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Effects of decreased calmodulin protein on the survival 1
mechanisms of alveolar macrophages during 2
Pneumocystis pneumonia 3
4
5
6
Mark E. Lasbury1*, Pamela J. Durant1, Chung-Ping Liao1, and Chao-Hung Lee1 7
8 1Department of Pathology and Laboratory Medicine 9
Indiana University School of Medicine 10
Indianapolis, IN 46202 11
USA 12
13
Running title: Calmodulin and alveolar macrophage survival 14
Abstract: 183 words 15
Manuscript: 50,740 characters with spaces (including abstract) 16
17
18 *Corresponding author: Mark E. Lasbury, Ph.D. 19
Department of Pathology and Laboratory Medicine 20
Indiana University School of Medicine 21
Fesler Hall, Room 406 22
1120 South Dr 23
Indianapolis, Indiana 46202-5112 24
Tel: (317) 274-4606 25
Fax: (317) 278-0643 26
e-mail: [email protected] 27
28
29
Keywords: Pneumocystis, calmodulin, apoptosis, granulocyte macrophage colony 30
stimulating factor, macrophage, AIDS-related opportunistic infections, innate immunity 31
32
33
Non-standard abbreviations: 34
Pcp, Pneumocystis pneumonia; Amø, alveolar macrophage; BAL, bronchoalveolar 35
lavage; ROS, reactive oxygen species; Dex, dexamethasone; Dex-Pc, dexamethasone-36
treated and Pneumocystis-infected; CD4-depl., CD4+ lymphocyte-depleted; CD4-depl.-37
Pc, CD4-depleted and Pneumocystis-infected 38
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00299-09 IAI Accepts, published online ahead of print on 1 June 2009
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Abstract 1
Pneumocystis infection causes increased intracellular levels of reactive oxygen species 2
(ROS) and the subsequent apoptosis of alveolar macrophages (Amø). Assessments of 3
key pro-survival molecules in Amø and bronchoalveolar lavage fluids from infected rats 4
and mice showed low levels of GM-CSF and reduced activation of the phosphoinositide-5
3 kinase (PI-3K). Protein and mRNA levels of the ubiquitous calcium-sensing protein, 6
calmodulin, were also reduced in Amø during Pneumocystis pneumonia (Pcp). 7
Calmodulin has been implicated in control of GM-CSF production and PI-3K activation 8
in other immune cell types. Experiments to determine the control of GM-CSF and PI-3K 9
by calmodulin in Amø showed that GM-CSF expression and PI-3K activation could not 10
be induced when calmodulin was inhibited. Calmodulin inhibition also led to increased 11
levels of ROS and apoptosis in cells exposed to bronchoalveolar lavage fluids from 12
infected animals. Supplementation of Amø with exogenous calmodulin increased 13
survival signaling via GM-CSF and PI-3K, and reduced ROS and apoptosis. These data 14
support the hypotheses that calmodulin levels at least partially control survival signaling 15
in Amø, and that restoration of GM-CSF or PI-3K signaling will improve host response 16
to the organism. 17
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Introduction 1
The alveolar macrophage (Amø) is an important cell type for the clearance of 2
Pneumocystis organisms from the lungs of animals and humans (33,35,38). Loss of 3
Amø renders animals susceptible to Pneumocystis pneumonia (Pcp) (47), while 4
increased Amø numbers retards progression of the disease (33, Lasbury submitted for 5
publication). Low Amø numbers in animals with Pcp are caused by increased apoptosis, 6
which is related to the catabolism of intracellular polyamines and production of 7
hydrogen peroxide (35,37). Reduced survival pathway signaling and antioxidant 8
expression also contribute to the apoptosis of Amø during Pcp (Lasbury, submitted for 9
publication). Elucidation of the mechanisms of reduced apoptotic resistance is 10
necessary to design immunomodulatory therapies to increase the host response to the 11
organism. 12
Many systems exist in mammalian cells that combat apoptotic stimulation via reactive 13
oxygen species (ROS), including GM-CSF and phosphoinositide kinase 3 (PI-3K). GM-14
CSF has anti-apoptotic and anti-Pneumocystis effects. Previous studies have shown 15
that GM-CSF knockout mice are prone to Pcp (54) and that GM-CSF is involved in the 16
adaptive immune response to Pneumocystis through augmentation in the killing ability 17
of CD8+ T lymphocytes (43) and expansion of CD4+ populations (51). GM-CSF over-18
expression in a CD4+ T lymphocyte-depleted and GM-CSF-/- mouse model of Pcp 19
resulted in less inflammation and reduced infection at four weeks (49), showing that 20
GM-CSF also plays a role in the innate immune response to the organism. 21
Phosphatidylinositol (3,4,5)-triphosphate (PIP3), the product of PI-3K enzymatic 22
activity, mediates Akt-1 (also called protein kinase B, PKB)(1,18,29) activation. Akt-1 23
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controls many pro-survival functions (9,10,11,13,23), making PI-3K activation a lynchpin 1
of survival signaling. Studies indicate that GM-CSF participates in the control of active 2
phospho-PI-3K (or pPI-3K) levels. Induction of PI-3K activation is lost if the cells are not 3
pre-treated with GM-CSF (30), and GM-CSF activates neutrophils via PI-3K (26). 4
Therefore, mechanisms that control GM-CSF production may also control survival 5
signaling. 6
Both GM-CSF expression and PI-3K activation are linked to the ubiquitous calcium-7
sensing molecule, calmodulin. However, calmodulin can both stimulate and inhibit these 8
molecules, depending on the cellular environment. For example, the action of a 9
calmodulin-dependent phosphatase, calcineurin, is required for GM-CSF transcription in 10
T lymphocytes (61), but elimination of a calmodulin-dependent kinase II binding site in 11
the Ets1 transcription factor actually enhanced GM-CSF transcription in T cells (39). 12
Similarly, inhibition of calmodulin prevents PI-3K-mediated phosphorylation of 13
phosphatidylinositol in Chinese hamster ovarian (CHO) cell lysates (24), but calmodulin 14
controls the PI-3K-mediated downstream phosphorylation of Raf1 at Ser338, which is 15
