Hyperplasia of Type 2 Pneumocytes Following 0.34 ppm
Nitrogen Dioxide Exposu re:
Quantitation by Image Analysis
RUSSELL P. SHERWIN, M.D.
VALDA RICHTERS, Ph.D.
Department of Pathology
University of Southern California
School of Medicine
2025 Zonal Ave.
Los Angeles, CA 90033
ABSTRACT. Swiss Webster male mice were exposed to intermittent 0.34 ppm
nitrogen dioxide for 6 wk. Quantitative image analysis showed increased Type 2
cell numbers in each of the three lobes measured, with and without adjustment
to alveolar wall measurements for lung volume normalization (e.g., P < .037 for
Type 2 cell number adjusted to alveolar wall perimeters, combined lobe analysis
of variance). The exposed animals dominated the upper quartile ranking of the
cell number/alveolar area ratio computations (P < .025), which implied the
presence of an especially susceptible subpopulation of animals. The Type 2 cell
increase is believed to result from damage and loss of Type 1 cells, the reversibili­
ty and progression of which are presently unknown. The data also suggest an in­
creased size of the Type 2 cell, and possibly slight atelectasis and/or edema of the
alveolar walls.
IN EARLIER STUDIES, a lactate dehydrogenase stain­
ing method was applied to the selection and quantita­
tion of Type, 2 pneumocytel~ tQllowing exposure of
guinea pigs t9 a supraambientT(:~cve'l of nitrogen dioxide
(N0 2 ).1,2 Subsequently, a se"miautomated image
analysis metQodology was applied to the same material
and a high level of correlation was found between the
manual and the automated quantitations. 3 . 4 An enlarge­
ment of the Type 2 cell was also shown to accompany
the increase in Type 2 cell number. s The increase in
Type 2 cell number, or Type 2 cell hyperplasia, implies
a corresponding damage to and loss of Type 1 cells. A
replacement of the thin Type 1 cell by cubiodal cells
has long been recognized as a common denominator
306
and early event for diverse kinds of destructive lung!'
diseases,6J and it is now well established that the Type
2 cell is a major contributor to this replacement. A
review of Type 2 cell hyperplasia in response to various
noxious agents has recently been presented by
WitschV while Evans and his associates have presented
data showing a correlation between the extent of Type
1 cell damage and Type 2 cell proliferation. 9
The present study is the first to test for a Type 2 cell
increase following an ambient level of N0 2 exposure
(0.34 ppm). It is also the first application of a computer
assisted image analysis methodology to a large volume
quantitation, i.e., approximately three million Type 2
cells counted, along with other measurements, in
0003-9896/82/3705-0306
Archives of Environmental Health
Sept
~
separate analyses of three lung lobes from 120 animals.
The short-term significance of finding a Type 2 cell
hyperplasia in the lungs of mice exposed to an ambient
level of N0 2 is some loss of functional reserves due to a
replacement of the ultrathin Type 1 cell by the relative­
ly thick Type 2 cell. The long-term significance is the
potential for irreversible damage to the Type 1 cell with
persistent Type 2 hyperplasia, as suggested by studies
of high level (12-18 ppm) N0 2 exposure,lO and
.ultimately a loss of alveolar function and/or structure
with progression of the l'lO 2 effects to the type 2 cell
and other components of the alveolus.
METHODS
A colony of 120 Swiss Webster male mice were
received as young adults having a mean weight of 20 g.
They were subdivided into two equal groups with ap­
proximately equal weight distributions. They were
housed in stainless steel environmental chambers that
were identical in construction and that provided a
horizontally directed laminar air flow. All handling pro­
cedures were common to each group, including cage
cleaning and changes of food and water supplies.
Animals were fed a standard mouse diet and were
given both food and water ad libitum. Chamber
temperatures were essentially identical at 22°C ± 2°C.
The relative humidity was that of the ambient environ­
ment, and was generally constant between 50% and
60%. Half of the animals in the colony were exposed to
0.34 ppm N0 2 for 6 hr/day, 5 days/wk, for 6 wk.
The details of the environmental chambers and the
N0 2 delivery system have been reported earlier, in­
cluding the use of a newly developed silicone fluid
method for supplying a highly controllable, constant
supply of N0 2 to the exposure chamber. 11 In brief,
N0 2 was applied to the air intake of the exposure
chamber through the continuous flow of a medical
grade 500 centistoke silicone fluid containing a stabi­
lized quantity of N0 2 • The inflow of air was filtered
(HEPA, fiberglass, and Purafil) and equally divided to
each chamber with a mixing device for the control
chamber to expose the incoming air to a pure silicone
fluid. The N0 2 levels in the chambers and in the room
were monitored through a fritted bubbler (Saltzman)
test, a continuous Saltzman fluid apparatus (Beckman),
and by a periodically calibrated chemiluminescent
detector (TECO).
