Full text of DOI:10.1093/gerona/56.12.M771

Journal of Gerontology: MEDICAL SCIENCES
Copyright 2001 by The Gerontological Society of America
2001, Vol. 56A, No. 12, M771–M774
The Characterization of Bronchoalveolar Lavage
Fluid in Very Elderly Patients With
Cerebrovascular Disease
Masayuki Kikawada,1 Tetsuo Oyama,1,2 Kimikazu Ogawa,1,2 Hisayuki Arai,1,2
Toshihiko Iwamoto,1 and Masaru Takasaki1
1Department of Geriatric Medicine, Tokyo Medical University, Japan.
2Department of Internal Medicine, YuYu-kenkoumura Hospital, Nagaoka, Japan.
Background. Elderly patients with cerebrovascular disease have a high frequency of pneumonia due to impaired im-
mune function and the occurrence of micro-aspiration.
Methods. We performed bronchoalveolar lavage in 11 very elderly subjects with cerebrovascular disease and 9
healthy volunteers to investigate whether there were changes of local immunity in the lungs of the elderly subjects. The
total cell count, the cell characteristics, and the lymphocyte subsets in bronchoalveolar lavage fluid were compared be-
tween the two groups.
Results. A significant increase in the total cell count as well as in the number of neutrophils and CD8ꢀ T cells was
observed in the elderly group. In addition, the mean CD4/CD8 lymphocyte ratio was lower in the elderly patients than in
the healthy volunteers.
Conclusions. These observations suggest that silent micro-aspiration occurs in many elderly individuals with cere-
brovascular disease and that pulmonary defenses decrease with age.
HE onset of cerebrovascular disease increases in el-
derly individuals, and such patients are more likely to
of them had previous cerebral infarction, six had multiple
lacunar infarcts, and one had Alzheimer’s disease. All pa-
tients had been bedridden for periods of 2 months to 9
years.
T
become bedridden and to be immunosuppressed. Some pre-
vious studies have shown that elderly patients with cere-
brovascular disease have depression of the swallowing and
cough reflexes (1,2) and suffer from micro-aspiration (3).
Thus, elderly patients with cerebrovascular disease may
have increased risk factors for pneumonia due to impaired
immune function or the existence of micro-aspiration.
Many studies have revealed that immune function in el-
derly persons changes as part of the aging process. How-
ever, there has been little investigation of immunological
change in the lungs of elderly subjects (4–8), and no study
has assessed pulmonary immunity in very elderly patients
with cerebrovascular disease.
None of the subjects had a history of bronchial asthma,
allergic rhinitis, or other allergic diseases. All of the healthy
volunteers had normal pulmonary spirometry findings.
None of the elderly patients or healthy volunteers was being
treated with corticosteroids or other immunosuppressive
therapy during the course of the study, and none had suf-
fered from a pulmonary infection within 1 month prior to
bronchofiberscopy and bronchoalveolar lavage (BAL). The
study conformed to the Declaration of Helsinki and was ap-
proved by the institutional Ethics Committee. Written in-
formed consent was obtained from each subject.
In the present study, we assessed the bronchoalveolar lav-
age fluid (BALF) in elderly patients who had cerebrovascu-
lar disease and compared the results with the findings ob-
tained in healthy subjects.
BAL and Flow Cytometry
Transoral bronchofiberscopy was performed (Olympus
BF, Type p-8; Olympus, Tokyo, Japan) after local anesthe-
sia of the upper airway with 4% lidocaine. Following obser-
vation of the airways, the tip of the fiberscope was wedged
into the subsegmental bronchus of the right middle lobe, af-
ter which 150 ml of normal saline was instilled in 50-ml ali-
quots through the fiberscope and aspirated under low suc-
tion using a sterile syringe after each instillation. The BALF
was filtered through two sheets of sterile gauze and centri-
fuged at 400 rpm for 3 minutes to prepare cells (Cytospin 2;
Shandon, Sewickley, PA). The cell pellets were later stained
by the May-Giemsa method, and differential counting was
METHODS
Subjects
The subjects were 11 very elderly patients with cere-
brovascular disease (1 man and 10 women with a mean age
of 90 years, all nonsmokers; see Table 1) and 9 healthy vol-
unteers (7 men and 2 women with a mean age of 43 years,
all nonsmokers). The patients had been admitted to a geriat-
ric hospital because of difficulty in managing at home. Four
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KIKAWADA ET AL.
