HCl (PDMP, 20 µM) (28-30). Cells were incubated with NB-DNJ,
PDMP, or lipid-free media alone for 72 h and subsequently
stimulated with PC
-glucan (5 × 106 particles/ml)
for 16 h. MIP-2 release was determined by ELISA. Toxicity to the
AECs induced by either NB-DNJ and PDMP was again determined using the
XTT viability assay (Roche Molecular Biochemicals). To further study
the effect of an independent stimulus, AECs were cultured with LPS (1.0 µg/ml), and MIP 2 release determined in the presence or absence of
NB-DNJ (400 µM).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Glucan Cell Wall Isolate Induces the Release of
MIP-2 from Alveolar Epithelial Cells--
AECs responded to
stimulation with P. carinii
-glucan by release of the
chemokine MIP-2. After 6 h of stimulation, MIP-2 production was
significantly increased with concentrations of PCBG as low as 1 × 106 particles/ml, which caused 165 ± 95-fold more
MIP-2 release compared with unstimulated controls (p = 0.003). The response of AECs occurred fairly rapidly after PCBG
stimulation (Fig. 1A). MIP-2
levels after PC
-glucan (5 × 106 particles/ml)
stimulation were significantly increased as early as 2 h post
stimulation. MIP-2 secretion was further enhanced at 6 through 24 h with maximal accumulation of MIP-2 in the media observed at 16 and
24 h. Thus, AECs respond to PCBG stimulation by release of MIP-2
in a graded time-dependent fashion.
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Fig. 1.
MIP-2 production from alveolar epithelial
cells following stimulation with P. carinii
-glucan cell wall isolate. A,
isolated AECs (3 × 105 cells/well in 200 µl) were
stimulated with PCBG (5 × 106 particles/ml) for
0 h (control), and 2, 6, 12, 16, or 24 h, and MIP-2 release
into the medium determined by ELISA. Data points are expressed as
mean ± S.E. (*, p < 0.05 compared with control).
B, to assess the relative potency of MIP-2 release from AECs
compared with alveolar macrophages (AMS), and to exclude the
possibility that residual macrophages in the AEC preparations
significantly contributed to the observed MIP-2 release, identical
numbers of AECs and alveolar macrophages (3 × 105
cells/well), as well as the maximal number of residual alveolar
macrophages in the AEC isolates (equal to 1.5 × 104)
were stimulated with PCBG (5 × 106 particles/ml) for
24 h. MIP-2 release into the medium was determined by ELISA (*,
p < 0.01 compared with 3 × 105
AECs).
-glucan compared with alveolar macrophages, equal numbers of AECs
and macrophages (3 × 105 cells/well) were cultured
with PC
-glucan for 24 h under identical conditions and the
level of MIP-2 in the medium determined by ELISA (Fig. 1B).
Under these conditions, the AECs release 7510 ± 376 pg/ml MIP-2.
In contrast, equal numbers of macrophages released only 2911 ± 532 pg/ml MIP-2 (p < 0.01). Thus, alveolar epithelial cells are a potent source of MIP-2 following challenge with this P. carinii cell wall component. To further exclude the
possibility that residual macrophages present in the AEC isolates were
responsible for the MIP-2 release in response to PC
-glucan, we
determined that the AEC preparations maximally contained 5% residual
macrophages at the time of isolation. This number of macrophages
(1.5 × 104 cells/well) was also concurrently cultured
with PC
-glucan and found to release only 89 ± 117 pg/ml MIP-2
(p < 0.001 compared with the AEC isolates challenged
with PCBG) (Fig. 1B). Thus, it is unlikely that residual
macrophages contributed significantly to the observed MIP-2 release in
the AEC preparations.
-Glucan Stimulates Enhanced Steady-state MIP-2
mRNA in Alveolar Epithelial Cells--
Next, ribonuclease
protection assays (RPAs) were performed to determine whether glucan
stimulation similarly increased MIP-2 RNA expression in primary
alveolar epithelial cells (Fig.
2A). Steady-state MIP-2
mRNA was significantly increased following 2 h of P. carinii
-glucan stimulation compared with unstimulated control.