critical for Raf1 activation in green monkey kidney cells (44). The role of calmodulin and 16
the downstream enzymes that are dependent on it in Amø GM-CSF expression and PI-17
3K activation have not been investigated. 18
In the current study, we hypothesized that Amø apoptosis during Pcp involves GM-19
CSF and the calmodulin-mediated mechanisms that control it. We also theorized that 20
changes in calmodulin and GM-CSF levels would affect downstream anti-apoptotic 21
molecules, such as PI3K. We found that GM-CSF, calmodulin, and pPI-3K levels were 22
low in Amø and bronchoalveolar lavage (BAL) fluids from rats and mice with Pcp. A 23
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calmodulin inhibitor reduced Amø expression of GM-CSF and PI-3K activation. Amø 1
incubated with BAL fluids from Pneumocystis-infected animals had higher levels of ROS 2
and apoptosis when calmodulin activity was inhibited. Exogenous calmodulin introduced 3
into the Amø reduced ROS and apoptosis, while also increasing GM-CSF and PI-3K 4
activation. These data indicate that Pcp-induced down-regulation of calmodulin plays a 5
role Amø susceptibility to apoptosis during the infection via GM-CSF and PI-3K. 6
7
Materials and Methods 8
Reagents and antibodies. ELISA for the measurement of total and phospho-PI-3K 9
(Active Motif, Carlsbad, CA) and GM-CSF (R&D Systems, Minneapolis, MN) for rat and 10
mouse was performed on Amø lysates per manufacturers’ instructions. Antibodies 11
against the following proteins were obtained from Abcam (Cambridge, MA): rat and 12
mouse cleaved caspase-3; rat and mouse calmodulin; rat and mouse GM-CSF. Anti-13
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Research 14
Diagnostics (Flanders, NJ). The calmodulin inhibitor, W-7 [N-(6-Aminohexyl)-5-chloro-1-15
naphthalenesulfonamide·HCl], was purchased from Biomol (Plymouth Meeting, PA), 16
and the PI-3K inhibitor, wortmannin, was purchased from Calbiochem (San Diego, CA), 17
while the antioxidant N-acetylcysteine (NAC) and bovine testis calmodulin protein were 18
obtained from Sigma-Aldrich (St. Louis, MO). Caspase-9 inhibitor was purchased from 19
R&D Systems. Chariot reagent for protein transfection was purchased from Active Motif. 20
The fluorescent probe 2’, 7’ dichlorodihydrofluorescein diacetate (H2DCFDA) for 21
assessment of ROS levels, as well as cell culture reagents, were purchased from 22
Invitrogen (Carlsbad, CA). 23
24
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Rodent models of Pcp and isolation of Pc organisms. All rodents (120-140 g 1
Sprague Dawley rats and 18-20g BALB/c mice) used were females, obtained from 2
Harlan (Indianapolis, IN), and were given antibiotics as previously described to prevent 3
extraneous infections (5,34). Mice were immunosuppressed by weekly intraperitoneal 4
injection of 0.3 mg anti-CD4 mAb (clone GK1.5, Harlan, Indianapolis IN), and 5
transtracheally inoculated with purified P. murina organisms as previously described 6
(31,34). Rats were treated continuously with 1.8 mg/liter dexamethasone in drinking 7
water and transtracheally inoculated with P. carinii organisms as previously described 8
(5). Animal studies were approved by the Indiana University Animal Care and Use 9
Committee and carried out under the supervision of veterinarians. Animals were housed 10
in the Indiana University Laboratory Animal Resource Center, an Association for 11
Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved facility. 12
13
Isolation of Amø and bronchoalveolar lavage fluids. Five milliliters of sterile saline 14
was used to lavage each rat (1 ml for mice) as previously described (34). Cells and 15
organisms were removed from the BAL fluids by centrifugation at 300 x g for 10 min, 16
and the clarified BAL fluids were stored at –70˚C until used. Amø were isolated from 17
BAL fluids, identified, and enumerated as previously described (31). Purified Amø were 18
used immediately for protein or RNA isolation, or were incubated in complete medium 19
(RPMI 1640 supplemented with 10% FBS, 1 mM pyruvate, 1% nonessential amino 20
acids, 14 mM glucose, 17.9 mM NaHCO3, 10 mM HEPES, 100 U/ml penicillin, and 0.1 21
mg/ml streptomycin) or BAL fluids, live organisms, heat-killed organisms (1 hr at 96˚C), 22
or chemical modulators for specific time periods in 24 well plates. 23
Calmodulin was introduced into Amø by first complexing the protein (1 µg in 100 µl 24
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phosphate buffered saline, pH 7.6) with 0.6 µl Chariot reagent in 100 µl H2O for 30 min 1
at 25 ˚C and then layering the complex over 1 x106 Amø in a well of a six well plate. 2
Serum-free medium (400 µl) was added to each well, and the plates were incubated for 3
3 hr at 37˚C/5% CO2. Test solutions were then added and incubated overnight. The 4
cells were released from the tissue culture plastic with xylocaine as previously 5
described (35), and harvested by gentle scraping. 6
7
RNA Isolation and Real-time RT-PCR. Total RNA was isolated from Amø using the 8
TRIzol reagent according to manufacturer’s instructions (Invitrogen). RNA concentration 9
and purity were determined by spectrophotometry. First strand cDNA was synthesized 10
from the total RNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) primed 11
by oligo-dT and random primers; 0.2 µg of total RNA was used for each reaction with a 12
total reaction volume of 20 µl. Two microliters of each cDNA product was used for 13
quantitative PCR analysis. Real-time RT-PCR for rat and mouse calmodulin 1, 2, and 3 14
were performed using the Assays-on-DemandTM gene expression kits (Applied 15
Biosystems, Foster City, CA) on a Smartcycler (Cepheid, Sunnyvale, CA). Ribosomal 16
protein S8 (RPS8) mRNA was assayed in an identical manner as a control, since its 17
mRNA levels are not affected by the infection (65). Primers for RPS8 PCR were as 18
described (65). Data were normalized to RPS8 mRNA levels in each sample and were 19
shown as fold increase relative to those of control cells from uninfected animals. 20
21