Table 1. Patient Characteristics
RESULTS
Patient
Number (y) (M/F)
Age Sex
Characteristics of BALF Cells
Disease
Nutrition
Duration
The total cell count and the differentiated count of BALF
cells are shown in Table 2. The total cell count was signifi-
cantly higher in the very elderly patients than in the healthy
volunteers. There were no significant differences in the per-
centages of macrophages or lymphocytes between the two
groups, but the mean absolute number of macrophages and
lymphocytes was higher in the elderly patients. The concen-
tration and percentage of neutrophils were also slightly
higher in BALF from the elderly patients compared with
BALF from the healthy volunteers, and the percentage of
eosinophils was slightly lower in the elderly group.
1
2
3
4
5
90
95
95
90
83
F
M
F
F
F
Multiple lacunar infarction Oral
Multiple lacunar infarction Oral
Multiple lacunar infarction Oral
3 mo
6 y
9 mo
4 mo
Alzheimer’s disease
Previous cerebral infarction
(right MCA)
Oral
Tube feeding
8 y
2 mo
6
7
93
97
F
F
Multiple lacunar infarction Oral
Previous cerebral infarction
(right MCA, left PCA)
Previous cerebral infarction
(right hemisphere)
Tube feeding
5 mo
8 mo
8
9
86
91
F
F
Tube feeding
Previous cerebral infarction
(left MCA)
Tube feeding
6 mo
2 mo
9 y
10
11
91
82
F
F
Multiple lacunar infarction IVH
Multiple lacunar infarction IVH
Two-Color Flow Cytometry
T-cell subsets in BALF are shown in Table 3. The per-
centage of CD3ꢀ T cells and CD4ꢀ T cells did not differ be-
tween the very elderly patients and the healthy volunteers,
but the percentage of CD8ꢀ T cells was higher in the elderly
group. The mean absolute number of CD4ꢀ T cells was also
higher in the elderly patients than in the healthy volunteers,
as was the mean absolute number of CD8ꢀ T cells. How-
ever, the CD4/CD8 ratio was lower in the elderly patients
than in the healthy volunteers. There were no significant
differences between the two groups in the percentage and
the mean absolute number of CD3ꢄCD56ꢀ cells (natural
killer cells) or CD4ꢀCD29ꢀ cells (memory cells).
Note: MCA ꢅ middle cerebral artery; PCA ꢅ posterior cerebral artery; IVH ꢅ
intravenous high calorie infusion.
performed on 500 cells under a light microscope. The remain-
ing BALF cells were resuspended in RPMI-1640 medium
(Gibco, Paisley, UK) supplemented with 10% heat-inacti-
vated fetal calf serum, washed twice in phosphate-buf-
fered saline (PBS), and finally adjusted to a concentration of
106/ml.
A total of 175 ꢁl of the BALF cell suspension was placed
into a polystyrene tube, and 4 ꢁl of a monoclonal antibody
was added. Fluorescein-isothiocyanate (FITC)-conjugated
anti-CD3 (Leu-4) and anti-CD4 (Leu-3a) antibodies and a
phycoerythrin (PE)-conjugated anti-CD8 (Leu-2a) anti-
body were purchased from Becton Dickinson (Mountain
View, CA). FITC-conjugated anti-CD29 (4B4) antibody
and PE-conjugated anti-CD56 (NKH-1) antibody were pur-
chased from Coulter Immunology (Hialeah, FL). For anti-
body staining, cells were incubated for 30 minutes in the
dark and then washed twice in cold PBS containing 0.1%
sodium azide. Next, the cells were resuspended in cold PBS
containing 0.5% paraformaldehyde and were analyzed us-
ing a flow cytometer. Data on 10,000 to 20,000 events were
stored in list mode files. A cell gate for lymphocytes was es-
tablished on the basis of forward and side light scatter, and a
computer system was used for data analysis.