A doublet hybridization pattern was observed for MIP-2. Further
increases in MIP-2 mRNA were also noted after 6 and 24 h of
stimulation. The total achieved level of MIP-2 RNA expression in AECs
was comparable and exceeded that elicited by 6 h of challenge with
TNF
(100 ng/ml), an established stimulant of MIP-2 expression. RNA
loading was verified by hybridization to the constitutively expressed
L32 and glyceraldehyde-3-phosphate dehydrogenase genes. In addition,
consistent with our prior studies, RPA on primary AECs further
demonstrated an increased level of TNF
and MCP-1 mRNA at both 6 and 24 h after stimulation with P. carinii
-glucan (3, 11).
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Fig. 2.
MIP-2 RNA expression in lung epithelial cells
after stimulation with P. carinii
-glucan. A, ribonuclease protection
assay of primary AEC cultures (5 × 106 cells/well in
3 ml) were performed following stimulation with PC
-glucan-rich cell
wall isolate (5 × 106 particles/well) for 2, 6, or
24 h. Control ("C") denotes unstimulated cultured
AECs. In parallel cultures, AECs were also cultured with TNF
(100 ng/ml), a known stimulant of MIP-2 expression for 6 h.
B, in a parallel fashion, the MLE-12 murine alveolar
epithelial cell line was assessed for chemokine RNA expression
following PCBG challenge for 2 or 4 h compared with unstimulated
control AECs. Again, AECs were also stimulated with TNF
(100 ng/ml),
for 4 h. RNA loading was determined by assessing
glyceraldehyde-3-phosphate dehydrogenase and L32 expression.
-glucan increase MIP-2 expression in MLE-12
cells, an immortalized alveolar epithelial cell line, free of any
macrophage contamination (Fig. 2B). RPA using MLE-12 cells
demonstrated that MIP-2 mRNA was increased 2 and 4 h after PC
-glucan stimulation compared with controls. Interestingly, an
increase in the murine chemokine TCA3 mRNA was also observed
following 2 and 4 h of PCBG stimulation in MLE12 cells. The MLE12
cell line exhibited MIP-2 expression following challenge with PCBG,
providing further confirmation that alveolar epithelial cells can
directly respond to PCBG with release of the MIP-2 chemokine in the
complete absence of macrophages.
-Glucan Stimulates MIP-2 mRNA Expression in
Alveolar Epithelial Cells in Vivo--
We have previously reported
that PC
-glucan induces substantial neutrophilic lung inflammation
and enhanced lung MIP-2 levels. Thus, we next sought to determine
whether PC
-glucan similarly enhanced MIP-2 gene expression in
alveolar epithelial cells following intratracheal instillation in
vivo (0.5 ml containing 1 × 107 PCBG particles).
Twenty-four hours following challenge, alveolar epithelial cells were
isolated from the rats, and MIP-2 expression was analyzed by RPA (Fig.
3). The 24-h time point was chosen as a
steady-state time point at which neutrophil recruitment and cytokine
expression are well established following PC
-glucan challenge in
our previous studies (3). MIP-2 mRNA expression was enhanced in the
PC
-glucan-stimulated animals compared with control animals. Minimal
basal MIP-2 RNA expression was found in the unstimulated controls. As
described above, RNA was immediately isolated after the alveolar cell
isolation procedure, and the AECs were not maintained in culture.
Again, CD45 fluorescence immunostaining of the AEC preparations
consistently confirmed less than 5% contamination with alveolar
macrophages. These data demonstrate that AECs express MIP-2 mRNA
following challenges in the intact host. Under such in vivo
conditions epithelial cells may be acting in direct responses to PC
-glucan, but MIP-2 responses may also be related to macrophage
activity, including the release of factors such as TNF
, which are
known to stimulate AECs.
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Fig. 3.
MIP-2 mRNA expression in alveolar
epithelial cells isolated from mice challenged intratracheally with
PC -glucan cell wall isolate. Twenty-four
hours after intratracheal injection, lungs were removed and alveolar
epithelial cells were immediately isolated. MIP-2 mRNA expression
was measured using RPA. Shown is a representative blot of three
separate experiments. The first lane contains control
unstimulated mouse lung RNA, the second lane contains
+PCBG-stimulated mouse lung RNA.
-Glucan
Receptor--
The integrin CD11b/CD18 is present on leukocytes and
initiates cell activation in response to
-glucans (12-14). Although
no prior reports of the presence of CD11b/CD18 on AECs could be
identified, we sought to independently confirm that this
-glucan
receptor was absent from primary AEC cultures (Fig.
