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* From the Health Effects Laboratory Division (Drs. Antonini, Yang, Ma, and Weissman, Ms. Roberts, and Mr. Barger), National Institute for Occupational Safety and Health, Morgantown, WV; and the Department of Environmental Health (Dr. Clarke), Harvard School of Public Health, Boston, MA.
Correspondence to: James M. Antonini, PhD, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Rd, Morgantown, WV 26505; e-mail: jga6{at}cdc.gov
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Abstract |
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Design: Male Fischer 344 rats, either 2.5 months or 20 months of age, were intratracheally inoculated with 5 x 103, 5 x 104, or 5 x 105 Listeria monocytogenes, and the effects on mortality, lung inflammation, pulmonary bacterial clearance, alveolar macrophage (AM) function, and T-lymphocyte characterization were determined.
Measurements and results: In noninfected control animals, the older rats had lower numbers of AMs on lavage and a lower percentage of total T, CD4+, and CD8+ cells. No difference was observed between noninfected young and old rats in AM function, assessing both chemiluminescence and nitric oxide (NO) production. After bacterial challenge, aged rats exhibited an increase in mortality, pulmonary infection, and edema, and lung lesions, which were more extensive than those observed in the younger rats. Interestingly, AM chemiluminescence was enhanced, while AM NO, a highly important antibacterial defense product, was abrogated in the aged rats as compared to the young rats.
Conclusions: This study demonstrated that advanced age is associated with alterations in lung defense mechanisms and increased susceptibility to pulmonary bacterial infection marked by elevated mortality, slowed pulmonary bacterial clearance, and altered AM function, specifically a decrease in NO production. These observations are indicative of reduced pulmonary defense function in an older population of rats.
Key Words: age • chemiluminescence • host defense • Listeria monocytogenes • macrophage • nitric oxide • pulmonary clearance
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the normal aging process, physical and biochemical changes occur which may affect the response of the lung to inhaled agents. Exposure to the oxidant gas NO2 resulted in more epithelial damage and altered tissue repair6 while O3 increased the proliferative response7 in the lungs of aged rats as compared to young rats. Other studies8 9 have demonstrated that older animals have a greater lung oxidative stress potential than do younger animals. An age-associated decrease in nitric oxide (NO), an important mediator in alveolar macrophage (AM) defense against infection, has been observed when studying aged rats.10 It has also been reported that elevated concentrations of nonheme iron (Fe3+), a factor associated with an enhanced risk of infection, increases with age in both humans and rats.11 In addition, elderly populations have been shown to be more susceptible to increased ambient particle levels as compared to the general population.12
In order to assess the pulmonary response to bacterial infection, a laboratory model using Listeria monocytogenes was employed. L monocytogenes is a Gram-positive, facultative intracellular bacterium that has been used in a number of animal studies13 14 15 16 to assess pulmonary host defense mechanisms. The initial immune response of the host to L monocytogenes is marked by macrophage activation and a rapid recruitment of neutrophils (polymorphonuclear leukocytes [PMNs]) to the site of infection.17 While the innate immune response is efficient at limiting the initial spread of infection, rapid clearance of L monocytogenes also depends on acquired T-cell–mediated immunity.18 It was the goal of the current investigation to study the lung defense mechanisms of young and aged rats before and after pulmonary challenge with a bacterial pathogen. Fischer 344 rats, young (2.5 months of age) and aged (20 months of age), were instilled intratracheally with three doses of L monocytogenes, and the effect on mortality, lung injury and inflammation, pulmonary bacterial clearance, AM function, and T-lymphocyte characterization was determined.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20 months of age and
weighed between 353 g and 512 g (mean, 429.6 ± 4.48 g)
comprised the "old" treatment group. The aged rats were in good
general health, showing no clinical or histopathologic evidence of
infection. They were acquired from colonies under the guidance of the
National Institute on Aging (National Institute of Health, Bethesda,
MD), which were maintained by Harlan Sprague Dawley, Inc. On arrival at
our facility, all rats received a conventional laboratory diet and tap
water ad libitum.