Flow cytometry. To determine ROS levels in Amø during Pcp, the cells were incubated 22
with 10 µM of H2DCFDA and fluorescence examined. H2DCFDA, a cell-permeable 23
indicator for ROS, is cleaved to a non-fluorescent diol by intracellular esterases and 24
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then oxidized by ROS to a fluorescent form. Cells were analyzed on an Epics 500 flow 1
cytometer (Beckman Coulter, Miami, FL) with a forward and side scatter gate applied to 2
select Amø. The median whole cell FITC fluorescence of oxidized H2DCFDA was 3
captured. 4
5
Immunoblotting. Amø isolated from animals were resuspended in 100 µl cold (4°C) 6
cell lysis buffer (150 mM NaCl, 1.0% Triton X-100, 1% deoxycholate, 5 mM EDTA, 10 7
mM Tris, pH 7.2) containing 1% protease inhibitor cocktail (Sigma) and sonicated for 4 x 8
10 s on ice. Soluble protein concentration was determined by using the Coomassie Plus 9
Protein Reagent (Pierce, Rockford, IL) per manufacturer’s instructions. Equal amounts 10
of soluble protein from each condition were electrophoresed in a 10% polyacrylamide 11
gel and then transferred onto a polyvinylidene difluoride membrane using the NuPAGE 12
System (Invitrogen). Membranes were blocked and then reacted with the primary 13
antibody in blocking buffer for 2 hr. After washing, the blots were reacted with a 14
secondary antibody anti-mouse IgG or anti-rabbit IgG conjugated with horseradish 15
peroxidase (HRP) for 1 hr at 25°C followed by chemiluminescence analysis with the 16
ECL Plus reagent kit (Amersham Biosciences, Piscataway, NJ). Densitometry analysis 17
of results on X-ray films was performed by using the ImageJ software 18
(http://rsb.info.nih.gov/ij/). 19
20
Statistics. Data are presented as mean ± standard deviation (S.D.) of the indicated 21
number of experiments. Differences between groups were determined using the two-tail 22
Student’s t-test and were considered statistically significant if p < 0.05. Comparisons 23
between three or more treatment groups were made by one-way ANOVA. Comparison 24
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tests as follow-up to ANOVA included both Bonferroni's test and multiple paired t-tests 1
for groups identified as statistically different. A 5% significance level was used for all 2
ANOVA tests and post-tests. 3
4
Results 5
GM-CSF levels in the BAL fluids of rats and mice with Pcp. To determine whether 6
Pneumocystis infection affects GM-CSF production, we assessed the levels of this 7
growth factor in the BAL fluids of rats and mice with Pcp. Alveolar lining fluids from 8
control and infected animals were recovered by bronchoalveolar lavage. Dilution of the 9
alveolar lining fluid was minimized by lavaging with small volumes of sterile saline (5 ml 10
for rats, 1 ml for mice). In an ELISA assay, normal rat BAL fluids showed low levels of 11
GM-CSF. BAL fluids from Dex rats had similar levels of GM-CSF, indicating that 12
immunosuppression did not significantly alter levels (Fig.1a). GM-CSF production was 13
found to be greatly increased (8 fold) in immunocompetent rats 48 hr after transtracheal 14
instillation of 1 µg IFN-γ (48) and 0.5 µg LPS (40). Transtracheal inoculation of normal 15
rats with 7.8 x 106 P. carinii organisms also greatly induced (10 fold increase) GM-CSF 16
production, measured seven days after the inoculation. However, four weeks of 17
Pneumocystis infection reduced the BAL fluid GM-CSF levels by 67% as compared to 18
the values from normal rats (p<0.05, Fig. 1a). 19
The infection had a similar effect on GM-CSF production in mice. Figure 1b shows 20
that GM-CSF levels in BAL fluids from immunocompetent mice were much higher than 21
those from mice infected with Pneumocystis for either four or six weeks (56% decrease 22
at four weeks and 69.5% at six weeks, p<0.05 for both time points versus normal). 23
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Immunosuppression by depletion of CD4+ lymphocytes did not have a significant effect 1
on GM-CSF levels; therefore, the infection was responsible for the losses noted. 2
3
GM-CSF levels in Amø from Pneumocystis-infected rats and mice. Alveolar 4
macrophages are a significant source of GM-CSF in the lung. To determine if a loss of 5
Amø GM-CSF production contributes to the low BAL fluid GM-CSF levels, we measured 6
GM-CSF levels in Amø from control and infected animals. In the sample of Amø from 7
normal rats, a single 14 kDa GM-CSF band was noted, as shown in Figure 2a for a 8
representative of six rats tested. In protein samples of Amø from Dex rats, two GM-CSF 9
bands were noted, and the total signal was slightly increased (Fig.2a, p>0.05 versus 10
normal). In blots of Amø protein from Dex-Pc rats, only the higher molecular weight 11
band was observed, but the intensity of the band was low. The average signal was 12
reduced by 65% (p<0.05 versus normal, Fig.2a) as assessed by image analysis of 13
independent immunoblots of four individual rats of each condition. Previous results 14
indicate that doublet or variable size bands for GM-CSF represent changes in 15
glycosylation of the protein (46). Our results suggest that the infection can induce 16
changes in GM-CSF glycosylation. 17
In the mouse model of infection, immunosuppression by CD4 lymphocyte depletion 18
did not have a significant effect on Amø GM-CSF levels. However, Western blotting of 19
the sample from pooled Amø of three mice for each condition showed a 69% to 71% 20
decrease in the amount of GM-CSF after four or six weeks of infection (Fig. 2b). Image 21
analysis of immunoblots from three independent trials showed that the decreases at 22
both time points were significant (p<0.05 versus normal, Fig. 2b). The immunoblot 23
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results revealed that Amø proteins from normal mice had a doublet of GM-CSF bands, 1
while those from infected mice showed only the lower band of the doublet. 2
3
GM-CSF in caspase-9 inhibitor-treated rats. Caspase-9 inhibitor treatment has been 4