4). Sequential immunoprecipitation and
immunoblot analysis clearly demonstrated that CD11b, although present on alveolar macrophages, was lacking on the alveolar epithelial cells, indicating that an alternate receptor must be mediating MIP-2
activation in response to PC
-glucan. This sequential
immunoprecipitation and immunoblot analysis further verified that
viable alveolar macrophages represented only a negligible component of
the primary AEC isolates.
View larger version (31K):
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Fig. 4.
Alveolar epithelial cells lack the
CD11b/CD18 -glucan receptor. Primary
alveolar epithelial cells and alveolar macrophages were isolated,
membrane proteins were extracted, and immunoprecipitated with
anti-CD11b antibodies. The precipitated products were separated by
SDS-PAGE, transferred, and analyzed with immunoblot using an antibody
recognizing the CD-11b integrin component receptor.
-Glucan--
As
anticipated from the preceding experiments, anti-CD11b did not
significantly inhibit MIP-2 production following stimulation with PC
-glucan, further indicating that AECs must utilize an alternate
-glucan receptor (Fig. 5A).
Lactosylceramide has recently been shown to function as such an
alternate
-glucan receptor (16). In contrast to anti-CD11b,
incubation of AECs with anti-CDw17, a mouse monoclonal IgM antibody,
markedly suppressed MIP-2 production as compared with isotype-matched
IgM non-immune control (Fig. 5A). The maximum degree of
inhibition was found with an antibody concentration of 200 µg/ml. In
contrast, anti-asialo-GM1, an antibody recognizing another
glycosphingolipid found in mammalian membranes, had no discernable
effect on MIP-2 release. Immunohistochemical staining confirmed strong
binding of both the anti-CDw17 and the anti-asialo-GM1 antibodies to
AEC cultures. Staining for both CDw17 and asialo-GM1 was eliminated
following digestion of the cells with endoglycosidase Hf
(data not shown).
View larger version (17K):
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Fig. 5.
Effect of antibodies to lactosylceramide on
MIP-2 release by alveolar epithelial cells incubated with P. carinii -glucan. A, isolated
AECs were incubated with media alone, non-immune mouse IgM, anti-CDw17,
anti-CD11b, or anti-asialoGM1 (200 µg/ml each) for 30 min before and
throughout a subsequent 16 h challenge with the PC
-glucan-rich
cell wall isolate (5 × 106 particles/ml). MIP-2
release under these conditions was determined by ELISA. B,
to further verify the specificity of anti-CDw17 on suppression of MIP-2
release by AECs in response to P. carinii
-glucan,
epithelial cells were stimulated in parallel with either PC
-glucan
(5 × 106 particles/ml) or LPS (1.0 µg/ml) in the
presence or absence of anti-CDw17 (200 µg/ml) and MIP-2 release
assessed by ELISA. Data points are expressed as mean ± S.E. (*,
p < 0.05, compared with control AECs incubated with
glucan alone).
-glucans (3). Notably,
AECs were well stimulated to release MIP-2 following challenge with
LPS, and this response was not altered by the presence of anti-CDw17.
Thus, antibodies to lactosylceramide (CDw17) specifically inhibit the
responses of AECs to PC
-glucan, yet the cells remain viable and
able to respond normally to an alternative stimulant such as LPS.
-glucan stimulation,
comparable to the effects of the whole IgM anti-CDw17 antibody (Fig.
6). Taken together, these data strongly implicate that lactosylceramide
(CDw17) participates in the membrane complex mediating AECs activation
to release MIP-2 following PCBG stimulation.
View larger version (14K):
[in a new window]
Fig. 6.
Incubation of AECs with either whole IgM or
IgG/rIgG anti-CDw17 both significantly decrease MIP-2 production
following PCBG stimulation. AECs were incubated with 200 µg/ml
either whole IgM anti-CDw17 or digested anti-CDw17 IgM (IgG and rIgG)
during challenge with PC -glucan and MIP-2 release determined by
ELISA. In parallel, anti-CDw17 (IgM) was preincubated with solubilized
lactosylceramide. Data points are expressed as mean ± S.E. (*,
p < 0.05, compared with control AECs incubated with
glucan alone; **, p < 0.05 comparing IgM anti-CDw17
and anti-CDw17 preincubated with lactosylceramide).
