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ABSTRACT |
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Poor growth, Pseudomonas aeruginosa endobronchitis, pulmonary inflammation, and decline of lung
function are hallmarks of cystic fibrosis (CF), yet the relationship between these features is poorly understood. Because animal models of chronic bronchopulmonary infection with P. aeruginosa used to
study pulmonary inflammation in CF have also been associated with weight loss, we sought to determine whether this weight loss was due to the inflammatory process and/or to changes in lung function. P. aeruginosa-laden agarose beads were instilled into the lungs of mice. Weight loss was greatest 3 d after Pseudomonas infection. Infected mice had a rapid though transient rise in absolute
neutrophil counts, mTNF-
, mIL-1
, mIL-6, mip-2, and KC in bronchoalveolar lavage fluid. There was
no difference in lung resistance or lung compliance measured by body plethysmography between infected and control mice. Weight loss did correlate with the concentration of proinflammatory cytokine levels 3 d after inoculation of mice with Pseudomonas, and body composition analysis revealed
loss of skeletal muscle mass. These results suggest that weight loss in P. aeruginosa-infected mice was
associated with the inflammatory process and not with altered pulmonary responsiveness. These
findings may provide insights into the cause of cachexia and weight loss seen in patients with CF.
van Heeckeren AM, Tscheikuna J, Walenga RW, Konstan MW, Davis PB, Erokwu B, Haxhiu
MA, Ferkol TW. Effect of Pseudomonas infection on weight loss, lung mechanics, and cytokines in mice.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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In cystic fibrosis (CF), defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells and submucosal glands results in chronic disease of the respiratory tract, manifested by airway obstruction and recurrent infections of the lung and paranasal sinuses (1), that begins early in life (2). The CF lung is particularly susceptible to Pseudomonas aeruginosa, and this organism plays a critical role in the development and progression of pulmonary disease in CF (1). Persons with CF acquire an endobronchial infection that elicits an intense inflammatory response, which appears to be excessive (3). Animal models of endobronchial infection with Pseudomonas aeruginosa have histopathologic features similar to those present in the lungs of patients with CF (4, 5), and they have been used to study pulmonary inflammation characteristic of this disease (6).
In addition, several investigators have noted that animals inoculated with Pseudomonas-laden agarose beads lost significantly more weight than did control animals treated with sterile beads (6, 7). The mechanism of weight loss in this animal model, however, is uncertain. Growth failure remains a significant problem in CF, and chronic malnutrition has also been associated with the progression of lung disease (9, 10). The factors associated with pulmonary exacerbations, such as infection, inflammation, and increased work of breathing may lead to increased energy expenditure and decreased caloric intake. Indeed, it has been suggested that such exacerbations result in further deterioration in the nutritional status and pulmonary function in patients with CF (11, 12). Because the same factors that are causing the cachexia in these animals may also be contributing to the weight loss observed during pulmonary exacerbations in patients with CF, we evaluated the relationship of infection, inflammation, and altered lung resistance in weight loss observed during the course of bronchopulmonary infection with Pseudomonas in mice.
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METHODS |
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Animals
Male C57BL/6 mice (6 to 8 wk of age) purchased from Charles River Laboratories (Bar Harbor, ME) were fed autoclaved Purina Mouse Chow 5010, and sterile water was available at all times. All mice were maintained in static microisolator units.
Experimental Model of Endobronchial Infection with Pseudomonas aeruginosa
The agarose bead model of chronic Pseudomonas endobronchial infection was developed by Cash and coworkers (4) and modified for mice by Starke and colleagues (5). This model was used to create chronic pulmonary infection in mice with several variations. Briefly, agarose was mixed with tryptic soy broth (TSB) for sterile beads, or TSB containing mucoid P. aeruginosa strain M57-15, a clinical isolate from a patient with CF, grown to late log phase. The agarose-broth mixture was added to mineral oil that was equilibrated at 50° C, rapidly stirred for 6 min at room temperature, then cooled over 10 min. The agarose beads were washed once with 0.5% deoxycholic acid, sodium salt (SDC) in phosphate-buffered saline at pH 7.4 (PBS), once with 0.25% SDC in PBS, and four times with PBS. The beads were measured to be 93 to 166 µm in diameter by light microscopy. Quantitative bacteriology was performed on an aliquot of homogenized bead slurry to determine the number of colony-forming units (CFU) per milliliter (approximately 1.3 × 107 CFU/ml slurry). Sterile bead preparations were confirmed to be sterile.