shown to increase Amø number in rats and mice with Pcp (33). To obtain further 5
evidence for the importance of Amø in alveolar GM-CSF production, caspase-9 inhibitor 6
was used to treat rats with Pcp. After three weeks of treatment, Amø and non-adherent 7
cells and cell-free BAL fluids were isolated from six untreated, infected rats and six 8
caspase-9 inhibitor-treated, infected rats. No Dex control was used, as both the 9
untreated and treated groups were dexamethasone-treated, and since previous results 10
indicated that dexamethasone had no effects on Amø apoptosis (33). Counting of Amø 11
revealed that caspase-9 inhibitor-treatment increased the numbers of the cells by 58.4 ± 12
4.1% [(1.94 ± 0.2) x 106 and 3.32 ± 0.1) x 106, untreated and caspase-9 inhibitor treated 13
animals, respectively], which agreed well with our previous results (33). Protein samples 14
from each rat were probed for GM-CSF. A representative immunoblot result is shown in 15
Figure 3. With suppression of Amø apoptosis and recovery of Amø numbers by 16
caspase-9 inhibitor (33), levels of GM-CSF in the cell-free BAL fluid and in the Amø 17
were increased in all trials. Image analysis of GM-CSF signal from Amø was normalized 18
to GAPDH signal and indicated a greater than 2 fold increase in cellular GM-CSF levels 19
(p<0.05 versus untreated control, Fig. 3). Non-adherent cell fractions from untreated 20
and caspase-9 inhibitor-treated animals had similar levels of GM-CSF, suggesting that 21
they contribute similar amounts of the growth factor. Image analysis was not carried out 22
for BALf or non-adherent fractions because no normalizing GAPDH signal was 23
detected; however, Fig. 3 shows a distinct increase in the level of BAL fluid GM-CSF 24
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between the samples and little or no change in GM-CSF signal in the non-adherent 1
fraction. This result suggests that Amø are a significant source of GM-CSF in BAL 2
fluids, and that Pcp-mediated effects on Amø are responsible for the low GM-CSF 3
levels seen during the pneumonia. 4
5
PI-3K activity levels in Amø from rats and mice with Pcp. GM-CSF has been shown 6
to control PI-3K activation (26,30). Low GM-CSF levels in the lungs and Amø of animals 7
with Pcp may suggest that PI-3K activation (phosphorylation) is low in Amø during Pcp. 8
To test this possibility, total and phospho-PI-3K (pPI-3K) levels in Amø from rats and 9
mice with Pcp were determined by ELISA. 10
After four weeks, both immunosuppression alone and Pcp conditions showed small 11
but significant increases in total Amø PI-3K protein levels (Fig. 4a). The level of pPI-3K 12
was also significantly higher in Amø from Dex rats, but Pcp negatively affected the 13
activation of PI-3K. pPI-3K level in Amø from Dex-Pc rats was 65.8% lower than in 14
those from Normal rats (Fig.4a, p<0.05), indicating that the infection inhibits the 15
activation of the PI-3K pathway, but not the expression of PI-3K protein. 16
In the CD4-depleted mouse model of Pcp, immunosuppression caused increased 17
levels of total PI-3K and pPI-3K, but the changes did not reach the level of statistical 18
significance. After four weeks of infection, there were small increases in total PI-3K 19
protein, but these increases were smaller than the changes in the rat model and not 20
statistically significant. In contrast, there was a 63.2% decrease in pPI-3K levels four 21
weeks after initiation of Pcp in mice. An additional 15.8% decrease was observed at six 22
weeks of infection (Fig. 4b, p<0.05 for both time points versus CD4-depl.). 23
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1
Calmodulin mRNA levels in Amø from rats and mice with Pcp. Calmodulin has 2
been implicated in the control of both GM-CSF expression and PI-3K activation; 3
therefore, the low levels of GM-CSF and PI-3K activation in Amø during Pcp suggest a 4
defect in calmodulin levels or activity. To investigate this possibility, we first assessed 5
calmodulin mRNA levels in Amø from rats and mice. 6
Mammalian calmodulin is encoded by three separate genes (CaM 1, CaM 2, and 7
CaM 3), but their gene products are identical (45). The mRNAs from the three different 8
genes can be discerned by real-time PCR with appropriate probes based on sequence 9
differences in codon usage (66). Differences in mRNA levels were assessed in six 10
individual rats of each condition in triplicate reactions, and average fold difference ± 11
S.D. from normal was calculated after normalization to the RPS8 mRNA level which 12
does not change during Pcp (65). Amø from Dex rats did not show a significant 13
difference in mRNA levels of any of the three calmodulin genes; however, Dex-Pc rat 14
Amø had lower levels of all three mRNAs (Table 1). The changes in the mRNA levels of 15
the CaM1 gene did not reach statistical significance, but changes for those of the CaM2 16
(70% decrease) and CaM3 (40% decrease) genes were significant (p<0.05 versus 17
normal). 18
In the mouse model, CD4-depletion did not change mRNA levels for any of the three 19
genes, based on average Ct values ± S.D. from triplicate reactions from five individual 20
mice. Mice infected for four weeks had large decreases in CaM 2 and CaM 3 mRNA 21
levels (60% decreases for each, p<0.05 for each versus normal, Table 1). Levels of 22
CaM1 mRNA were also decreased, but as was the case in rats, this change did not 23
reach the level of statistical significance. 24
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To determine if the factor that negatively affects calmodulin mRNAs was present in 1
the alveolar lining fluid, normal rat Amø were incubated with BAL fluids from control or 2
infected rats for 18 hr and then assessed for CaM mRNA levels. Real time PCR for 3
each of the three calmodulin genes showed that only BAL fluids from infected rats were 4
capable of decreasing the calmodulin mRNA levels (Table 2). Similar to Amø from 5
infected animals, Amø incubated with BAL fluids from infected animals showed a 6