Intratracheal Bacteria Inoculation
L monocytogenes (strain 10403S, serotype 1) was a
donation from Rosana Schafer of the Department of Microbiology and
Immunology at West Virginia University. L monocytogenes was
cultured overnight in brain heart infusion broth (Difco Laboratories;
Detroit, MI) at 37°C in a shaking incubator. Following incubation,
the bacteria concentration was determined via spectrophotometry at an
optical density of 600 nm and diluted with sterile saline solution to
the desired concentrations.
The rats were lightly anesthetized by an intraperitoneal injection of 0.6 mL of a 1% solution of sodium methohexital (Brevital; Eli Lilly; Indianapolis, IN) and inoculated intratracheally with 5 x 103 (low dose), 5 x 104 (middle dose), or 5 x 105 (high dose) L monocytogenes in 500 µL of sterile saline solution, according to the method of Brain et al.19 Animals in the vehicle control noninfected group were dosed intratracheally with 500 µL of sterile saline solution.
Mortality/Histopathology
Animal weights and mortality were monitored daily over the
course of the treatment period. Histopathologic analysis was also
performed on rats from each group. The rats were killed with sodium
pentobarbital, and the lungs were preserved with 10% buffered formalin
by airway fixation at total lung capacity. The lobes of the lungs were
removed, sectioned, embedded in paraffin, and stained with hematoxylin
and eosin. Histopathologic analysis was performed by Dr. Val Vallyathan
(National Institute for Occupational Safety and Health [NIOSH],
Morgantown, WV), who was unaware of the experimental design and blinded
to the treatment groups of the study.
BAL
At 3 days, 5 days, and 7 days after bacteria instillation, the
rats were deeply anesthetized with an overdose of sodium pentobarbital
and then exsanguinated by severing the abdominal aorta. The left main
bronchus was clamped off, and BAL was performed on the right lungs of
rats from each group. Lavage was performed on their right lungs first
with a 4-mL aliquot of calcium-free and magnesium-free phosphate buffer
solution (PBS), pH 7.4. This first BAL fluid sample was centrifuged at
500g for 10 min and filtered with 0.22 µm sterile filters,
and the resultant cell-free supernatant was analyzed for various
biochemical parameters. Subsequently, lung lavage employed 6-mL
aliquots of PBS until 50 mL was collected. These samples were also
centrifuged for 10 min at 500g, and the cell-free BAL fluid
was discarded. The cell pellets from all washes for each rat were
combined, washed, and resuspended in 1 mL of PBS buffer and evaluated.
Cellular Evaluation
Total cell numbers were determined using a Coulter Multisizer II
and AccuComp software (Coulter Electronics; Hialeah, FL). Cells were
differentiated using a Cytospin 3 centrifuge (Shandon Life Sciences
International; Cheshire, England); 1 x 105
cells were spun for 5 min at 800 revolutions per minute and pelleted
onto a slide. Cells (200 cells per rat) were identified on
cytocentrifuge-prepared slides after labeling with Leukostat stain
(Fisher Scientific; Pittsburgh, PA). The number of cells per lung
volume was also determined by dividing the total cell number by total
lung capacity. Total lung capacity equaled 12.5 mL multiplied by rat
body weight in grams divided by 300.20
Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from lung and lung-associated lymph node
(LALN) tissue by guanidine isothiocyanate lysis (Trizol; Life
Technologies; Rockville, MD). Tissues were processed immediately after
sacrifice. Reverse transcription was performed using 5 µg total RNA,
oligo(dT)12–18 primer, and Moloney murine leukemia virus
reverse transcriptase (Superscript First-Strand complementary DNA
Synthesis Kit; Life Technologies).
Polymerase chain reaction (PCR) was performed using complementary DNA derived from 0.4 µg RNA, primers as listed, Taq polymerase (Sigma-Aldrich, St. Louis, MO), deoxynucleoside triphosphate mix, 10X PCR buffer, MgCl2, and H2O. PCR conditions involved denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 90 s. The initial cycle contained a 4-min denaturation at 94°C, and the final cycle contained a 7-min extension at 72°C. Thirty-seven cycles were performed.