Mice were anesthetized with intraperitoneal injections of 2.5%
Avertin (2,2,2-tribromoethanol and tert-amyl alcohol in 0.9% NaCl
administered at a dose of 0.015 ml/g body weight), and placed in dorsal recumbency, and the ventral cervical area was surgically prepared.
A transverse skin incision was made, and the trachea was visualized
by blunt dissection. Transtracheal insertion of a 22-G 1 inch intravenous catheter was used to instill 50 µl of a 1:10 dilution of the bead
slurry into the right lung (approximately 6.5 × 104 CFU/mouse). Mice
were then killed at different times by carbon dioxide narcosis and exsanguinated by cardiac puncture. Serum samples were collected for
cytokine and urea analysis. Bronchoalveolar lavage (BAL) was performed by cannulating the trachea in situ with a 22-G 1.5 inch bead-tipped feeding needle, instilling three aliquots of 1.0 ml sterile PBS
and collecting the fluid by gentle aspiration. The total lavage fluid
recovered from all animals was greater than 2 ml. After addition of
100 M phenylmethylsulfonyl fluoride (PMSF) and 5 mM ethylenediaminetetraacetic acid (EDTA), the fluid was centrifuged for 10 min at
4° C, and the supernatants were sterile-filtered and stored at 
80° C. The pellet was resuspended in 1.0 ml of PBS, and a total leukocyte
cell count was performed using a hemacytometer. A differential cell
count was performed on cytocentrifuge preparations (Cytospin 3;
Shandon, Pittsburgh, PA) stained with hematoxylin-eosin (H&E) using standard methods. The lungs of cohorts of mice were processed
for either quantitative bacteriology or histopathology.
A total of eight experiments were performed to evaluate weight loss, bacteriology, and inflammatory parameters. The results of these experiments have been combined. Not all outcome measures were the same for each experiment. A total of 18 untreated mice were killed. Seventy-seven mice were inoculated with sterile beads, of which three died from postoperative complications. Ninety-six mice were inoculated with Pseudomonas-laden agarose beads. Six of these died from postoperative complications, seven were improperly infected because of technical difficulties, two were killed 4 d after infection because of continued weight loss and poor clinical signs, and three were found dead 6 to 7 d after infection as a result of massive pulmonary involvement.
Measurements of Pulmonary Resistance and Dynamic Compliance
The pulmonary hyperresponsiveness of mice was also examined 2, 7, 14, or 21 d after inoculation with agarose beads. Seven experiments were performed, the results of which were combined. Animals were anesthetized with intraperitoneal injections of urethane (1.2 g/kg) and placed in a body plethysmograph for measurement of lung resistance. The lungs of these mice were artificially ventilated through a tight-fitting endotracheal tube, with a side opening for measurements of the inflation pressure and connected to a volume ventilator (Harvard Apparatus, Holliston, MA), delivering a cycled volume of 10 ml/kg from a bag containing 100% O2 at a ventilatory rate of 160 strokes/min. The level of anesthesia was monitored to ensure that there were no withdrawal responses to nociceptive stimuli. The body temperature was maintained between 37.5 and 38.5° C by placing a heating pad under the plethysmograph. An external jugular vein was cannulated for administration of a paralytic agent (gallamine triethiodide, 20 mg/kg). Gallamine was used because in addition to its paralytic actions, via blockade of N2 receptors, it reduces the effects of cholinergic agonists on heart rate and consequently cardiac output. Lung resistance (RL) and dynamic compliance (Cdyn) were measured using a specifically designed body plethysmograph and computerized program (Buxco, New York, NY). Noncumulative concentration-response curves for carbachol, a cholinergic agonist, were performed. Increasing concentrations (range, 1 to 100 µg/100 g body weight) of carbachol were administered intravenously in a sequential fashion. The lungs were inflated by occluding the expiratory line of the ventilator for two to three consecutive volume cycles after the mouse received a given concentration of carbachol. The next higher concentration was then administered 5 min after the previous dose. Noncumulative concentration- response curves for carbachol were constructed, and changes in RL and Cdyn were plotted as a function of a given concentration. After administration of the highest concentration of carbachol, mice were exsanguinated and BAL was performed and analyzed as described above.