significant decrease in the mRNA levels of CaM2 (50% decrease) and CaM3 (30% 7
decrease) (p<0.05, Table 2). CaM1 mRNAs were low, but not significantly so, also 8
similar to Amø from infected animals. BAL fluids from Dex-treated, but uninfected rats 9
did not alter calmodulin mRNA levels, indicating that dexamethasone was not 10
responsible for the altered calmodulin levels (Table 2), and similar results were obtained 11
from infected rats and infected mice despite different immunosuppressive regimens, 12
suggesting that the method of immunosuppression did not play a role in the effect. 13
Since remnants of lysed and dead organisms are present in cell-free BAL fluids, we 14
also sought to determine if these were responsible for the calmodulin defects. Normal 15
Amø (1 x106) were incubated with viable or heat killed P. carinii organisms (5 x106) for 16
18 hr before real time PCR determination of calmodulin mRNA levels. In six 17
independent incubation trials, heat killed organisms had no effect on the mRNA levels 18
from any of the CaM genes. Likewise, viable organisms incubated with Amø for 18 hr 19
did not alter the Amø calmodulin mRNA levels (p>0.05 for CaM1, CaM2, and CaM3 20
versus no organism control, n=6). 21
22
Calmodulin protein levels in Amø from rats and mice with Pcp. We next determined 23
if reduced calmodulin mRNA levels were indicative of reduced calmodulin protein levels. 24
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Rat Amø proteins were blotted and probed for calmodulin. With GAPDH probing to 1
ensure equal protein loading from each condition, calmodulin signal was compared 2
between normal rats, immunosuppressed rats, and rats with Pcp. As shown in Figure 3
5a, Amø from normal and Dex rats had similar levels of calmodulin protein, but Dex-Pc 4
rat Amø had very low levels of calmodulin. 5
Representative immunoblots of mouse Amø showed a similar decrease in calmodulin 6
protein levels. Amø from normal and CD4-depl. mice had high levels of calmodulin, 7
while Amø from infected mice, at both four weeks and seven weeks of infection, showed 8
substantial decreases in the level of calmodulin protein (Fig. 5b). At four weeks of 9
infection, the number of Amø in mice with Pcp has not dropped significantly (35), but the 10
loss of calmodulin correlates well with the level of apoptosis noted in these cells during 11
Pcp (33,35). 12
13
NAC treatment of Amø alters calmodulin levels. Low levels of calmodulin in the Amø 14
could be due solely to decreased expression. The results showing low calmodulin 15
mRNA levels (Table 1) and protein levels (Fig. 5) suggest that down-regulation of 16
expression contributes to the decrease. However, increased calmodulin degradation by 17
the 26S proteasome through oxidation of methionines by ROS would also contribute to 18
lower calmodulin levels (16,59). To investigate whether ROS may contribute to low 19
calmodulin levels, we treated rat Amø with BAL fluids from normal, Dex, or Dex-Pc rats 20
for 18 hr. Some Dex-Pc BAL fluids were supplemented with 2.5 mM NAC. As shown in 21
Figure 6, Amø incubated with BAL fluids from Dex-Pc rats had 60% lower levels of 22
calmodulin protein as compared to Amø incubated with BAL fluids from normal or 23
immunosuppressed animals. NAC treatment resulted in significantly increased 24
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calmodulin protein levels in the Amø, a 3.2 fold increase compared to Amø that did not 1
get the oxidant, and 0.3 fold higher than control macrophages (p<0.05 versus control, 2
Fig. 6). This result shows that calmodulin protein levels are positively affected by 3
reduction of ROS in the Amø, so ROS may contribute to the low calmodulin levels in 4
Amø during Pcp. 5
6
Effects of calmodulin inhibition on GM-CSF. Our results described above suggest 7
that calmodulin and GM-CSF levels are low in Amø during Pcp. Published results 8
suggest that calmodulin can control GM-CSF expression in some cell types (61). 9
Therefore, we tested if inhibition of calmodulin could affect GM-CSF production in Amø. 10
Normal rat Amø were incubated with the calmodulin inhibitor W-7 [N-(6-aminohexyl)-5-11
chloro-1-naphtalenesulfonamide] (120 nM) for 30 min (58), with subsequent addition of 12
saline or 100 ng/ml LPS and IFN-γ (36) or 200 nM acetylcholine (53) for 2 hr to induce 13
GM-CSF production. Total cellular protein was then probed for GM-CSF on immunoblot. 14
The growth factor was induced in Amø incubated with IFN-γ/LPS or with acetylcholine, 15
resulting in a two-fold increase in GM-CSF signal when normalized for GAPDH signal 16
(p<0.05 versus unstimulated, Fig. 7). However, no increase in GM-CSF was noted when 17
Amø were pre-treated with the calmodulin inhibitor prior to stimulation with IFN-γ/LPS or 18
acetylcholine (p>0.05 versus unstimulated, Fig.7). These results indicate that 19
calmodulin controls GM-CSF protein levels in Amø, as in other cell types (55, 61). 20
21
Effects of calmodulin on apoptosis of Amø incubated with Dex-Pc BAL fluids. 22
Amø were incubated with BAL fluids from immunosuppressed or infected rats, with or 23
without a calmodulin inhibitor. In each of three trials, Dex BAL fluids did not increase 24
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apoptosis or decrease pPI-3K levels. BAL fluids from infected rats had a significant 1
effect on apoptosis as assessed by image analysis of activated caspase-3 immunoblots 2
(increased 9.3 fold, p<0.05 versus Dex BAL fluids). These same BAL fluids also 3
reduced pPI-3K levels in Amø by 31.8% as measured by ELISA (p<0.05 versus control). 4
Further inhibition of calmodulin by inclusion of the inhibitor W-7 significantly 5
increased apoptosis (3.5 fold increase over Dex-Pc BAL fluid-treated samples, p<0.05). 6
pPI-3K levels were reduced an additional 39.1% by calmodulin inhibition in addition to 7
Dex-Pc BAL fluids (p<0.05). These data suggest that calmodulin plays a role in survival 8