DISCUSSION
Morphological and physiological studies on the aging of
the human lungs have been reported by many researchers.
Aging is characterized by loss of elastic recoil (9), loss of
elastin fibers (10), and dilatation of the air spaces (11), and
the chief physiological abnormality associated with aging is
a decreased diffusing capacity (12). Examination of the
BALF is a useful method for investigating inflammatory
cells, proteins, and cytokines in many pulmonary disorders.
However, few studies have assessed the cell characteristics
and immunological state of the lower airways using BAL in
normal elderly subjects (4–8), and there has been no assess-
ment of pulmonary immunity in elderly subjects with cere-
brovascular disease, especially among bedridden patients.
In the present study, we performed BAL in very elderly
subjects with cerebrovascular disease to investigate age-
related immunological changes of the lungs. We demon-
strated that the total cell count as well as the absolute and
relative neutrophil count were increased in BALF from the
All results are expressed as the mean ꢂ SD. Analysis of
variance or Scheffe’s test was used for comparison of me-
dian values between the groups. A p value of ꢃ.05 was con-
sidered significant.
Table 2. Cell Differentiation in Bronchoalveolar Lavage Fluid
Total Cells
(ꢆ105/ml)
Macrophages (%)
(ꢆ104/ml)
Lymphocytes (%)
(ꢆ104/ml)
Neutrophils (%)
(ꢆ104/ml)
Eosinophils (%)
(ꢆ104/ml)
Group
Control (n ꢅ 9)
0.97 ꢂ 0.37
90.79 ꢂ 5.02
8.84 ꢂ 3.33
87.00 ꢂ 8.01
22.92 ꢂ 8.81**
7.16 ꢂ 3.52
0.70 ꢂ 0.44
10.91 ꢂ 7.01
3.19 ꢂ 2.61*
0.78 ꢂ 0.48
0.09 ꢂ 0.09
1.91 ꢂ 1.30*
0.58 ꢂ 0.54*
0.49 ꢂ 0.53
0.06 ꢂ 0.08
0.09 ꢂ 0.30*
0.03 ꢂ 0.09
Pt (n ꢅ 11)
2.68 ꢂ 1.06**
Notes: Values are means ꢂ SD. Pt ꢅ very elderly subjects with cerebrovascular disease.
*p ꢃ .05 and **p ꢃ .001 as compared with the values obtained from the normal control subjects.

BALF IN VERY ELDERLY PATIENTS
M773
Table 3. T-Cell Subsets in Bronchoalveolar Lavage Fluid
CD3ꢀ (%)
(ꢆ104/ml)
CD3ꢄCD56ꢀ (%)
CD4ꢀ (%)
(ꢆ104/ml)
CD4ꢀCD29ꢀ (%)
CD8ꢀ (%)
(ꢆ104/ml)
Group
(ꢆ104/ml)
(ꢆ104/ml)
CD4/8
Control (n ꢅ 9)
70.13 ꢂ 13.06
0.53 ꢂ 0.39
75.69 ꢂ 11.89
2.45 ꢂ 2.08*
4.40 ꢂ 1.76
0.02 ꢂ 0.02
4.12 ꢂ 2.58
0.13 ꢂ 0.18
48.08 ꢂ 13.48
0.29 ꢂ 0.26
45.25 ꢂ 12.88
1.17 ꢂ 1.08*
41.06 ꢂ 13.33
0.07 ꢂ 0.08
43.35 ꢂ 13.31
0.57 ꢂ 0.67
19.54 ꢂ 6.51
0.11 ꢂ 0.09
30.98 ꢂ 8.47**
0.86 ꢂ 0.84*
2.59 ꢂ 0.70
Pt (n ꢅ 11)
1.61 ꢂ 0.77**
Notes: Values are means ꢂ SD. Pt ꢅ very elderly subjects with cerebrovascular disease.