Following the 6-wk exposure period, mice were
killed in groups of 15 pairs (30 animals) each day by an
intraperitoneal injection of 0.5 ml (60 mg/ml) sodium
pentobarbital. Each lung was removed from the chest
cavity with the trachea intact and placed on a dry ice
cooled Petri dish. The lungs were slowly, and by gentle
perfusion of the tracheobronchial tree, inflated with a
6% gelatin solution (pH = 6.8-7.0) until the lung
volume approximated that of the thoracic cage. The
lung was placed in a refrigerator to allow the gelatin to
solidify and then the lobes were separated, wrapped in
aluminum foil, and. quick frozen in liquid nitrogen. The
frozen lungs were transported on dry ice to a deep
freeze where they were stored at - 85°C. Frozen sec­
September/October 1982 [Vol. 37 (No. 5)J
tions (15 J-L) of each lobe (ten sections for each lobe)
were obtained and lyophilized for 1 to 1.5 hr. After
lyophilization, the slides were brought to room
temperature and a lactate dehydrogenase (LDH) reac­
tion carried out for eight of the ten sections. As a rule,
240 slide preparations, representing three lobes from
four pairs of animals, were obtained with each section­
ing procedure. The details of the LDH reaction have
been reported earlier. J,21n brief, after a reaction time Qf
generally 15 to 20 min, the incubating medium was
drained, and the gelatin removed by immersing the
slides in 38°C distilled water. The slides were then fixed
in 10% calcium formol, rinsed several times in distilled
water, air dried, and mounted in glycerin jelly.
The present study is based on three independent
quantitative image analyses of left lung, right upper
lobe, and right lower lobe. Each slide was divided into
four quadrants by two intersecting lines, and the
quadrants consistently numbered from one to four
clockwise. Orientation of apex and base was kept cons­
tant and the first of the eight sections taken was the
most lateral in the sagittal plane of the lung. With 120
animals, eight slides per lobe, and four fields per slide,
a total of 3840 fields per lobe were available for image
analysis, and for the th ree lobes a grand total of 11,520
fields.
Image analysis. One field within each of the four
previously demarcated quadrants of each lung section
was selected for analysis. The first field acceptable to
the one person responsible for the quantitation (K. K.)
was used for analysis, i.e., the field was considered to
be technically satisfactory for lung structure integrity
and for LDH staining of the Type 2 pneumocytes. Bron­
chi and vessels were moved out of the counting area if
sufficiently peripheral, or were deleted through the use
of an image editor. Focal areas of the lung section that
were technically unsatisfactory were also deleted.
The gray values for detection were selected as
follows. (1) For the Type 2 pneumocytes, one of two
discriminators was set at a level which resulted in no
change between the area of a Type 2 pneumocyte as
seen in the video display of the microscopic image, and
that observed in the positive electron image displayed
by the detector system. The numbers of Type 2 cells
detected were "fine-tuned" by observing how well the
electronic detection of the Type 2 cells was matched to
the "flagging" signal on the video display. Also, the
detected image and the field as seen through th'e
microscope were compared. Checking the accuracy of .
detection was facilitated through the selection of
reduced field areas (accept mode) and comparing hand
counts with the electronic counts. In addition, detec­
tion and measurement functions were checked at the
beginning and end of analytical "runs" through the use
of test grids supplied by the manufacturer. (2) A second
discriminator was used for the measurement of wall
area, alveolar wall perimeters, and linear intercepts
with respect to alveolar walls. Comparisons between
microscopic fields and electronic images were made to
effect the best representation of the alveolar wall area,
and at a level where background "noise" was minimal.
307
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The measurements of wall area were made with and
without a "sizing" factor (~1O p- thickness). A com­
plete listing of the measurements made and the com­
putations that were applied is provided in Table 1.
Type 2 cell identification. As stated in the initial
report, 1 all cells of the alveolar wall show some LDH
activity. However, an essentially specific selection of
the very strongly reacting Type 2 cell can be achieved
for image analysis quantitation 3- 5 by controlling the en­
zyme reaction time and the gray value settings of the
image analyzer. While rounded macrophageswithin
the alveolar lumina may also be strongly positive for
LDH, they are rarely present in the lung sections
analyzed since the gelatin inflation procedure used
washes out most of the luminal contents. Elongated
macrophages in the alveolar interstitium are excluded
by the relatively weak LDH response of the stretched
out cytoplasm and by a sizing factor of the image
analyzer. A comparison of Figure 1A (frozen section of
mouse lung and LDH reaction) with Figure 1B (1 p­
epoxy embedded section of mouse lung and LDH reac­
tion) shows that the LDH positive cells are Type 2 cells.