signaling and apoptosis resistance. However, in each case, incubation with W-7 alone 9
did not negatively affect apoptosis or pPI-3K levels, suggesting that calmodulin 10
inhibition or low pPI-3K levels alone do not induce cell death in the absence of an 11
apoptotic stimulus. 12
To confirm the importance of calmodulin in apoptosis of Amø during Pcp we 13
introduced exogenous calmodulin protein into Amø three hours prior to incubation with 14
BAL fluids from infected rats. Some samples also included 120 nM calmodulin inhibitor 15
W-7 or 50 ng/ml wortmannin (47) which inhibits PI-3K activity. Some cells were 16
incubated with BAL fluids and Chariot reagent only as a control. Amø were harvested 17
after 12 hr and assessed for calmodulin, cleaved caspase-3, GAPDH, and GM-CSF 18
protein levels, as well as intracellular ROS and pPI3K. Amø from three wells of the 19
same condition were pooled for Western blotting, while individual wells were used for 20
ROS and pPI-3K assessment. 21
As shown in Fig.8a, cells incubated with Dex-Pc BAL fluid had low levels of 22
calmodulin and GM-CSF proteins but high levels of cleaved caspase-3. Chariot alone 23
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and other controls had no effect on the parameters assessed. Pretreatment of Amø with 1
calmodulin protein increased intracellular calmodulin levels, as shown by the three 2
calmodulin-treated lanes in Fig. 8a. Exogenous calmodulin also increased GM-CSF 3
levels and decreased active caspase-3 levels, while inhibition of calmodulin eliminated 4
these effects (Fig. 8a). This result indicates that calmodulin can affect GM-CSF 5
production in Amø. In cells incubated with calmodulin and a PI-3K inhibitor, calmodulin 6
and GM-CSF levels were unchanged, but the levels of cleaved caspase-3 were 7
increased (Fig. 8a). These results indicate that active PI-3K was necessary for inhibition 8
of some apoptosis, but did not control calmodulin or GM-CSF protein levels. 9
ROS levels were high in Amø incubated with BAL fluids from infected animals, but 10
were reduced by 42% when the cells were pretreated with calmodulin protein (p<0.05 11
versus Dex-Pc BAL fluids alone, Fig. 8b). Inhibition of calmodulin eliminated the 12
suppressive effect of calmodulin on ROS production and resulted in a significantly 13
higher level of ROS in these cells than those incubated with Dex-Pc BAL fluids alone 14
(p<0.05 versus Dex-Pc BAL fluids only, Fig. 8b). Inhibition of PI-3K with wortmannin 15
also eliminated the positive effect of exogenous calmodulin, but not to the same degree 16
as calmodulin inhibition. These data indicate that both calmodulin and pPI-3K play a 17
role in control of ROS production. 18
Finally, pPI-3K levels were significantly increased with calmodulin transfection 19
(increased 1.54 ± 0.2 fold, p<0.05 versus Dex-Pc BAL fluid-treated, Fig. 8c). This 20
increase was lost with inhibition of calmodulin or PI-3K (p>0.05 versus Dex-Pc BAL 21
fluid-treated), suggesting that calmodulin controls PI-3K activation in Amø. 22
23
Discussion 24
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GM-CSF is important for host response to Pneumocystis in cytokine production (41) 1
and clearance of organisms by Amø (51), but our data show that GM-CSF levels in BAL 2
fluids (Fig. 1) and Amø (Fig. 2) are reduced during Pcp. Previous reports have 3
suggested that fibroblasts and alveolar epithelial cells are producers of GM-CSF (7,19). 4
We have shown that suppression of Amø apoptosis by inhibition of caspase-9 activation 5
leads to increased GM-CSF production (Fig. 3), suggesting that Amø are also an 6
important source of GM-CSF in the lung. This implies that the loss of Amø due to 7
apoptosis during Pcp (33,35) has a deleterious effect on GM-CSF levels in the alveolar 8
space, which in turn may contribute to Pcp progression. This data also suggests that the 9
loss of Amø-produced GM-CSF is not compensated for through increased GM-CSF 10
production by pulmonary or inflammatory cells, rendering the alveolar environment 11
unable to respond to infection with normal GM-CSF signaling events. 12
Amø defects during Pcp are similar to those in primary alveolar proteinosis (PAP), a 13
condition in which GM-CSF levels and actions are reduced. PAP is caused by 14
circulating GM-CSF-neutralizing antibodies or defective GM-CSF receptor activity 15
(14,27). During both Pcp and PAP, phagocytosis and recycling of surfactant lipids and 16
surfactant proteins by Amø are defective. Surfactant proteins and lipid build up in the 17
alveolar spaces, and there are losses of Amø functions in both Pcp (4,32,35) and PAP 18
(21). 19
Pcp is often found in association with secondary PAP (56,60), which may be due to 20
dysfunction in surfactant uptake or low GM-CSF levels leading to decreased Amø 21
survival. Administration of GM-CSF leads to increased macrophage survival (8,20,62) 22
and organism clearance in neonate mice with Pcp (51). Data presented in this study 23
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correlating reduced GM-CSF levels with decreased Amø survival signaling, increased 1
intracellular Amø ROS levels (Fig. 8), and increased apoptosis suggest that Pcp 2
pathogenesis is linked to Amø survival. 3
Our data also show that PI-3K activation is low in Amø during Pcp (Fig. 4). This is 4
important because PI-3K plays a central role in cell survival signaling (13), and survival 5
signaling in Amø during Pcp is reduced (Lasbury, submitted for publication). However, 6
low PI-3K signaling is not the source of Amø apoptosis, since PI-3K inhibition by 7
wortmannin alone does not induce apoptosis (52). 8
Since the calcium sensing protein, calmodulin, has been shown to be involved in the 9
control of both GM-CSF levels (61) and PI-3K activation (44), we determined calmodulin 10
levels in Amø from animals with Pcp. During Pcp, protein levels of calmodulin were 11
down (Fig. 5), and this change was seen in both rat and mouse models of infection. We 12
also assessed calmodulin mRNAs in Amø during Pcp. Calmodulin is coded for by three 13