There were 12 untreated mice tested for lung mechanics, 10 of which survived the procedure. Of the 33 mice that were successfully inoculated with sterile beads, 26 were successfully tested, and of the 38 mice that were successfully inoculated with P. aeruginosa-laden agarose beads, 33 completed the test satisfactorily. Mice that did not survive the testing procedure died either because of problems with the anesthesia or because of an inability to cannulate the trachea caused by technical difficulties. These mice were not included in the analysis.
Clinical Signs, Pair-feeding, and Body Composition Analysis
Body weight, core body temperature, and respiratory rate were determined before and after inoculation of three mice with sterile agarose beads and six mice with infected beads. In a second cohort of mice, food and water consumption was determined by weighing the food daily (grams) and measuring the amount of water (milliliters) consumed daily in eight mice inoculated with sterile beads and seven mice inoculated with Pseudomonas-laden beads. Measurements were taken at the same time of day. There was no appreciable loss of food particles in the cage. Mice were housed one to two per cage. In order to determine whether or not weight loss could be attributed solely to a decrease in food intake, another set of eight control mice were pair-fed and weighed daily. Pair-feeding involves feeding control mice the same amount of food that infected mice eat in the preceding 24-h period. Food was given once daily. Body weights of these animals were determined and compared with those of infected mice. Mice were killed 3 d after treatment by carbon dioxide narcosis. Gross lung pathology was examined. The animals were skinned and the left gastrocnemius and sartorius muscles (leg muscles) were removed. The entire carcass was reweighed to determine the wet weight. The carcasses were placed in a food dehydrator (Mr. Coffee, Inc., Bedford Heights, OH) for at least 24 h until the weights remained constant (dry weight). The water weight of the mice was determined by subtracting the dry weight from the wet weight. The leg muscles were dehydrated using a microwave oven (Panasonic, Secaucus, NJ) at 50% power for a minute at a time until the weight remained constant to determine skeletal muscle mass. The animal research protocol was reviewed and approved by the Case Western Reserve University Institutional Animal Care and Use Committee.
Lung Histopathology
After BAL, the lungs were inflation-fixed in 2% paraformaldehyde in PBS for at least 48 h, then cut once midsagittally and embedded in paraffin. Tissue sections were cut throughout the entire tissue block such that 5-µm sections were taken at regular intervals that encompassed the full craniocaudal range of sections. Sections were stained with hematoxylin-eosin using standard techniques and examined for histopathologic changes.
Quantitative Bacteriology
The lungs of mice inoculated with agarose beads embedded with P. aeruginosa were excised aseptically and homogenized in 40 ml normal saline at pH 7.4, and samples of the lung and BAL homogenates were cultured quantitatively by serial dilution on MacConkey agar plates. Spleens were removed and homogenized in 1.0 ml sterile PBS. A 10-µl aliquot of the splenic homogenate was applied to tryptic soy agar (TSA) plates. Blood was also collected and streaked on TSA plates. The lungs of mice inoculated with sterile beads were excised aseptically, homogenized in 20 ml of sterile PBS at pH 7.4, and plated on TSA plates in duplicate. The plates were incubated at 37° C and inspected for P. aeruginosa colonies after at least 24 h.