Identification of the Type 2 cells in the epoxy sections is
in accord with the criteria defined by Kauffman and her
associates. 12 A more detailed investigation of the Type 2
cell and macrophage populations based on ultrastruc­
tural cytochemistry will be reported separately.13 Of
pertinence, the latter study has shown that the excep­
tionally strong LDH response of theType 2 cell reflects
not only its large size and rounaed configuration, but
also a very strong LDH activity of mitochondria,
nuclear heterochromatin, and less consistently,
lamellar bodies. Of further pertinence, horseradish
peroxidase (HRP) labeling of macrophages, combined
with the LDH reaction, has shown a negative HRP up­
take by the LDH positive Type 2 cells and a random
distribution pattern for the HRP positive macro­
phages. 13 Cell population studies of the mouse lung are
very limited, but Haies and his co-workers have
reported a 3.2% macrophage population for the rat
lung as compared to 14.5% for the Type 2 cell. 14
RESULTS
The principal finding of the study is an increase in the
numbers of Type 2 pneumocytes in the lungs of mice
exposed intermittently to 0.34 ppm N0 2 for 6 wk. The
"Type 2 cell increa.?e was found in all three of the lung
compartments analyzed, i.e., the left lung, the right up­
per lobe, and the right lower lobe. Table 2 is a sum­
mary of the data analysis for representative measure­
ments of the left lung. Most measurements of Type 2
cell numbers showed increases for the exposed animals
at highly significant (P - 0) le\!els. The increases were
found with and without normalization to wall area, i.e.,
mean numbers of Type 2 cells per field, or mean
numbers of Type 2 cells per field divided by the
amount of wall area. Highly significant increases in
Type 2 cell numbers were also found for the right upper
lobe and the right lower lobe, as indicated in Table 3,
"all data by fields (t test)."
In addition to tests based on fields, an analysis of
308
Table I.-Measurements by Image Analyzer
"A.
Direct Measurements
1-3)
B.
Numbers of Type 2 cells, ~ 8 fl, 10 fl, and 12 fl in
diameter
4)
Area of Type 2 cells (~1O fl)
5)
Alveolar wall area (+ 10)
6)
Alveolar wall area (~1O fl sizing) minus Type 2 cell
area
7)
Perimeter of alveolar walls
8)
Linear intercepts of alveolar walls
Ratio Measurements
9-11)
Ratio of numbers of Type 2 cells to alveolar wall
area (Type 2 sizing of ~ 8 fl, ~ 10 fl, and ~ 12 fl)
12-14)
Ratio of numbers of Type 2 cells to area modified
alveolar wall area (3 Type 2 cell sizings with wall
area minus Type 2 cell area)
"
15-16)
Ratio of numbers of Type 2 cells to sizing modified
alveolar wall area (Type 2 cell ~ 10 fl and wall area
with ~ 10 fl sizing, with and without subtraction
of Type 2 cell area)
17)
Ratio of Type 2 cell area (~1O fl) to Type 2 cell
(~1O fl) number
18)
Ratio of Type 2 cell number (~1 0 fl) to alveolar wall
perimeter
19)
Ratio of alveolar wall perimeter to alveolar wall area
NOTE: Except for numbers of Type 2 cells, values are expressed in
picture point units per field. One picture point (Pixel) unit ~ 3.176
square micra.
variance (Table 3) was carried out for each of the three
lung lobes based on animal. The numbers of Type 2
cells (with and without adjustment to wall area) were
again higher for the N0 2 -exposed animals, but the dif­
ferences were statistically significant only for the right ".
lower lobe (Table 3). When the data from all three
lobes were combined and subjected to an analysis of
variance based on animal (Table 6), the increase of
Type 2 cells for the exposed animals was highly signifi­
cant for Type 2 cell numbers without alveolar wall nor­
malization (P < .001) and with normalization to
alveolar wall perimeter (P < .037), and of borderline
significance for normalization to wall area (P < .092).
To test for especially susceptible animals, or high
responders to N0 2 , Type 2 cell numbers were
evaluated according to upper quartiles by animal and
upper five percentiles by field. The levels of statistical
significance are presented in Table 3. For all tests, the_
exposed group of animals was dominant for high Type
2 cell/alveolar wall ratios, i.e., increased numbers of
cells per alveolus. The dominance was highly signifi­
cant (P < .05 to P - 0) for all analyses by field; the up­
per quartile analysis by animal was also highly signifi­
cant bufonly for the left lung (Table 4; P < .025).