genes; they diverge in the 5’- and 3’-UTRs and other non-coding sequences, but are 14
translated to form the same protein (17). The CaM1 and CAM3 genes have more than 15
one polyadenylation site, and a total of five calmodulin transcripts may be produced 16
(45). Evidence shows that these transcripts are differentially regulated and transcribed 17
because their promoter sequences possess different combinations of transcription 18
factor binding sites (3,22,57). Only CaM2 and CaM3 genes showed changes in Amø 19
mRNA levels (Tables 1 and 2) during Pcp, suggesting that there is differential control of 20
calmodulin transcription in Amø. 21
Aged calmodulin, that is calmodulin with oxidized C-terminal methionine residues, is 22
targeted for proteasomal degradation at a higher rate than reduced calmodulin or 23
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calmodulin bound by calcium (16,59). Since exogenous antioxidants increased the 1
levels of calmodulin protein in Amø (Fig. 6), the low calmodulin pools in Amø during Pcp 2
may also be due to an increase in calmodulin degradation, not merely reduced 3
production. 4
Cell-free BAL fluids from infected animals were capable of inducing the changes in 5
calmodulin mRNA levels (Table 2), while purified organisms could not. Viability of the 6
organisms was also not a factor in altering calmodulin mRNA levels; therefore, 7
molecule(s) responsible for down-regulating the expression of calmodulin may be 8
soluble factors from host or organism. Previous reports have implicated β-glucan 9
(15,37), the major surface glycoprotein (6,34), and polyamines from the organism 10
(35,37) in modulation of host cell functions. However, our data showed that incubation 11
of organisms with Amø for 18 hr did not induce changes in CaM mRNA levels, 12
suggesting that if an organism excreted substance is responsible, the levels of this 13
agent are not sufficiently high in the media after 18 hr to produce change. Concentration 14
differences of candidate molecules in BAL fluids from infected animals, which can 15
induce CaM mRNA reductions, and conditioned media from Pneumocystis and Amø co-16
incubations, which cannot alter CaM mRNA levels, may identify the responsible agent. 17
Calmodulin is important to Amø response during Pcp, as shown by both inhibition 18
(Fig. 7) and add-back experiments (Fig. 8). This is the first data showing that calmodulin 19
mediates GM-CSF expression in alveolar macrophages, having previously be shown 20
only in T lymphocytes and fibroblasts (55,61). In our studies, calmodulin controlled GM-21
CSF protein levels (Fig. 7, Fig. 8) and PI-3K activation (Fig. 8), and these changes 22
correlated with changes in Amø ROS and apoptosis levels (Fig. 8). Suppression of 23
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apoptosis by calmodulin is a specific response to the apoptotic stimulus induced by Pcp 1
or Dex-Pc BAL fluids, since calmodulin alone did not alter apoptosis or pPI-3K levels in 2
the absence of an apoptotic stimulus (data not shown). Whether calmodulin addition 3
mediates its effects as a general response to apoptosis was not assessed but is 4
possible, since previous data indicates that other apoptosis-stimulating mechanisms 5
(Fas, staurosporine) involve calmodulin-mediated events (50,64). 6
Our data indicated that calmodulin is an important signaling molecule for survival 7
and ROS control in Amø during Pcp. However, modulation or over-expression of 8
calmodulin is not an attractive therapeutic target because of the myriad systems in 9
which it acts (2,12,25,28,42,63). Therefore, stimulation of calmodulin or calcium 10
signaling as a possible treatment for Pcp carries the high probability of disruption of 11
other delicately balanced regulatory systems. In vitro evidence in this study indicated 12
that factors that increase GM-CSF and PI-3K activation result in reduced ROS and 13
apoptosis, and suggest that further investigations into the signaling that led to their 14
down-regulation and methods for increasing their activity will lead to new treatments for 15
Pcp. 16
17
Acknowledgements 18
This study was supported in part by funds from the National Institutes of Health (RO1 19
HL 65170 and RO1 A1062259). The authors have no conflicting financial interests. 20 21
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1
2
Figure Legends 3
4
Figure 1. Levels of GM-CSF in BAL fluid. Soluble rat or mouse BAL fluid proteins 5
were assessed by ELISA for total GM-CSF. All GM-CSF values are averages ± S.D 6
from triplicate samples of six individual rats or eight individual mice from each condition. 7
A. Normal rats were immunocompetent, Dex rats were immunosuppressed with 8
dexamethasone, and Dex-Pc rats were immunosuppressed and Pneumocystis infected 9
for four weeks. Normal-Pc rats were immunocompetent, transtracheally inoculated with 10
P. carinii, and sacrificed seven days later. Normal, IFN-γ + LPS rats were intranasally 11
instilled with 1 µg IFN-γ and 0.5 µg LPS, and the animals were sacrificed 48 hr later. B. 12
The same ELISA kit was used to assess soluble Amø protein from mice. Normal mice 13
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were immunocompetent, CD4-depl mice were immunosuppressed by anti-CD4 antibody 1
administration, and CD4-depl-Pc mice were immunosuppressed and infected for four or 2
six weeks. For both panels, *p<0.05 versus normal condition. 3
4
Figure 2. GM-CSF protein in Amø during Pcp. Soluble proteins from rat or mice Amø 5
were blotted and probed for GM-CSF. A. Conditions for rats are as described for 6
Normal, Dex, and Dex-Pc in Fig. 1 legend. B. Conditions for mice are as described in 7
the legend of Fig. 1. For both panels, representative immunoblots are shown from four 8
trials of individual rats and three independent trials of Amø pooled from three animals of 9
each condition for mice. Fold change from normal values are average signal strength ± 10