Primers used for reverse transcription-PCR (RT-PCR) were as follows:
interferon (IFN)-
5'-ATCTGGAGGAACTGGCAAAAGGACG-3' and
5'-CCTTAGGCTAGATTCTGGTGACAGC- 3', which amplify a 288-base pair (bp)
fragment; interleukin (IL)-4, 5'-ACCTTGCTGTCACCCTGTTCTGC-3' and
5'-GTTGTGAGCGTGGACTCATTCACG-3', which amplify a 352-bp fragment;
TNF-
5'-TACTGAACTTCGGGGTGATTGGTCC-3' and
5'-CAGGCTTGTCCCTTGAAGAGAACC-3', which amplify a 295-bp fragment; IL-6,
5'-CAAGAGACTTCCAGCCAGTTGC-3' and 5'-TTGCCGAGTAGACCTCATAGTGACC-3',
which amplify a 614-bp fragment; and G3PDH,
5'-TGAAGGTCGGTGTCAACGGATTTGGC-3' and 5'-CATGTAGGCCATGAGGTCCACCAC-3',
which amplify a 983-bp fragment. With the exception of IL-4, all primer
sequences were obtained commercially (Clontech; Palo Alto, CA). PCR
products were visualized in ethidium bromide-stained agarose gels.
Inspection of glyceraldehyde 3-phosphate dehydrogenase PCR product was
used to document equal loading.
Pulmonary Clearance of L monocytogenes
At 3 days, 5 days, and 7 days after bacteria instillation,
left lungs, which had not undergone lavage, were removed from all rats
in each treatment group. The excised tissues were suspended in 10 mL of
sterile water, homogenized using a Polytron 2100 homogenizer (Brinkmann
Instruments; Westbury, NY), and cultured quantitatively on brain heart
infusion agar plates (Becton Dickinson; Cockeysville, MD). The number
of viable colony forming units were counted after an overnight
incubation at 37°C.
Luminol-Dependent Chemiluminescence
Luminol-dependent chemiluminescence, a measure of light
generation representing reactive oxidant species (ROS) production, was
performed with an automated Berthold Autolumat LB 953 luminometer
(Wallace; Gaithersburg, MD) as described previously.21
Resting chemiluminescence was determined by incubating
0.5 x 106 BAL cells at 37°C for
10 min in 0.008 mg/dL (weight/volume) luminol in a total volume of 0.5
mL of 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid buffer
(Sigma Chemical; St. Louis, MO) followed by the measurement of
chemiluminescence for 15 min. Luminol is used as an amplifier to
enhance detection of the light. Nonopsonized, insoluble zymosan (2
mg/mL; Sigma Chemical) was used as a stimulant and was added to the
assay immediately prior to measurement of chemiluminescence. Since PMNs
do not respond to unopsonized zymosan, the zymosan-stimulated
chemiluminescence produced is from AMs.22
Measurement of
chemiluminescence was recorded for 15 min at 37°C, and the integral
of counts per minute (cpm) vs time was calculated. Chemiluminescence
was calculated as the cpm of stimulated cells minus the cpm of the
corresponding resting cells.
AM NO Production
BAL cells were suspended at a concentration of
1.0 x 106 cells/mL in Essential Minimum Eagle
Medium (EMEM) [BioWhittaker; Walkersville, MD] supplemented with 2 mM
glutamine, 100 g/mL streptomycin, 100 U/mL penicillin, and 10%
heat-activated fetal calf serum, and seeded onto each well of a 24-well
tissue culture plate. BAL cells were allowed to adhere to the plates
for 2 h at 37°C at 5% CO2. After the
incubation, nonadherent cells were removed by washing three times with
EMEM media. The adherent cells, which were found to be > 90% AMs
were then incubated in fresh EMEM for 18 h at 37°C at 5%
CO2. The AM-conditioned media was collected,
centrifuged, and stored at - 70°C until analysis. The production of
NO was determined as an accumulation of nitrite by a modified
microplate assay using the Griess reagent.23
Briefly, the
samples were incubated with an equal volume of the Griess reagent at
room temperature for 10 min. The absorbance at 550 nm was determined
with a microplate spectrophotometer reader (SpectraMax 250; Molecular
Devices; Sunnyvale, CA). Sodium nitrite (Sigma Chemical) was used as a
standard. The results were expressed as µmol
nitrite/106 AMs.