The mean area per field of the Type 2 cells, Le., total
area of Type 2 cells per field divided by total number of
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Fig. lA. Lactate dehydrogenase activity of mouse lung. The Type 2 cells of the lung from a control animal are
located primarily at the corners of alveoli and have a characteristic perinuclear deposit of formazan pigment.
Ultrastructural histochemical studies have shown that the deposits are largely mitochondrial in location, and
that a heterochromatin selectivity for the nucleus contributes to the perinuclear emphasis. Other cells of the
alveolus, including macrophages in the walls, are weakly active for LDH and are not detected by the gray value
setting of the image analyzer. (X64 @ 35 mm negative)
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Type 2 cells per field, was also greater for the exposed
animal group but the level of significance was border­
line for the most part (Table 3), P < .02 (RUt) and P <
.1 (LL & RLU. In Table 5, an analysis of the combined
data from all lobes by animal, the Type 2 cell area dif­
ference is at a borderline level of significance (P <
.136).
The exposed animals also had a greater amount of
alveolar wall area, the increase being statistically signifi­
cant for analyses by field (Tables 2 and 3), but falling
short of significance by animal (Tables 3 and 4). When
the area of Type 2 cells was subtracted from the wall
area in the computations, the level of significance for
the difference was generally but not invariably
lowered, or resulted in differences that were not signifi­
cant. Note that in Table 4, Upper Quartile Analysis by
Animal,' there was no significant wall area difference,
but at the same time the ratio of wall area to number of
Type 2 cells (the inverted ratio was used to avoid small
fractions) was high Iy sign ificant.
The linear intercept measurement received very
limited analytical study; in general, the findings tended
to parallel those of the alveolar wall (Table 2).
The data analysis also included an evaluation of the
influence of lung site on the cell and wall findings.
Table 6 provides two examples of the quantitations.
September/October 1982 [Vol. 37 (No.5)]
Each lobe of the lung was cut in a consistent fashion,
beginning with the most lateral or peripheral surface of
the lung (No.1 slide) and ending with the most medial
or central lung section (No.8 slide). The eight slides
generally represented a total thickness of lung of ap­
proximately 150 J-t, i.e., usually 15 slices were made to
obtain the eight slides needed. While the tissue
thickness of the 16 slides represents a relatively small
percentage of the lung volume, a broad sampling of dif­
ferent parts of the lung is obtained, i.e., whole lung sec­
tions in the sagittal plane with the four sampling sites
being apical anterior and posterior, and basal anterior
and posterior of each lobe. A search for topographical
differences was limited to comparisons of the eight
slides, with the four fields of each slide averaged. The
analysis (Table 7) indicated that different levels of the
lung, from the periphery(lateral) towards the
hilum(medial), varied significantly in Type 2 cell
numbers, but not cell area, and varied significantly in
wall area and p_erimeters. The ratio of wall area to Type
2 cell number (inverted to avoid the small fraction of
cell number to wall area) was not different between
levels. The control and exposed animals had similar dif­
ferences, i.e., there were no differences with respect to
group. Of interest, statistically significant group-slide in­
teractions were noted with wall area (P = .034) and
309
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Fig. 1B. Epoxy embedded (1 IL) section of mouse lung. This preparation permits identification of the Type 2 cells
through finer cytostructural detail, in particular a surface or interstitial location of the Type 2 cell and rounded,
osmiophilic bodies that are often within vocuoles (arrows). Compare with the lDH section, and not that some
alveolar corners do not exhibit Type 2 cells in the plane of section. (X252 @ 35 mm negative)
with Type 2 cell numbers for those cells equal to or
greater than 12 It in diameter.
DISCUSSION
This is the first demonstration of an increase in Type 2
cells in the lungs of mice exposed to an ambient level
(0.34 ppm) of N0 2 • Earlier studies reported hyperplasia
and hypertrophy following the exposure of guinea pigs
to a supraam bient level (2 ppm NO 2).2-5 The increase in
Type 2 cell number was consistently present in the
three separate analyses of left lung, right upper lobe,
and right lower lobe (Tables 2 and 3). The strongest
support comes from the combined analysis of variance
(all three lobes; Table 6, notably: (1) increased
numbers of Type 2 cells per field (P < .001); (2) in­
creased numbers of Type 2 cells adjusted to alveolar
wall perimeter (P = .037); and (3) increased numbers
of Type 2 cells adjusted to wall area (P = .092).
Moreover, there appeared t6 be a subpopulation of
animals that was especially susceptible to the effects of
N0 2 • A ranking of numbers of Type 2 cells adjusted to
wall area showed the left lung of the exposed animals
to be clearly dominant (Table 4, Chi square (x 2 ) by
animal; P < .025).