Analysis of Cytokines in Bronchoalveolar Lavage Fluid
The protease inhibitors PMSF and EDTA were added to the BAL
samples immediately after collection, as above. Murine tumor necrosis factor- (mTNF-), interleukin-1 (mIL-1), interleukin-6 (mIL-6), macrophage inflammatory protein-2 (mip-2), and KC/N51 (KC)
were measured using commercially available sandwich enzyme immunoassays (EIA) according to the manufacturer's recommended protocols (R&D Systems, Minneapolis, MN). The limits of detection for the
cytokines in these assays were < 5.1 pg/ml, < 3 pg/ml, < 3.1 pg/ml,
< 1.5 pg/ml, and < 2 pg/ml, respectively. Values that fell below the limits of detection were assigned a value of 0 pg/ml. The BAL supernatants were assayed in duplicate and compared with known standards, and the values were corrected for the respiratory epithelial lining fluid (ELF) volume recovered by measuring urea dilution (13).
Statistical Analysis
Data are expressed as the mean ± standard error of the mean (SEM).
Kruskall-Wallis one-way ANOVA using Dunn's method to compare differences between groups, Mann-Whitney rank sum test, and correlation and linear regression analysis were performed using the statistical package in SigmaStat V2.03 (Jandel Scientific, San Rafael, CA). The criterion for statistical significance was p 
0.05.
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RESULTS |
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INTRODUCTION
METHODS
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DISCUSSION
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Clinical Findings
C57BL/6 mice lost a significant amount of weight after inoculation with Pseudomonas-laden agarose beads (Figure 1). The
greatest amount of weight loss was observed 3 d after inoculation with infected beads (13.7 ± 1.3%), after which time
most mice gradually regained their body weight. Some infected mice displayed mild physical signs of ill health, manifested as an unkempt or scruffy hair coat, and a few displayed
more severe clinical signs such as dehydration and exhibiting
signs of pain. Mice that displayed severe clinical signs and/or
continued weight loss during the course of the experiment
were killed and no data were obtained from them. Mice that
were inoculated with sterile agarose beads lost some weight
(4.6 ± 0.7 g) 3 d after inoculation, but appeared healthy, and
mice treated with anesthesia alone initially lost weight the first
day after inoculation (1.5 ± 0.5%), but gained weight thereafter. Untreated mice gained weight during the course of the
experiment (data not shown). Also, core body temperature remained within normal limits for uninfected (37.0 ± 0.3° C) and infected mice (36.8 ± 0.1° C) 3 d after inoculation with beads. Fever was not detected in the infected mice at any time point studied. The mice were noted to be somewhat tachypneic 1 d
after inoculation with infected beads as compared with control
mice, but respiratory rates were within normal limits.
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Significantly different from mice treated with sterile agarose beads.
Quantitative Lung Bacteriology
Pulmonary bacterial counts were maximal 3 d after mice were inoculated with P. aeruginosa (Figure 2) and declined at 4 d but Pseudomonas was still isolated 7 d after inoculation with infected beads. Only three mice treated with sterile beads had a single bacterial colony on culture; the rest were culture-negative. BAL from seven untreated animals were culture-negative. No mice had blood cultures that were positive for P. aeruginosa (n = 11). Only one mouse had a spleen homogenate that was positive for Pseudomonas 3 d after inoculation with infected beads.
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Inflammatory Mediator Levels in Bronchoalveolar Lavage (BAL) Fluid and Sera
Proinflammatory mediators were significantly (p < 0.05) elevated in BAL fluid of Pseudomonas-infected mice compared
with those in control mice (Figure 3). Except for mIL-1, untreated mice and mice treated with sterile beads had little to
no detectable levels of proinflammatory mediators in the epithelial lining fluid. Low, basal concentrations of mIL-1 were
detected in untreated mice (0.8 ± 0.1 ng/ml BAL fluid) and in
mice 3 d after inoculation with sterile beads (1.1 ± 0.5 ng/ml
BAL fluid). Results showed that mTNF-, mIL-1, and mIL-6
concentrations in the epithelial lining fluid peaked 2 to 3 d after inoculation with Pseudomonas-laden agarose beads. The
neutrophil chemokine KC peaked 3 to 4 h after infection,
whereas the concentration of another neutrophil chemokine mip-2 peaked 3 d after inoculation. All of these cytokines fell considerably at 4 d after inoculation, and most reached near
baseline levels by 7 to 10 d after infection with Pseudomonas.