-Glucan--
A number of
agents have been produced that inhibit the synthesis of
glycosphingolipids (28-30). Such compounds may represent novel means
to alter P. carinii-induced lung inflammation. To test this,
cellular generation of lactosylceramide (CDw17) in AECs was inhibited
with NB-DNJ and PDMP, two potent inhibitors of glycosphingolipid
synthesis (Fig. 7A). After
72-h incubation with NB-DNJ (400 µM), there was
significantly reduced MIP-2 release following
-glucan stimulation
compared with control cells incubated in lipid-free media alone.
Similarly, incubation of AECs with PDMP (20 µM) also
resulted in significantly decreased levels of MIP-2 production from
AECs following P. carinii glucan challenge. Neither NB-DNJ
nor PDMP altered cellular viability as measured by the XTT
viability assay.
View larger version (16K):
[in a new window]
Fig. 7.
Effect of glycosphingolipid biosynthesis
inhibitors on -glucan induced MIP-2 generation
from alveolar epithelial cells. A, AECs were incubated
with either PDMP (20 µM) or NB-DNJ (200-400
µM) for 72 h, challenged with PC
-glucan for a
subsequent 18 h, and MIP-2 release determined. B, to
further verify specificity of the effect of inhibiting
glycosphingolipid synthesis on suppression of MIP-2 release by AECs,
epithelial cells were stimulated in parallel with either PC
-glucan
(5 × 106 particles/ml) or LPS (1.0 µg/ml) in the
presence or absence of NB-DNJ (400 µM). Data points are
expressed as mean ± S.E. (*, p < 0.05 compared
with AECs incubated with PC
-glucan alone).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucans strongly stimulate neutrophilic lung inflammation (3, 11).
Nonspecific anti-inflammatory therapies, such as adjunctive
corticosteroids, have significantly decreased mortality during severe
PC pneumonia (31). Thus, a better understanding of glucan-induced
inflammation may provide novel means by which lung inflammation can be
selectively modulated during this infection.
and MIP-2, which are potent
inducers of neutrophil migration and activation (3). It is, however,
becoming more evident that alveolar epithelial cells are also active
participants in the host's inflammatory response (20, 21, 32). For
instance, Standiford reported that A549 alveolar epithelial cells
produce significant quantities of IL-8, the human functional homologue
of MIP-2, when stimulated with TNF
(32). However, A549s did not
express IL-8 in response to LPS alone, and these authors postulated the
existence of a cytokine network model whereby alveolar epithelial cells
are stimulated by activated alveolar macrophages (32). In contrast,
primary alveolar epithelial cell cultures have been shown to produce
both TNF
and MIP-2 in direct response to LPS (14). The current study is the first to demonstrate that the
-glucan cell wall fraction from
P. carinii can induce production of significant quantities of the chemokine, MIP-2, from alveolar epithelial cells, further implicating the alveolar epithelium as an important participant in host
inflammation during lung infection.
-glucan stimulation of the AEC cultures were
related to the few residual macrophages in these isolates. Furthermore,
the murine alveolar epithelial cell line, MLE-12, which is free of any
other cell population, also demonstrated a significant increase in
MIP-2 mRNA after PC
-glucan stimulation. In light of the greater
numbers of alveolar epithelial cells in the lung contrasted to
macrophages and other cells, the direct response of AECs to
-glucan
components of the PC cell wall likely represents a significant source
of MIP-2 during this infection. It does, however, remain possible that
macrophages may also release factors that augment epithelial cell
release of chemokines such as MIP-2 during in vivo challenge.
and
MCP-1 following PCBG stimulation. Prior studies by our group and others
have documented that TNF
is essential for optimal clearance of
P. carinii infection (3, 11, 33-35). Additional studies
indicate that MCP-1 is also significantly expressed in rodent lungs
during P. carinii infection and by A549 cells stimulated
with P. carinii major surface glycoprotein (36, 37). We
instead have focused on the stimulatory effects of P. carinii
-glucan, because our prior studies have indicated this
component to principally mediate lung inflammatory responses during
P. carinii infection (3, 11). In addition to observing
enhanced expression of MIP-2, we also observed that TCA3 mRNA
expression was increased after PC
-glucan stimulation in MLE12
cells. TCA3 is an activation-specific cytokine product previously
described to be produced by T cells and Mast cells and to function as a
potent chemotactic agent for neutrophils and macrophages (38, 39). We
believe this is the first demonstration that epithelial cells express
TCA3. These studies strongly implicate lung epithelial cells as an
integral component in the recognition and response of the host to
P. carinii, with epithelial cell-derived MIP-2 serving to
promote neutrophil recruitment during infection. Moreover, because
-glucans are common throughout the pathogenic fungi, this innate
response may represent a generalized response of the epithelium to
fungal infection.