The use of a ratio.computation of numbers of Type 2
cells divided by the amount of wall area, or perimeters,
310
is a means of controlling for variation in the final
volume, i.e., gelatin inflation of the lung was not strictly
controlled for a constant final volume, and field editing
varied with the needs of each lung section, However,
there are other controls for the evaluation of Type 2 cell
numbers, in particular the alternate processing and
analysis of 120 control and exposed animal lungs.
Variations due to technical factors would be expected
to cancel out over the 3840 fields (32 per animal)
analyzed. The analysis has shown that both the direct
measurements of Type 2 cell number and the ratio
computations indicate a Type 2 cell hyperplasia in the
lungs of the exposed animals. In fact, the ratio com­
putations understate the statistically significant in~
creases in Type 2 cell number since there was a trend
towards a greater amount of alveolar wall (area, in­
tercepts, and perimeters) for the exposed group of
animals (Tables 2, 3, 4, and 6).
The trend towards a greater amount of alveolar wall
area, although not statistically significant at times when
the Type 2 cell increase was highly significant (e.g:,
Table 4, upper quartile analysis) and although other­
wise not consistently increased at statistically significant
levels, -is nevertheless probably real. Table 3 shows a
consistent increase for all three lobes of the lung (t test
analysis), Also, the analysis of variance for the com-
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Table 2.-Partial Listing of Data Analysis of Left Lung
Control Animals
Mean
SO
Exposed Animals
Mean
SO
T
Value
df
Prob.2-Tail
-0
Direct Measurements
'­
229.2
108.2
250.9
123.2
5.31
3192
Area of Type 2 cells
2722.6
2265.4
3129.1
2885.2
4.42
3210
-0
Area of alveolar wall
4076.4
2352.1
4236.7
2351.6
1.93
3211
0.05
Perimeters of alveolar walls
2622.4
1180.6
2747.9
1231.4
2.95
3211
0.003
Linear intercepts of alveolar walls
7786.0
3526.6
8229.4
3749.4
3.45
3210
0.001
Number of Type 2 cells
Computer Ratios
Alveolar wall area -<­
Type 2 cell number
88.61
34.43
82.84
28.82
-5.16
3207
-0
Type 2 cell area .;­
Type 2 cell number
10.64
3.99
1091
4.94
1.70
3206
0.09
Alveolar wall perimeter -<­
Alveolar wall area
6.86
1.04
6.88
0.96
0.54
3210
0.59
Alveolar wall perimeter .;­
Number Type 2 cells
120. 8
-4.12
3072
-0
37.3
115.7
32.0
NOTES: Direct Measurements for this table: 1) Type 2 Cells 2: 101" (longest diameter). However, significance was the same for Type 2
Cells 2: 8 I" and 2: 121". 2) Alveolar wall area -<- 10. When alveolar wall areas were "sized:' i.e., wall thickness reduced by 10 1", or
reduced by subtracting Type 2 cell area, no significant differences were found, P < .44 and P < .14, respectively.
Ratio Measurements for this table: Type 2 cells 2: 101" (longest diameter) and alveolar wall area with thickness reduced by 10 1", i.e.,
same sizing for cells and wall area. However, all ratios of wall area to cell number, regardless of sizing for cells and wall area, gave
equivalent results.
-
Table 3.-Summary of Key Statistical Analysis'
.
Lung Lobes
Type of Analysis
Type 2
Number
Type 2 Cell No.
Wall Area
Type 2 Area
Type 2 No.
Wall Area
Alveolar
Perimeter
BV (ield.'
t-Test
All data
LL II
RUL#
RLL **
-0
-0
-0
-0
< .003
-0
<.1
< .02
<.1
< .05
-0
-0
<.003
-0
-0
Upper quartile t
RUL
RLL
-0
-0
-0
-0
-0
-0
<.1
-0
<.001
-0
Upper 5%
LL
RLL
RUL
-0
-0
<.002
-0
< .05
<.01
-0
< .003
< .8(NS)
< .18(NS)
-0
<.007
< .44(NS)
-0
-0
< .42(NS)
< .42(NS)
By animal
Chi square
Upper quartilet
LL
<.05
<.025
Analysis of variance§
RLL
< .002
NS
<.1
< .07
NS
<.01
• AII,results: findings for exposed animals greater than those of controls.
t See Upper quartile by Animal, below, for Left Lung Analysis.
t For RUL and RLL, no significant differences for the most part.
§ For RUL and LL, no significant differences.