Regardless of treatment, mTNF- and mIL-1 were undetectable in the serum of most mice, though a few mice had low levels of these cytokines (data not shown). Serum mIL-6, however, peaked 3 h after inoculation with P. aeruginosa-laden
agarose beads and decreased to normal levels by 7 d postinoculation.
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Neutrophil Numbers in BAL Fluid
The percentage of neutrophils peaked at 2 to 3 d after inoculation with infected beads (81 ± 2 and 82 ± 2%, respectively), as shown in Figure 4A, whereas the highest percentage of neutrophils in mice that received sterile beads was 1 d after inoculation (54.9 ± 6.8%). The absolute number of neutrophils in the BAL fluid rose in a similar fashion to that of inflammatory mediators, peaking at 3 d after inoculation with Pseudomonas-laden agarose beads (3.7 × 106 ± 0.7 × 106 cells/ml BAL) and falling 4 d postinoculation (Figure 4B). Mice treated with sterile beads had a maximal absolute number of 4.7 × 104 ± 0.5 × 104 cells/ml BAL 1 d after inoculation.
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Histopathology
The cellular response of mice to the presence of sterile agarose beads and Pseudomonas-laden agarose beads 3 and 10 d after inoculation with beads is shown in Figure 5. The response of mice to sterile beads (closed arrows) was mild, focal, and consisted primarily of a mononuclear cellular infiltrate directly surrounding the beads (Figures 5A and 5B). Mice inoculated with Pseudomonas-laden beads (open arrows) had an intense neutrophilic infiltration within and around the small and medium-sized bronchi, as well as a perivascular cellular infiltrate. Some extension of this infiltrate into the parenchyma was present in several sections (Figures 5C and 5D).
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Pulmonary Hyperresponsiveness to Carbachol
There were no differences in baseline total lung resistance between untreated mice (1.23 ± 0.06 cm H2O/ml/s) or mice inoculated with sterile or P. aeruginosa-laden agarose beads 2 d after treatment (1.13 ± 0.08 cm H2O/ml/s and 2.95 ± 1.32 cm H2O/ml/s, respectively), or at any other time point studied. The effect of intravenous administration of carbachol on lung reactivity 2, 7, 14, or 21 d is illustrated in Figures 6A, 6B, 6C, and 6D, respectively, after inoculation of animals with sterile (n = 12, 4, 8, 3, respectively) or Pseudomonas-laden (n = 10, 10, 8, 6, respectively) agarose beads. There were no significant differences in RL or Cdyn between mice treated with Pseudomonas-laden agarose beads and mice treated with sterile agarose beads at any of the time points studied. The highest concentration of carbachol administered (1 µg/g body weight) elicited a modest increase in RL and decrease in Cdyn in untreated mice, and there was no difference between untreated mice and mice inoculated with sterile agarose beads (data not shown).
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The Relationship of Weight Loss and Lung Responsiveness with Pulmonary Inflammation
Linear regression analysis (Table 1) was performed comparing
change in body weight to the concentration of inflammatory
mediators and absolute neutrophil number in BAL fluid of
mice 3 d after inoculation with P. aeruginosa-laden beads.
Weight loss directly correlated with the levels of mTNF- (R = 0.678), mIL-1 (R = 0.774), mip-2 (R = 0.902), KC (R = 0.835), and absolute neutrophil number (R = 0.752) in
BAL. There was also a direct relationship between absolute
neutrophil number and concentrations of mIL-1 (R = 0.825),
mip-2 (R = 0.781), and KC (R = 0.779) in the BAL fluid.