-glucan receptors have been reported in the
literature. The most widely studied is the
2 integrin
CD11b/CD18, which possesses a lectin-binding site on CD11b that has
specificity for
-glucan. This receptor is cross-linked by high
molecular weight soluble and particulate
-glucans leading to the
activation of phagocytic cells (40, 41). The current study
demonstrates, however, that CD11b/CD18 is not present on epithelial
cells to any significant extent and does not function as a glucan
receptor mediating AEC activation. Very recent studies have also
documented an additional
-glucan receptor, namely dectin-1, which is
largely present on macrophages and dendritic cells (42, 43). Its
presence in epithelial and other cells has not yet been established.
Instead, our study instead implicates lactosylceramide as an epithelial cell receptor participating in AEC activation to release MIP-2 in
response to P. carinii
-glucan components.
-glucan derived from the cell wall of
Saccharomyces cerevisiae (15). The binding of PGG-glucan to
LacCer activates a NF-
B-like nuclear transcription factor in human
polymorphonuclear cells (16). Herein, we demonstrate that antibodies to
cell membrane LacCer (CDw17) and inhibitors of LacCer synthesis
significantly attenuate the secretion of MIP-2 after PC
-glucan
challenge in alveolar epithelial cells. The glycosphingolipid
biosynthesis inhibitors N-butyldeoxynojirimycin (NB-DNJ)
inhibits the first step in glycosphingolipid biosynthesis, where
glucose is transferred to ceramide. Culture of cells with NB-DNJ
causes a depletion of glycosphingolipids, including LacCer (28, 29). In
contrast, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol
(PDMP) is an inhibitor of glycosyl transferase (29). Incubation of AECs
with either NB-DNJ or PDMP causes a significant decrease in MIP-2
release after stimulation with PC glucan. Such compounds might
represent a novel means to similarly modulate P. carinii-induced inflammation in vivo.
-glucan in microdomains may result in concerted uptake of
-glucan particles and recruitment and activation of signaling
kinases within these microdomains.
-glucan components of the
P. carinii cell wall are able to stimulate alveolar
epithelial cells to produce MIP-2. These investigations further
demonstrate that the membrane glycosphingolipid lactosylceramide
participates in the receptor complex mediating PC
-glucan activation
of AECs. MIP-2 produced by AECs and macrophages significantly promotes neutrophilic lung inflammation during severe Pneumocystis
pneumonia. Emerging strategies to attenuate glucan-mediated
inflammatory signaling, including inhibition of lactosylceramide-based
signaling, may hold therapeutic benefit to reduce lung injury during
P. carinii pneumonia.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Zvezdana Vuk-Pavlovic, Robert Vassallo, and David Marks for many helpful discussions. We also thank Kathy Streich for assistance during the final preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL55934, HL57125, and HL62150 (to A. H. L.) and GM22942 (to R. E. P.) and a grant from the Robert N. Brewer Family Foundation to Thoracic Disease Research Unit.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.
¶ To whom correspondence should be addressed: Thoracic Diseases Research Unit, 8-24 Stabile Bldg., Mayo Clinic and Foundation, Rochester, MN 55905.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209715200
2
GM1,
Gal1
3GalNAc
1
4(NeuAc
2
3)Gal
1
4Glc
1-Cer.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PC, P.
carinii;
TNF, tumor necrosis factor;
MIP-2, macrophage
inflammatory protein-2;
LPS, lipopolysaccharide;
IL-8, interleukin-8;
FITC, fluorescein isothiocyanate;
DMEM, Dulbecco's modified Eagle's
medium;
PCBG, PC -glucan rich cell wall isolate;
ELISA, enzyme-linked immunosorbent assay;
NB-DNJ, N-butyldeoxynojirimycin;
AMS, alveolar macrophages;
RPA, ribonuclease protection assays;
XTT, sodium-3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic
acid hydrate;
LacCer, lactosylceramide;
PDMP, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-HCl;
rIgG, reverse immunoglobulin G.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Thomas, C. F., Jr., and Limper, A. H. (1998) Semin. Respir. Infect. 13, 289-295[Medline] [Order article via Infotrieve] |
2. | Limper, A. H., Offord, K. P., Smith, T. F., and Martin, W. J., 2nd (1989) Am. Rev. Respir. Dis. 140, 1204-1209[Medline] [Order article via Infotrieve] |
3. |
Vassallo, R.,
Standing, J. E.,
and Limper, A. H.