II LL: Left Lung
# RUL: Right Upper Lobe
•• RLL: Right Lower Lobe
September/October 1982 [Vol. 37 (No.5)]
311
Table 4.-Chi-Square Analyses of Contingency Tables' (left lung)
Alveolar Wall Area +
Type 2 Cell Numbers
(> 10 ,,):1:
Number of Type 2 Cells
(~8
,,)t
l
Alveolaf Wall Area
Total
-­
U
-­
-­
-­
l
-U­
13
X
38
19
57
X
44
C
47
10
57
-­
C
41
85
29
114
Total
-- -­
Total
X' ~ 3.75 (P
<
.05)
16
-­ -­
X'
85
29
~
0.42 (NS)
Total
-L­
-­
Total
-­
U
-­
57
X
48
57
-­
C
37
-­
20
-­
57
-­
114
Total
85
29
114
5.60 (P
<
X'
~
9
57
.025)
, Animals are classified as being part of the upper quartile (U) or the remainder (L). A significant X indicates an
association between being in a particular group and belonging to the upper quartile. Measurements are mean
values per field.
t
For Type 2 cell (~1O ,,) and Type 2 cells (~12 ,,) of the left lung, (,05
<
P
<
.1).
:t: For right upper and lower lobes, P - O.
Table 5.-Examples of Data from left lung of One Exposed and One Control Animal'
Number of Type 2 Cells (~8 " Diameter)
Slide
Exposed (N ~ 50)
Control (N - 44)
1
413.21 ± 130.68
±
Alveolar Wall Area + 10
Slide
Exposed (N ~ 50)
373.95 ± 136.80
1
5153.46 ± 2389.66
4780.49
±
2471.45
4868.93
±
±
2046.57
4958.29
±
2144.32
2220.94
5125.63 ± 2443.20
Control (N = 44)
2
396.83
124.03
381.24
±
122.92
2
3
396.24 ± 152.46
386.21
±
135.83
3
4887.84
4
381.38 ± 146.46
368.42 ± 130.55
4
4724.43 ± 2266.62
4811.73
±
2227.94
5
381.46
±
129.49
380.45 ± 120.08
5
4693.89 ± 2065.82
5049.82
±
2199.71
6
370.63
±
133.03
±
117.47
6
4616.77 ± 2043.88
4593.34 ± 2121.44
7
396.43 ± 146.23
340.69 ± 104.91
7
4922.87 ± 2421.41
4342.06
±
1796.00
8
384.75 ± 141.29
369.32 ± 106.83
8
4696.41 ± 2100.02
4694.45
±
1773.50
350.02
Mean, Slides 1 - 8
390.10
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4820.42
4794.45
,
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NOTES: The alveolar wall area is given in pixel or picture point units. 1 pixel unit ~ 3.2 ,,'; conversion to area in
square micra requires a correction factor for area "erosion" by the 8 " sizing factor used.
, Mean values for each of eight slides (four fields per slide) for control and exposed animals. The lungs were sec­
tioned consistently in a lateral and medial order, with the first slide the most lateral and the eighth slide the most
medial.
bined three lobes showed an increase at a borderline
level of statistical significance (P < .07; Table 6). Un­
fortunately, limitations of the initial programming ob­
viate further evaluation of the wall thickening. Also, the
question of slight atelectasis cannot be answered. In
the latter respect, the almost identical ratios of
perimeters to wall area for the two groups, especially
for a t test analysis with a very high degree of freedom
(Table 2), may reflect slight atelectasis, in addition to
wall thickening, contributing to increases in both
perimeters and area. Speculatively, some increase in
wall thickness would be expected in view of demon­
strated increases in both the protein content of alveolar
312
lavage fluid 1s ,16 and of the lung tissue in general",'B
following ozone and N0 2 exposures.
One final consideration in support of an increase in
the numbers of Type 2 cells is a topographical factor.
There is strong evidence that the proximal alveoli, and
associated bronchioles, are the main sites of oxidant
damage. 9 Assuming that relatively little damage has .oc­
curred in the distal alveoli of the N0 2 exposed animals
of this study, the Type 2 cell increase that we report is
again conservative since the unselective analysis would
resuft in a "diluting out" effect by the distal alveoli.