There was no correlation, however, between cytokine concentration in BAL fluid and lung response to 0.75 mg/kg carbachol administered intravenously (p < 0.05) 2 d after inoculation with Pseudomonas beads (Table 2).
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Pair-feeding and Body Composition Analysis
Mice treated with sterile or infected beads ate less food 1 d after inoculation (81 and 69%, respectively), but then regained their appetite (Figure 7A). Water consumption also declined 1 d after inoculation for both cohorts, but mice inoculated with sterile beads drank more water than infected mice did thereafter (Figure 7B). Infected mice lost significantly (p < 0.05) more weight than pair-fed mice did 2 and 3 d after treatment (Figure 7C). The breakdown of the body composition analysis is shown in Table 3. There were significant differences in the absolute wet weights of infected mice compared with those in untreated animals (p = 0.024). There were no significant differences in absolute water weight between any of the groups, although infected mice had a greater percentage of water relative to their wet weight compared with untreated mice (p = 0.004) and mice inoculated with sterile beads (p = 0.026). Infected mice had significantly less muscle mass than did untreated mice (p < 0.006) or pair-fed animals (p = 0.027).
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DISCUSSION |
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INTRODUCTION
METHODS
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Patients with CF, in the midst of an acute pulmonary exacerbation, frequently have substantial weight loss. Several investigators have shown that weight loss during acute pulmonary
exacerbations is the result of greater resting energy expenditure (11, 12, 14), yet it is unclear if this change in resting energy expenditure is caused by altered pulmonary function, abnormal body composition because of fat malabsorption, chronic
or acute infection, or inflammation related to the presence of
proinflammatory mediators in the lung. In experiments using
an animal model of chronic bronchopulmonary infection, which
closely resembles the lung pathology seen in patients with CF,
mice infected with mucoid P. aeruginosa had significant weight
loss 3 d after inoculation as compared with control animals
(7). In this study, the change in body weight correlated with
BAL concentrations of mTNF, mIL-1, mip-2, and KC and
absolute neutrophil counts 3 d after inoculation with P. aeruginosa-laden agarose beads. Moreover, the weight loss directly correlated with the degree of pulmonary inflammation and
not changes in pulmonary function.
In this study, C57BL/6 mice rapidly developed pulmonary inflammation in response to P. aeruginosa. This intense inflammatory reaction, as measured by cytokine concentrations and absolute neutrophil numbers in bronchoalveolar lavage (BAL) fluid, was transient and reached near baseline levels by 7 to 10 d after inoculation. No mice tested 3 d after Pseudomonas inoculation had clinical evidence of septicemia, although a spleen homogenate from one mouse yielded P. aeruginosa. Inflammation was still present in mice 10 d after infection, however, as indicated by the presence of pulmonary histopathology and an altered leukocyte differential in BAL fluid. The concentration of the neutrophil chemokine, mip-2, in BAL fluid mirrored the number of neutrophils in the BAL fluid. Both neutrophil number and mip-2 concentration peaked 3 d after infection with Pseudomonas. Absolute neutrophil number and mip-2 concentration in BAL fluid were positively correlated (R = 0.791, p < 0.0006) 3 d after infection. Another neutrophil chemokine, KC, peaked 3 to 4 h after infection, and this value also positively correlated (R = 0.779, p < 0.0007) with neutrophil counts 3 d after inoculation. Thus, it appears that in this model system the early release of KC initiated neutrophil migration into the infected lung, and the inflammatory response was perpetuated by the release of mip-2, which acted as the driving force for the chronic influx of neutrophils into the lung.