S.D. for all trials, after normalization of GM-CSF to the GAPDH control signal. *p<0.05 11
versus normal condition. 12
13
Figure 3. GM-CSF protein level after caspase-9 inhibition. Dex-Pc rats were treated 14
with caspase-9 inhibitor (33) or diluent only for three weeks. BALf samples are cell-free 15
BAL fluids. Amø were isolated as described in the Materials section. Non-adh. refers to 16
the non-adherent fraction of the BAL fluid particulate matter after isolation of Amø. 17
Blotted proteins were probed for GM-CSF and GAPDH. GAPDH was detected in Amø 18
fractions only. The BAL fluid fraction was cell free, and the cell number in the non-adh 19
fraction was too low to allow for detection of GAPDH. Fold change in GM-CSF signal 20
strength in Amø, normalized to GAPDH signal was calculated from average ± S.D. 21
Results are representative of three separate trials, each trial composed of samples 22
pooled from two rats of each condition. 23
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1
Figure 4. Levels of total and phosphorylated PI-3K in Amø during Pcp. Soluble rat 2
or mouse Amø proteins were assessed by ELISA for total and active PI-3K (pPI-3K). 3
Triplicate samples were assessed for each of six individual rats or eight individual mice, 4
and average values ± S.D for arbitrary ELISA units are shown. A. Rat conditions were 5
as described for Normal, Dex, and Dex-Pc in Fig. 1. B. The same ELISA kit was used to 6
assess soluble Amø protein from mice. Condition for mice were as described in the 7
legend of Fig. 1. For both panels, *p<0.05 versus normal condition of same target, total 8
or pPI-3K. 9
10
Figure 5. Calmodulin protein in Amø during Pcp. Soluble proteins from rat or mice 11
Amø were blotted and probed for calmodulin. A. Conditions for rats are as described for 12
Normal, Dex, and Dex-Pc in Fig. 1 legend. B. Conditions for mice are as described in 13
the legend of Fig. 1, except that mice were assessed after four and seven weeks of 14
infection. For both panels, representative immunoblots are shown from four trials of 15
individual rats and three independent trials of Amø pooled from three animals of each 16
condition for mice. Fold change from normal values are average signal strength ± S.D. 17
for all trials, after normalization of GM-CSF to the GAPDH control signal. *p<0.05 18
versus normal condition. 19
20
Figure 6. Calmodulin protein in rat Amø after treatment with antioxidant. Normal 21
Amø were incubated with BAL fluids from Normal, Dex, or Dex-Pc rats for 18 hr. Some 22
Amø were incubated with BAL fluids from Dex-Pc rats supplemented with 2.5 mM N-23
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acetylcysteine (NAC), an antioxidant. Soluble Amø proteins were probed for GAPDH 1
and calmodulin. Fold change in calmodulin signal strength, normalized to GAPDH signal 2
was calculated from average ± S.D. Signal strength values were from three individual 3
trials. *p<0.05 versus Amø incubated with Normal BAL fluids. $p<0.05 versus Amø 4
incubated with Dex-Pc BAL fluids. 5
6
Figure 7. Amø GM-CSF protein levels after calmodulin inhibition. Normal Amø 7
were incubated with saline, IFN-γ, LPS, or acetylcholine (Ach) for 2 hr at the 8
concentrations stated. Some samples were also treated with the calmodulin inhibitor W-9
7. Soluble Amø proteins were probed for GAPDH and GM-CSF detection. Fold change 10
in GM-CSF signal strength, normalized to GAPDH signal was calculated from average ± 11
S.D. Signal strength values were from three individual trials; each trial consisted of Amø 12
pooled from four rats. *p<0.05 versus Amø incubated with saline. 13
14
Figure 8. Effects of exogenous calmodulin on survival signaling and apoptosis. 15
Normal rat Amø (1 x 106/well) were incubated with 1 µg calmodulin protein complexed 16
to Chariot transfection reagent. After 3 hr, the cells were exposed to Dex-Pc BAL fluids, 17
and some were exposed to chemical inhibitors as shown. A. Representative immunoblot 18
analysis of Amø protein pooled from three wells of each condition. Two independent 19
trials were conducted. B. ROS levels were assessed as described in Materials section. 20
One well of Amø was used for assessment for each condition. Results are averages ± 21
S.D. for two independent trials. *p<0.05 versus Dex-Pc BAL fluid-treated alone, $p<0.05 22
versus Dex-Pc BAL fluids + 1 µg calmodulin. C. ELISA-based assessment of pPI-3K 23
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levels from individual wells of Amø treated as described. Results shown are average 1
fold change ± S.D. from two independent trials. The level of pPI-3K in Dex-Pc BAL fluid-2
treated cells was arbitrarily set to 1.0. *p<0.05 versus Dex-Pc BAL fluids alone, $p<0.05 3
versus Dex-Pc BAL fluids + 1 µg calmodulin. 4
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Table 1. Calmodulin mRNA levels in Amø during Pcp.
1 Results are average ± S.D. for six rats or eight mice of each condition
2 Immunocompetent animals
3 Immunosuppressed animals
4 Immunosuppressed and Pneumocystis-infected animals
5 individual calmodulin genes
6 p<0.05 versus normal condition of same species
Table 2. Effects of BAL fluids on Amø calmodulin mRNAs.
1 Results are average ± S.D. for six independent assays of
each condition 2 Immunocompetent animals
3 Immunosuppressed animals
4 Immunosuppressed and Pneumocystis-infected animals
5 individual calmodulin genes
6 p<0.05 versus normal condition of same species
Species Condition CaM 15 CaM 25 CaM 35
Rat Normal2
1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 Rat Dex
3 1.3 ± 0.3 0.9 ± 0.1 0.9 ± 0.4
Rat Dex-Pc4
0.7 ± 0.2 0.3 ± 0.26
0.6 ± 0.26
Mouse Normal2
1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 Mouse CD4-depl.
3 1.1 ± 0.2 1.0 ± 0.1 1.1 ± 0.1
Mouse CD4-depl.-Pc4
0.7 ± 0.4 0.4 ± 0.36
0.4 ± 0.26
BAL fluid Source1 CaM 15 CaM 25 CaM 35
Normal2
1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 Dex
3 1.2 ± 0.1 1.0 ± 0.1 1.0 ± 0.0
Dex-Pc4
0.8 ± 0.2 0.5 ± 0.26
0.7 ± 0.26
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