(2000)
J. Immunol.
164,
3755-3763 |
4. | Edman, J. C., Kovacs, J. A., Masur, H., Santi, D. V., Elwood, H. J., and Sogin, M. L. (1988) Nature 334, 519-522[CrossRef][Medline] [Order article via Infotrieve] |
5. | Matsumoto, Y., Matsuda, S., and Tegoshi, T. (1989) J. Protozool. 36, 21S-22S[Medline] [Order article via Infotrieve] |
6. |
Kollar, R.,
Petrakova, E.,
Ashwell, G.,
Robbins, P. W.,
and Cabib, E.
(1995)
J. Biol. Chem.
270,
1170-1178 |
7. | Nollstadt, K. H., Powles, M. A., Fujioka, H., Aikawa, M., and Schmatz, D. M. (1994) Antimicrob. Agents Chemother. 38, 2258-2265[Abstract] |
8. | Abel, G., and Czop, J. K. (1992) Int. J. Immunopharmacol. 14, 1363-1373[CrossRef][Medline] [Order article via Infotrieve] |
9. | Castro, M., Morgenthaler, T. I., Hoffman, O. A., Standing, J. E., Rohrbach, M. S., and Limper, A. H (1993) Am. J. Respir. Cell Mol. Biol. 9, 73-81[Medline] [Order article via Infotrieve] |
10. | Olson, E. J., Standing, J. E., Griego-Harper, N., Hoffman, O. A., and Limper, A. H. (1996) Infect. Immun. 64, 3548-3554[Abstract] |
11. |
Hoffman, O. A.,
Standing, J. E.,
and Limper, A. H.
(1993)
J. Immunol.
150,
3932-3940 |
12. | Thornton, B. P., Vetvicka, V., Pitman, M., Goldman, R. C., and Ross, G. D. (1996) J. Immunol. 156, 1235-1246[Abstract] |
13. | Ross, G. D. (2000) Crit. Rev. Immunol. 20, 197-222[Medline] [Order article via Infotrieve] |
14. |
Vetvicka, V.,
Thornton, B. P.,
and Ross, G. D.
(1996)
J. Clin. Invest.
98,
50-61 |
15. |
Zimmerman, J. W.,
Lindermuth, J.,
Fish, P. A.,
Palace, G. P.,
Stevenson, T. T.,
and DeMong, D. E.
(1998)
J. Biol. Chem.
273,
22014-22020 |
16. | Wakshull, E., Brunke-Reese, D., Lindermuth, J., Fisette, L., Nathans, R. S., Crowley, J. J., Tufts, J. C., Zimmerman, J., Mackin, W., and Adams, D. S. (1999) Immunopharmacology 41, 89-107[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Limper, A. H.,
Edens, M.,
Anders, R. A.,
and Leof, E. B.
(1998)
J. Clin. Invest.
101,
1148-1155 |
18. | Kallenberg, C. G., Schilizzi, B. M., Beaumont, F., Poppema, S., De, Leij, L., and The, T. H. (1987) Clin. Exp. Immunol. 67, 182-190[Medline] [Order article via Infotrieve] |
19. | Schneeberger, E. E., DeFerrari, M., Skoskiewicz, M. J., Russell, P. S., and Colvin, R. B. (1986) Lab. Invest. 55, 138-144[Medline] [Order article via Infotrieve] |
20. |
Standiford, T. J.,
Kunkel, S. L.,
Phan, S. H.,
Rollins, B. J.,
and Strieter, R. M.
(1991)
J. Biol. Chem.
266,
9912-9918 |
21. |
Xavier, A. M.,
Isowa, N.,
Cai, L.,
Dziak, E.,
Opas, M.,
McRitchie, D. I.,
Slutsky, A. S.,
Keshavjee, S. H.,
and Liu, M.
(1999)
Am. J. Respir. Cell Mol. Biol.