Lymphocyte Differentiation
At 7 days after inoculation with L monocytogenes,
LALNs were excised and homogenized in PBS to count and differentiate T
cells and T-cell subsets—CD4+ helper and CD8+ cytotoxic cells. The
respective cell types were labeled with an appropriate monoclonal
antibody (MoAb) that was conjugated with a fluorescent probe for
visualization according to the method of Luster et al.24
Single-cell suspensions were prepared from excised lung-associated
lymph nodes. The cells were collected by centrifugation and suspended
in PBS, pH 7.4, containing 1% bovine serum albumin and 0.1% sodium
azide to a cell density of 1.5 x 106/mL. For
T-cell enumeration, anti-rat CD5RA MoAb was conjugated to fluorescein
isothiocyanate. For T-cell subsets, anti-rat CD4 MoAb was conjugated
with fluorescein isothiocyanate, and antimouse CD8 MoAb was conjugated
with dipalmitoylphosphatidylethanolamine. The cells were also
incubated with their respective isotype control to correct for
autofluorescence. After incubation with the conjugated MoAb, the cells
were washed once with the staining buffer and incubated for 5 min with
propidium iodide as a viability stain. The cells were again washed and
then enumerated on a Becton Dickinson FACS Vantage Flow Cytometer
(Becton Dickenson). Fluorescence was gated on propidium iodide to
eliminate dead cells. The values are expressed as the percent of gated
live cells and the absolute number of cells staining positive for each
surface marker.
Statistical Analysis
Results are expressed as means ± standard error of
measurement (SE). Statistical analyses were carried out using a
statistical program (JMP IN; SAS Institute; Cary, NC). The significance
of the interaction among the different treatment groups for the
different parameters at each time point was assessed using an analysis
of variance. The significance of difference between individual groups
was analyzed using the Tukey-Kramer post hoc test. For all
analyses, the criterion of significance was set at p < 0.05.
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Results |
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Age Comparison of Rats Infected With L
monocytogenes
Following intratracheal inoculation with three different doses of
L monocytogenes (low dose, 5 x 103
bacteria; middle dose, 5 x 104 bacteria; high
dose, 5 x 105 bacteria), cumulative survival
among young and old rats was determined (data not shown). The low-dose
inoculum had no effect on survival for either young or old rats. One of
the old rats exposed intratracheally to the middle dose of bacteria
died 4 days after exposure, while none of the young rats died. In
contrast, after treatment with the high dose, 40% of the old rats had
died within 3 days after intratracheal inoculation with the bacteria;
none of the young rats had died up to that point. Beyond 3 days,
survival continued to decline with time in the old rats. By 6 days
after high-dose bacteria instillation, all of the old rats had died.
For the young rats, 80% of the animals were still alive at 4 days, but
by 7 days postinfection, only 28% remained alive. At 8 days after
high-dose bacteria instillation, all of the young rats had also died.
Histopathologic analyses were performed on the lungs of rats from each treatment group (Fig 1 ). Lungs appeared normal for both the noninfected young and old groups (data not shown). A severe pneumonitis, characterized by a peribronchiolar accumulation of PMNs, and the appearance of multiple granulomatous lesions were observed throughout the lungs of both the young and old rats 5 days after inoculated intratracheally with the low 5 x 103 dose of L monocytogenes (Fig 1 , top left, A, and top right, B). Five days after intratracheal instillation of the middle 5 x 104 dose, severe edema, inflammation with significant infiltration of PMNs, and many granulomatous lesions with amorphous tissue debris were observed (Fig 1 , bottom left, C, and bottom right, D). The inflammation, edema, and lesions that were observed in the old rats inoculated intratracheally with both the low and middle bacteria doses were more extensive and more pronounced than those observed in the young rats.