Similarly, clustering (2 or more Type 2 cells abutting
each other) understates the hyperplasia. A more de­
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Table 6.-Key Findings of Combined Data Analysis: left lUAg, Right Upper lobe, and Right lower lobe
df
F*
Signif.
of F
.Numbers of Type 2 cells (~8 fl)
Group
lobe
Group x lobe
1
2
2
10.268
14.925
0.690
0.001
0.000
0.502
Alveolar wall area
Group
lobe
Group x lobe
1
2
2
3.294
59.592
0.796
0.070
0.000
0.452
Numbers of Type 2 cells (~8 fl) + alveolar wall area
(cell/area ratio)
Group
lobe
Group x lobe
1
2
2
2.854
48.097
0.008
0.092
0.000
0.992
Numbers of Type 2 cells (~8 fl) + alveolar wall perimeter
(cell/perimeter ratio)
Group
lobe
Group x lobe
1
2
2
4.378
47.531
0.326
0.037
0.000
0.722
Type 2 cell area
Group
lobe
Group x lobe
1
2
2
6.957
12.103
0.801
0.009
0.000
0.450
Type 2 cell area + Type 2 cell number
Group
lobe
Group x lobe
1
2
2
2.237
13.066
0.683
0.136
0.000
0.506
NOTES: For all analyses, the values were greater for the exposed animal group. The ratios of Type 2 cells to alveolar
wall area and to alveolar wall perimeters were calculated as inverse functions to avoid very small fractions, and conver­
sion gave increased values for the exposed group.
For all tests, residual was 327 and total was 352.
* F = F statistic.
Table 7.-left lung: Microscopic Slide as a Repeated Measure*
P Values
GS Interaction §
P Values
Group (G}t
P Values
Slide (S):I:
~lOfl
~ 12 fl
.371
.406
.514
.008
.014
.041
.134
.109
.070
Type 2 cell area
.431
.457
.101
Type 2 cell numbers
~
8fl
Wall area
.949
.036
.034
Alveolar wall perimeters
.819
.041
.100
.327
.120
.693 (d. Fig. 2)
Wall area
(~8
+
No. Type 2 cells
fl)
* Two-factor analysis of variance.
t G indicates whether significant differences exist between Exposed and Control Animals.
:I: S shows differences according to slide number (1-8).
§ GS indicates interactions between Sand G.
tailed area sizing is needed to "erode" the aggregates
for the evaluation of clustering.
In earlier studies/os an increase in Type 2 cell size as
well as number was found after N0 2 exposure. This
was also true for the present study, but at borderline
levels of statistical significance for the most part (Tables
3 and 5). An increase in size is in accord with cell
edema and other manifestations of cell damage recent-
September/October 1982 [Vol. 37 (No.5)]
Iy reported at the ultrastructural level. 19,20 Damage to
the Type 2 cell has serious implications for the ~ever­
sibility ofType 1 cell damage since the Type 2 cell is the
progenitor cell for the Type 1 cell.
The generally large variation in the data can be at­
tributed, in part, to technical factors mentioned earlier.
Many are being ameliorated in ongoing studies, as, for
example, the use of a shading corrector for the
313
analyzer and a detailed analysis of total and edited
fields. However, a large part of the variation is believed
to be due to individuality of lung sampling sites as well
as to the animals themselves. Table 7 demonstrates that
most of the measurements showed statistically signifi­
cant differences between the eight slides', i.e., com­
parisons of slide one for each of the 120 animals, and
two through eight thereafter as repeated measures.
There did not appear to be any group differences, but
there were statistically significant interactions. While
the limited analysis of sites does not permit conclu­
sions, it can be expected that the topographical dif­
ferences in structure and function that are well
recognized for the human lung will also be found to
some extent in all mammalian lungs.
Another major factor contributing to the variation of
the data is the presence of indigenous organisms in the
mammalian lung and the varied degree of opportunis­
tic expression of these organisms, Of the diverse kinds
of infectious and parasitic agents present in the mouse
lung as clinically silent or opportunistically expressed
pathogens, a relatively overlooked one is the unbigui­
tous retrovirus. The virus is well known for its relation­
ship to various types of neoplastic diseases, but there is
relatively little information on its potential for non­
neoplastic pathogen ic effects/D,22 and there is little if
any data on its relationship to lung disease in the
mouse. Germanely, we have noted a relationship be­
tween levels of retrovirus expression (high and low ex­
pressor strains of mice) and the frequency of macro­
phage congregation on lung cells in culture,23,24 and
N0 2 exposure has been shown to facilitate the expres­
sion of retrovirus in the spleen albeit not in the lung. 2s It
should be emphasized that specific-pathogen-free (SPF)
mice harbor the retrovirus, and that the SPF state is dif­
ficult to maintain in transit from the vendor and in the
laboratory. Moreover, there are many unresolved
problems concerning murine inflammations of the
lung, in particular the extent of subclinical lung disease
in the mouse and the agent or agents responsible for
endemic murine pneumonia. 26
The significance of the cell population change found
after 0.34 ppm NO 2 exposure, a level slightly higher
than the present air quality standard for California (0.25
ppm 1 hr average), is the potential of air pollution to ac­
celerate the depletion of structural and functional
reserves of the lung, That there is widespread lung
reserve depletion in the well population is evident from
just two aspects of the growing problem of lung
disease. There is a well recognized deterioration in
lung function with time, 27 and emphysema, one of
many Silently destructive lung diseases, is present in
more than trace amounts in the majority of adult lungs
from hospital 2B- 30 and Medical Examiner 30 autopsies,
While emphysema and chronic lung disease in general
are unquestionably exacerbated by air pollution, the
influence of pollution is believedto be relatively minor
compared to the role of cigarette smoking. 3D However,
Ishikawa and his associates have reported a marked in­
crease in the severity of emphysema that they attribute
to a syneristic effect with smoking. 31 The cell popula­
314
tion shift implied by the present findings, and by other
reports of Type 2 cell hyperplasia due to diverse kinds
of noxious agents, B are also in accord with a synergistic
effect of poor air quality on emphysema and lung
disease in general. In effect, a cell population shift is a
common denominator and early lesion for emphysema
and other destructive lung diseases. Further, em­
physema is basically an irreversible depletion of lung
reserves ("vanishing lung disease") that progresses
covertly from subtle cellular alterations to clinical
manifestations. Thus, it is generally agreed that symp­
toms and signs of emphysema are not reliably diagnos­
tic until at least 50% of lung reserves have been lost.