The cytokines mTNF-, mIL-1, and mIL-6 are known to
initiate a cascade of various inflammatory responses. Among
their many effects, mIL-6, mIL-1, and mTNF- play a role in
mediating weight loss, net protein catabolism, anorexia and
fever (15). Fever was not detected in any of the mice in this
study, regardless of treatment. Of note, patients with CF are
chronically colonized and/or infected with a variety of pathogens, most frequently P. aeruginosa, yet are rarely febrile despite the intense pulmonary inflammation (1). Several investigators have evaluated the role that circulating levels of TNF-
and IL-1 play in the pathophysiology of patients with CF-associated cachexia and lung disease with variable results. Elborn and
colleagues (18) reported that resting energy expenditure was
correlated to TNF- levels in the circulation of CF, whereas
Brown and coworkers (19) indicated that circulating TNF-
was inconsistently detected in patients with CF and IL-1 was
not detected at all. In this study, mice inoculated with P. aeruginosa-laden agarose beads did not produce consistent amounts of circulating mTNF- or mIL-1, regardless of treatment.
Infected mice did have detectable levels of circulating mIL-6
after infection with P. aeruginosa, which peaked early in the course of the disease. Circulating IL-6 has been detected in
patients with CF, and the measured concentrations of this cytokine appeared to be related to the extent of pulmonary involvement. Nixon and colleagues (20) showed that blood concentrations of this cytokine declined after treatment with systemic
antibiotics, thus implicating pulmonary infection and inflammation with the elevated circulating levels of IL-6. One day after
inoculation of mice with Pseudomonas beads, however, there
was no correlation between weight loss and serum IL-6 concentration (R = 0.684, n = 6). Although IL-6 has been considered
a proinflammatory mediator by mediating cachexia and the
acute phase response (21), it also has been more recently reported to have anti-inflammatory properties by regulating the proinflammatory mediators TNF- and IL-1 yet having no effect on the anti-inflammatory cytokine IL-10 (22).
Lung resistance and compliance in mice chronically infected with P. aeruginosa has not previously been investigated. Pseudomonas infection or the inflammatory response to it does not cause an increase in lung reactivity to carbachol in mice since the administration of sterile beads caused the same effect. We have shown that there are no significant changes in lung resistance or lung compliance in control and infected mice early in the course of the disease when weight loss occurs. There was no correlation between lung responsiveness to carbachol and cytokine concentration in BAL fluid. Thus, it appears that impaired lung resistance or compliance during Pseudomonas infection plays no role in the weight loss seen early in the course of infection. Because mice later in the course of the infection, measured as late as 21 d postinoculation with agarose beads, did not show a difference in the lung compliance and responsiveness to carbachol, it is unlikely that lung function in this model is significantly affected by infection with P. aeruginosa. Nevertheless, it is important to note that endobronchial inflammation in this model system is not uniform, and the impairment of lung function in these animals may be underestimated because of the sensitivity of our assay. Furthermore, this model may not mimic the lung disease that is characteristic of CF closely enough in order to be able to show such changes in lung function. Finally, it also is possible, though, that patients with CF could inherently have airway hyperresponsiveness that is independent of pulmonary inflammation. In future studies, we will investigate changes in pulmonary function in mice with CF compared with littermate control mice.
The amount of food consumed by infected and control mice was similar, although infected mice drank less water than mice inoculated with sterile beads after the first day of treatment. Mice inoculated with either sterile or P. aeruginosa-laden beads did consume less food and water 1 d after inoculation with beads. It is unlikely that the weight loss observed was caused by decreased food intake alone since infected mice lost significantly more weight than pair-fed control mice. Body composition analysis indicated that weight loss was also due in part to a reduction in muscle mass. Individual cytokines have been studied in their role in inducing weight loss, directly and indirectly. Matthys and Billiau (23) noted in 1997 that cachexia can rarely, if ever, be attributed to one single cytokine but rather a set of cytokines that work in concert. They also assert that even if individual cytokines are not measurable systemically, they could be in sufficient amounts to act synergistically to cause cachexia.
Because the inflammatory response to bronchopulmonary infection with P. aeruginosa appears to play a role in weight loss in mice, we need to further investigate the mechanism by which local inflammation in the lung leads to these constitutional effects. The relative importance of pulmonary inflammation in causing weight loss was confirmed by Konstan and colleagues (24), who showed that in patients with CF and mild lung disease, treatment with the anti-inflammatory agent ibuprofen slowed the progression of their lung disease and decreased the weight loss compared with placebo-treated patients. Also, Chmiel and colleagues (25) examined the effects of the immunomodulatory cytokine, human interleukin-10, on mice infected with P. aeruginosa. Animals inoculated with Pseudomonas-laden beads then treated with repeated intraperitoneal injection of human interleukin-10 had significantly less pulmonary inflammation and lost less weight than did untreated control mice in this model.