21,
510-520 |
22. |
Lin, Y.,
Zhang, M.,
and Barnes, P. F.
(1998)
Infect. Immun.
66,
1121-1126 |
23. |
Wickremasinghe, M. I.,
Thomas, L. H.,
and Friedland, J. S.
(1999)
J. Immunol.
163,
3936 |
24. |
Wolpe, S. D.,
and Cerami, A.
(1989)
FASEB J.
3,
2565-2573 |
25. | Schmal, H., Shanley, T. P., Jones, M. L., Friedl, H. P., and Ward, P. A. (1996) J. Immunol. 156, 1963-1972[Abstract] |
26. | Driscoll, K. E., Hassenbein, D. G., Howard, B. W., Isfort, R. J., Cody, D., Tindal, M. H., Suchanek, M., and Carter, J. M. (1995) J. Leukocyte Biol. 58, 359-364[Abstract] |
27. | Dobbs, L. G., Gonzalez, R., and Williams, M. C. (1986) Am. Rev. Respir. Dis. 134, 141-145[Medline] [Order article via Infotrieve] |
28. | Andersson, U., Butters, T. D., Dwek, R. A., and Platt, F. M. (2000) Biochem. Pharmacol. 59, 821-829[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Platt, F. M.,
Reinkensmeier, G.,
Dwek, R. A.,
and Butters, T. D.
(1997)
J. Biol. Chem.
272,
19365-19372 |
30. | Kovacs, P., Pinter, M., and Csaba, G. (2000) Cell Biochem. Funct. 18, 269-280[CrossRef][Medline] [Order article via Infotrieve] |
31. | Bozzette, S. A., Sattler, F. R., Chiu, J., Wu, A. W., Gluckstein, D., Kemper, C., Bartok, A., Niosi, J., Abramson, I., and Coffman, J. (1991) N. Engl. J. Med. 323, 1451-1457[Abstract] |
32. | Standiford, T. J., Kunkel, S. L., Basha, M. A., Chensue, S. W., Lynch, J. P., 3rd, Toews, G. B., Westwick, J., and Strieter, R. M. (1990) J. Clin. Invest. 86, 1945-1953[Medline] [Order article via Infotrieve] |
33. | Chen, W., Havell, E. A., and Harmsen, A. G. (1992) Infect. Immun. 60, 1279-1284[Abstract] |
34. | Kolls, J. K., Lei, D., Vazquez, C., Odom, G., Summer, W. R., Nelson, S., and Shellito, J. (1997) Am. J. Respir. Cell Mol. Biol. 16, 112-118[Abstract] |
35. |
Rudmann, D. G.,
Preston, A. M.,
Moore, M. W.,
and Beck, J. M.
(1998)
J. Immunol.
161,
360-366 |
36. |
Wright, T. W.,
Johnston, C. J.,
Harmsen, A. G.,
and Finkelstein, J. N.
(1999)
Infect. Immun.
67,
3452-3460 |
37. | Benfield, T. L., Lundgren, B., Shelhamer, J. H., and Lundgren, J. D. (1999) Eur. J. Clin. Invest. 29, 717-722[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Luo, Y.,
Laning, J.,
and Dorf, M. E.
(1993)
J. Immunol.
150,
971-979 |
39. |
Luo, Y.,
Laning, J.,
Devi, S.,
Mak, J.,
Schall, T. J.,
and Dorf, M. E.
(1994)
J. Immunol.
153,
4616-4624 |
40. |
Yan, J.,
Vetvicka, V.,
Xia, Y.,
Coxon, A.,
Carroll, M. C.,
Mayadas, T. N.,
and Ross, G. D.
(1999)
J. Immunol.
163,
3045-3052 |
41. | Jimenez-Lucho, V., Ginsburg, V., and Krivan, H. C. (1990) Infect. Immun. 58, 2085-2090[Medline] [Order article via Infotrieve] |
42. | Brown, G. D., and Gordon, S. (2001) Nature 413, 36-37[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Willment, J. A.,
Gordon, S.,
and Brown, G. D.
(2001)
J. Biol. Chem.
276,
43818-43823 |
44. | Simons, K., and Ikonen, E. (1997) Nature 387, 569-572[CrossRef][Medline] [Order article via Infotrieve] |
45. | Varma, R., and Mayor, S. (1998) Nature 394, 798-801[CrossRef][Medline] [Order article via Infotrieve] |