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and IL-6, as well as the Th1 cytokine, IFN-, and the Th2
cytokine, IL-4, in lung and LALN tissue (Fig 4
). Seven days after infection with a low dose of L
monocytogenes (5 x 103 organisms),
greater levels of mRNA encoding the proinflammatory cytokines TNF-
and IL-6 were detected in the lungs of old rats as compared to the
young animals. Lung levels of IFN- and IL-4 did not differ between
young and old animals at this time point. The pattern of cytokine gene
expression was different in LALN tissue. In LALN tissue, TNF- mRNA
levels were greater in young animals. Other LALN tissue cytokine mRNA
levels evaluated did not differ between the two groups.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that the noninfected old rats had a significantly lower number of lavagable AMs as compared to the noninfected young rats. When the number of cells per lung volume was determined because the lungs of the aged rats were nearly twice the size of the lungs of young rats, there was a significant decrease in both AMs and PMNs in the older rats. It might be expected that due to the larger lungs of the older animals, more pulmonary cells would be recovered. However, decreased retrieval of pulmonary cells by BAL in aged animals have been observed in a previous study.25 Likewise, age-related decreases in AMs were observed after exposure to ozone.26 Also, Clarke et al27 observed a decrease in total cells and AMs retrieved by BAL in aged rats as compared to young rats. This observation of decreased pulmonary cells in aged rats suggests that these animals have suppressed lung defenses because AMs play a key role in the clearance of pathogens from the lungs, so altered AM number or function might well contribute to impaired clearance of pulmonary infection. It has been reported that impaired mucociliary clearance from the airways of aged individuals may be another factor that might predispose elderly to respiratory infection.28
In the assessment of AM function, we observed no difference in chemiluminescence and NO production when comparing noninfected aged and young rats. Wyde et al29 also noted no differences in the ability of phagocytes from young or aged noninfected animals to phagocytize or kill bacteria in vitro, suggesting that no innate deficit in function exists with an increase in age. Others have concluded that macrophage function appears to be largely unaffected by aging.2 Elder et al9 have shown that the respiratory burst activity of recovered BAL cells as measured by chemiluminescence production is actually elevated in old rats as compared to young rats. However, the respiratory burst activity of the PMNs was also included in their measurement, which likely accounted for the observed increased activity. PMN respiratory burst activity has been reported to be impaired with aging.30
There were no significant differences in the number of total T, CD4+ T-helper, and CD8+ T-cytotoxic cells recovered from the noninfected aged and young rats; however, the LALNs from the older rats were slightly bigger. In an attempt to normalize the data, we calculated the percentage of T cells in the total lymphocytes recovered. We found that there was a significantly lower percentage of total T, CD4+, and CD8+ cells recovered in the aged rats as compared to the young rats. Cell-mediated immunity has been shown to be highly vulnerable to the effects of aging as evidenced by involution of the thymus with aging, diminished delayed-type hypersensitivity response due to decreased T-cell proliferation, a reduction in CD4+ cells, and defects in signal transduction among various protein kinases in senescent T cells.30 31 Therefore, the decrease in the percentages of T cells and T-cell subsets we observed in the aged rats suggests the possibility of an impairment of cellular immune function.
After intratracheal instillation with the bacterial pathogen L monocytogenes, there was an increase in mortality and pulmonary inflammation for the aged rats. Lung edema and lesions observed in this group were more extensive and more pronounced than those observed in the young rats. There were significant elevations in PMNs, total T cells, and CD4+ cells, and in some cases AMs, recovered from the aged animals. Also, pulmonary clearance mechanisms had become significantly compromised in the aged rats after bacterial exposure. With the severity and extent of lung injury observed in the aged group, this observation is not surprising. Lesions were seen throughout the pulmonary interstitium, and the alveoli had become flooded with fluid, indicating possible lymphatic obstruction and inhibition of lung liquid clearance. A similar result was also observed in mice,29 using doses comparable to the ones chosen for this current study. It was shown that a significantly greater number of two different species of bacteria was isolated from the lungs of aged animals when compared with young animals.