Clearly, there is a need for more sensitive pulmonary
function tests and for pathological measurements of '.~
lung reserve depletion, In the latter respect, the quan"i'
titation of Type 2 cell hyperplasia by image analysis afel'
fords a very high level of sensitivity for the measureJi~
ment of early adverse effects. For end stage quantita·~i
tion, e.g., inventories of alveoli according to absolute:
numbers and relatively structural integrity, there is
special need to extend image analysis quantitation to
the establishment of reversibility and the rate of reserve
depletion.
(1J
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* * * * * * * * * *
We thank Kestutis Kuraitis for technical assistance with the
analysis. Michael Jones, Nancy Chang, and Dr. Stanley Azen
with the data analysis, Dr. Arnis Richters with the NO, exposure, and
the Medical Science Imaging Group of the University of Southern
California (Robert Erbe and Dr. Werner Freil with the data processing.
This study was supported by Contract A6-218-30 from the State of
California Air Resources Board.
Submitted for publication April 23, 1982; revised; accepted for
publication August 3, 1982.
Requests for reprints should be sent to: Russell P. Sherwin, M.D.,
Department of Pathology, University of Southern California School of
Medicine, 2025 Zonal Avenue, Los Angeles, CA 90033.
**********
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,
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Health 27: 90-93.
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20. Gil, j., and Thurnheer, U. 1971. Morphometric evaluation of
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21. Bishop, j. M. 1980. The molecular biology of RNA tumor vi ruses:
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digenous retrovirus expression. Fed Proc 39: 621.
24. Richters, V.; Elliott, G.; and Sherwin, R. P. 1978. Influence of 0.5
ppm nitrogen dioxide exposure of mice on macrophage con­
gregation in the lungs. In Vitro 14: 458-64.
25. Roy-Burman, P.; Pattengale, P. K.; and Sherwin, R. P. 1982. Effect
of low levels of nitrogen dioxide inhalation on endogenous
retrovirus gene expression. Exp Mol Pathol 36: 144-55.
26. Lindsey, j. R.; Baker, H. j.; Overcash, R. G.; Cassell, G. H.; and
Hunt, C. E. 1971. Murine chronic respiratory disease.
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27. Bosse, R.; Sparrow, D.; Rose, C. L.; and Weiss, S. T. 1981.
Longitudinal effect of age and smoking cessation on pulmonary
function. Am Rev Respir Dis 123: 378-81.
28. Mitchell, R. S.; Walker, S. H.; Silvers, G. W.; Dart, G.; and
Maisel, j. C. 1969. Frequency and severity of anatomic' em­
physema in men over 40 dying in two Denver hospitals. Arch En­
viron Health 18: 667-70.
29. Roberts, G. H., and Scott, K. W. M. 1972. A necropsy study of
pulmonary emphysema in Glasgow. Thorax 27: 28-32.
30. Thurlbeck, W. M.; Ryder, R. c.; and Stern by, N. 1974. A com­
parative study 0f the severity of emphysema in necropsy popula­
tions in three different countries. Am Rev Respir Dis 109: 239-48.
31. Ishikawa, S.; Bowden, D. H.; Fisher, V.; and Wyatt, j. P. 1969.
The "emphysema profile" in two midwestern cities in North
America. Arch Environ Health 18: 660-66.
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September/October 1982 [Vol. 37 (No.5)]
315