*p ꢃ .05 and **p ꢃ .001 as compared with the values obtained from the normal control subjects.
elderly subjects compared with BALF from younger volun-
teers. Wallace and colleagues (5) described an increase of
alveolar macrophages in the lungs of nonsmokers with age
in a study using tissue sections. In addition, Meyer and col-
leagues (6,7) and Thompson and colleagues (4) reported an
increase of neutrophils in the BALF of elderly individuals.
These researchers concluded that low-grade inflammation
exists asymptomatically in the lower respiratory tract of
normal elderly individuals. Although these studies were
similar to our study, the mean age of the elderly subjects
was slightly higher in the present investigation (all subjects
were over 80 years old) compared with the other studies,
and all of the patients were bedridden and had cerebrovas-
cular disease. Generally, elderly patients with cerebrovascu-
lar disease have depression of the swallowing and cough re-
flexes and may suffer from a high frequency of aspiration
pneumonia. Therefore, it may be considered that, in elderly
persons, an increase of neutrophils in the BALF suggests
continuous inflammation of the lower respiratory tract due
to silent microaspiration.
viduals showed some overlap with the features of peripheral
airway disease associated with smoking or COPD. There-
fore, it is possible that irritants such as air pollution may
cause chronic inflammation in the lower respiratory tract of
elderly individuals and that these inflammatory changes
may increase with aging.
In summary, we investigated the characteristics of BALF
in very elderly subjects with cerebrovascular disease, and
we demonstrated an increased percentage of neutrophils, an
increased number and percentage of CD8ꢀ T cells, and a de-
crease of the CD4/CD8 ratio when compared with younger
volunteers. It is unclear whether these BALF data from our
elderly subjects suggest all alterations of the lungs related to
aging. However, our findings suggest that subjects with
cerebrovascular disease may have continuous inflammation
of the lower respiratory tract due to silent micro-aspiration.
There may also be a possibility that host pulmonary de-
fenses vary with the aging process.
Acknowledgment
We also found a high percentage of CD8ꢀ T cells and a
decreased CD4/CD8 ratio in BALF from the elderly group
compared with BALF from the healthy volunteers. On the
other hand, Meyer and Soergel (8) described an increase
of CD4ꢀ T lymphocytes and an increase in the CD4/CD8
ratio in BALF from older subjects. Some researchers have
reported a decrease in the absolute number of peripheral
blood lymphocytes and CD3ꢀ T lymphocytes, including
both CD4ꢀ and CD8ꢀ subsets (13,14). In addition, the per-
centage and absolute number of CD3ꢀHLA-DRꢀ cells,
CD4ꢀCD29ꢀ cells, and natural killer cells have been shown
to increase with age (14–16). However, another investiga-
tion revealed that the percentage of high CD8ꢀ T lympho-
cytes and low CD4ꢀ T lymphocytes was associated with
mortality in very elderly individuals (17). In the present
study, our findings about CD4ꢀ and CD8ꢀ T-lymphocyte
subsets in the BALF of elderly individuals did not concur
with the results of Meyer and coworkers (6,8). This discrep-
ancy of BALF findings may be due to differences in the
mean age of the subjects in elderly group (82–97 years vs
65–78 years).
Address correspondence to M. Kikawada, Department of Geriatric
Medicine, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku,
Tokyo 160-0023, Japan. E-mail: kikawada@tokyo-med.ac.jp
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Received January 1, 2001
Accepted January 2, 2001
Decision Editor: John E. Morley, MB, BCh