It is also important to note that at baseline, there were greater proportions of T lymphocytes, CD4+ cells, and CD8+ cells in noninfected younger animals as compared to the old rats. But after infection, the older animals had influxes of T cells capable of equalizing the proportions of T lymphocytes and CD4+ cells, but insufficient to equalize the proportions of CD8+ cells. Thus, the older rats appeared to be better able to mount a CD4+ response than a CD8+ response. Given the important role of CD8+ cells in combating infection due to intracellular pathogens, this potentially is of mechanistic importance in explaining the impairment in ability of the aged rats to clear the intrapulmonary L monocytogenes infection.
With regard to RT-PCR results, increased levels of proinflammatory
cytokine gene expression in the lungs of older rats at 7 days after
infection most likely reflects continued pulmonary infection and
resulting inflammation in the older animals. More complete clearance of
infection in the younger animals is associated with lower levels of
TNF- and IL-6 mRNA in the lungs of these animals. IFN- plays a
key role in host defense against intracellular
pathogens.32
33
The presence of similar IFN- mRNA
levels in the lungs and LALN of young and old rats suggests that
delayed clearance of infection in the older animals is not primarily
due to impaired IFN- response. Additionally, the presence of similar
IL-4 mRNA levels suggests that a switch toward "Th2-like" cytokine
production also fails to explain impaired clearance of L
monocytogenes from the lungs of older rats.
While lavage reveals a lower number of AMs present in the lungs of aged rats, these AMs appear to be more responsive after in vivo challenge with bacteria than AMs collected from young rats. A significant elevation in ROS production as measured by chemiluminescence was observed for the AMs recovered from the old rats. This response was highly consistent for each dose and at each time point after L monocytogenes inoculation. This indicates that the AMs may have adapted to an acute challenge with a pulmonary toxicant to improve the survivability of the aged animals. Elder et al9 have demonstrated that aged mice and rats undergo adaptation after inhaling low doses of lipopolysaccharide subsequent to a high-dose challenge. Even with this elevation in the production of these highly ROS, there seemed to be little effect on bacterial killing. However, this elevation in oxidant generation may be responsible in mediating pneumonia-associated lung injury that led to the excess in morbidity and mortality observed in the older rats. It has been well established that activation of AMs and the subsequent release of ROS is one of the primary mechanisms by which inhaled substances damage the lungs.34
Interestingly, NO production by AMs recovered from the aged rats was significantly decreased as compared to the AMs of the young rats. NO is a highly reactive short-lived radical secreted by AMs and plays an important role in AM-mediated defense against infections.35 NO has been shown to promote the cytotoxic and mitochondrial activities of AMs and modulate cell-mediated immunity.36 NO has been demonstrated to react with superoxide anion to form the highly reactive substance peroxynitrite.37 Hickman-Davis et al38 have concluded that peroxynitrite generation by AMs is necessary for the killing of bacterial pathogens in the lungs. The suppression in pulmonary clearance of the bacteria observed in our current study then may be partially explained by the decrease in NO production by AMs recovered from aged rats after L monocytogenes challenge. Koike et al10 demonstrated that AM NO production activated by cells from lymph nodes in BAL fluid of old rats was significantly decreased when compared with cells in BAL fluid of young rats, concluding that inhibition in NO production suppresses defense against infections in the elderly.
In summary, the present study demonstrated that aged rats have altered lung defenses, making them more susceptible to lung injury and inflammation after pulmonary bacterial challenge. There appears to be a dramatic alteration in pulmonary clearance mechanisms of aged rats as compared to young rats, which involves a decrease in NO production by AMs. Also, this work established an aged animal model that will be used in future studies to assess the effect of aging on pulmonary-infection responses in the lung.
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Acknowledgements |
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Footnotes |
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Received for publication July 10, 2000. Accepted for publication January 4, 2001.
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References |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4 regulate the growth and differentiation of CD8+ T cells into subpopulations with distinct cytokine profiles. J Immunol 155,2928-2937[Abstract]
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