In conclusion, proinflammatory mediators and not changes in lung resistance or compliance appear to play a role in the weight loss associated with P. aeruginosa infection in mice. This suggests that the presence of locally produced proinflammatory cytokines in the lung has profound systemic effects and may have clinical implications in patients with CF. The actual mechanism of this effect remains to be elucidated and is currently under active investigation. Fully understanding the mechanism by which local inflammation in the lung leads to these constitutional effects may allow us to discover new targets for anti-inflammatory therapy in the treatment of pulmonary disease in CF.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Anna M. van Heeckeren, D.V.M., M.S., Case Western Reserve University School of Medicine, Biomedical Research Building 827, 2109 Adelbert Road, Cleveland, OH 44106-4948. E-mail: amv2{at}po.cwru.edu
(Received in original form March 1, 1999 and in revised form July 21, 1999).
Acknowledgments: The writers wish to express their appreciation to Chris Statt, Heidi Carroll, Lisa Shyjka, Jay Hilliard, Jeanne Knesebeck, Jerry Chipuk, Lisa Hogue, Christiaan van Heeckeren, and Alma Wilson for providing their expert technical support.
Supported by Grants DK48996, DK43999, DK48994, HL-50527, and P30-DK-27651 from the National Institutes of Health and by a Research Development Program Grant from the Cystic Fibrosis Foundation.
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References |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
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1. Konstan, M. W., and M. Berger. 1993. Infection and inflammation of the lung in cystic fibrosis. In P. B. Davis, editor. Cystic Fibrosis. Marcel Dekker, New York. 219-276.
2. Khan, T. Z., J. S. Wagener, T. Bost, J. Martinez, F. J. Accurso, and D. W. Riches. 1995. Early pulmonary inflammation in infants with cystic fibrosis. Am. J. Respir. Crit. Care Med. 151: 1075-1082 [Abstract].
3. Noah, T. L., H. R. Black, P. W. Cheng, R. E. Wood, and M. W. Leigh. 1997. Nasal and bronchoalveolar lavage fluid cytokines in early cystic fibrosis. J. Infect. Dis. 175: 638-647 [Medline].
4. Cash, H. A., D. E. Woods, B. McCullough, W. G. Johanson Jr., and J. A. Bass. 1979. A rat model of chronic respiratory infection with Pseudomonas aeruginosa. Am. Rev. Respir. Dis. 119: 453-459 [Medline].
5. Starke, J. R., M. S. Edwards, C. Langston, and C. J. Baker. 1987. A mouse model of chronic pulmonary infection with Pseudomonas aeruginosa and Pseudomonas cepacia. Pediatr. Res. 22: 698-702 [Medline].
6. Konstan, M. W., K. M. Vargo, and P. B. Davis. 1990. Ibuprofen attenuates the inflammatory response to Pseudomonas aeruginosa in a rat model of chronic pulmonary infection: implications for antiinflammatory therapy in cystic fibrosis. Am. Rev. Respir. Dis. 141: 186-192 [Medline].
7. van Heeckeren, A., R. Walenga, M. W. Konstan, T. Bonfield, P. B. Davis, and T. Ferkol. 1997. Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa. J. Clin. Invest. 100: 2810-2815 [Medline].
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Gosselin, D.,
M. M. Stevenson,
E. A. Cowley,
U. Griesenbach,
D. H. Eidelman,
M. Boule,
M. F. Tam,
G. Kent,
E. Skamene,
L. C. Tsui, and
D. Radzioch.
1998.
Impaired ability of CFTR knockout mice to control lung infection with Pseudomonas aeruginosa.
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