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Morphometric characterisation of the fine structure of human type II pneumocytes.

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THE ANATOMICAL RECORD 243~49-62 (1995)
Morphometric Characterisation of the Fine Structure
of Human Type II Pneumocytes
HEINZ FEHRENBACH, ANDREAS SCHMIEDL THORSTEN WAHLERS,
STEFAN w. HIRT, FRANK BRASCH, DORTE RIEMANN,
AND JOACHIM RICHTER
Abteilung Elektronenmikroskopie, Zentrum Anatomie, Uniuersitat Gottingen, Gottingen
J.R.), Klinik fur Thorax-, Herz- und Gefapchirurgie, Medizinische
(H.F., A.S., F.B., D.R.,
Hochschule Hannouer, Hannouer (T.W.), and Klinik fur Herz- und Gefapchirurgie,
Christian Albrechts Uniuersitat, Kiel (S.W.H.), Germany
ABSTRACT
Background: Pulmonary type I1 pneumocytes have been
examined by scanning electron microscopy (SEM), transmission electron
microscopy (TEM), and morphometry in numerous mammals. Until now,
the fine structure of the human type I1 pneumocyte has not been studied by
means of morphometry.
Methods: Eleven human donor lungs, which could not be made available
for a suitable recipient, were preserved with Euro Collins solution (ECS)
according to clinical organ preservation techniques. The lungs were fixed
via the airways. Systematic random samples were analyzed by SEM, TEM,
and classical stereological methods.
Results: Type I1 pneumocytes showed normal fine structural characteristics. Morphometry revealed that although inter-individual variation due
to some oedematous swelling was present, the cells were in a normal size
range as indicated by an estimated mean volume of 763 k 64 pm3. The
volume densities were: nucleus 21.9 & 2.2%,mitochondria 5.8 k 0.9%,lamellar bodies 9.8 f 3.6%, and remaining cytoplasmic components 62.4 2.9%of
the cell volume. Since the inter-individual variations in the volume densities referred to the cell may, to variable degrees, reflect the variation in the
reference space, the volume densities referred to the constant test point
system and the respective volume-to-surface ratios were used for interindividual comparisons. These parameters indicate that lamellar bodies
were independent of cellular swelling, while mitochondria < nucleus <
remaining cytoplasmic components increased in size with increasing cell
size.
Conclusions:Two to 7.5 hours of cold ischemia following ECS preservation do not deteriorate the fine structure of type I1 pneumocytes of human
donor lungs. For reliable assessment of fine structural variations, morphometric parameters are required that are independent of variations in cell
size. 0 1995 Wiley-Liss, Inc.
*
Key words: Human lung, Type I1 pneumocytes, Lamellar bodies, Surfactant, Electron microscopy, Morphometry, Organ preservation,
Euro Collins solution
The fine structure of pulmonary type I1 pneumocytes of all major components of the alveolar lining material
has qualitatively been described in detail both by termed surfactant (Askin and Kuhn, 1971; Chevalier
means of scanning (SEM) and transmission electron and Collet, 1972; Voorhout et al., 1993; Young et al.,
microscopy (TEM) in various mammalian species (e.g., 1993). The surfactant material, which is composed of
Stratton, 1978; Takaro et al., 1979; Crapo et al., 1982; about 90-95% lipid, predominantly phospholipids, and
Kliewer et al., 1985; Shimura et al., 1986; Vincent and
Nadeau, 1987; Ten Have-Opbroek et al., 1988; Shibamot0 et al., 1989; Snyder and Magliato, 1991; Lakritz
et al., 1992) including man (e.g., Weibel, 1963; McDouReceived December 14, 1994; accepted April 6, 1995.
gall and Smith, 1975; Stratton, 1984; Weibel and TayAddress reprint requests to Dr. Heinz Fehrenbach, Abt. Eleklor, 1988). Autoradiographic and biochemical studies tronenmikroskopie, Zentrum Anatomie, Universitat Gottingen,
clearly showed that type I1 pneumocytes are the source Kreuzbergring 36, D-37075 Gottingen, Germany.
0 1995 WILEY-LISS, INC
50
H. FEHRENBACH ET AL.
5 -10% protein (Hawgood, 19911, is intracellularly deposited in special storage organelles termed lamellar
bodies, and is excreted upon various stimuli (Wright
and Dobbs, 1991; Van Golde et al., 1994).
As alveolar surfactant plays a major role in the
maintenance of the normal respiratory function of the
mammalian lung, pathogenetic alterations of type I1
pneumocytes have been claimed in some instances to
be responsible for the development of surfactant related respiratory dysfunction (e.g., Holm and Matalon,
1989; Seeger et al., 1990; Gregory et al., 1991; Uhlig et
al., 1995). Recent studies indicate that impairment of
the surfactant system might be one of the factors that
are involved in limiting the respiratory function
achieved after lung transplantation (Klepetko et al.,
1990; Veldhuizen et al., 1993; Erasmus et al., 1994).
Hence, surfactant replacement therapy in lung transplantation (Novick et al., 1994) and in adult respiratory distress syndrome (Haslam et al., 1994, Heikinheimo et al., 1994) is coming into focus.
Although a number of morphometric studies of human lungs have been performed (Weibel, 1963,1979b;
Cassan et al., 1974; Gehr et al., 1978; Crapo et al.,
1982;Zeltner et al., 1987; Stone et al., 1992a,b),human
type II pneumocytes, however, have not yet been studied in detail by means of morphometry. As TEM based
morphometry has recently been shown to be a helpful
tool in the assessment of human donor lung quality
(Fehrenbach et al., 19941, this study aims at the morphometric characterization of the type I1 pneumocyte of
human donor lungs. Today, donor lung preservation by
means of flush perfusion of the pulmonary artery with
cold modified Euro Collins solution is one of the most
widely and successfully applied procedures for transplantation purposes allowing periods of ischemia of
about 7 hours (e.g., Wahlers et al., 1991). Ischemic
stress is well known to result in cellular membrane
damage in various organs (e.g., Gutierrez, 1991). Since
the cell and its various components may subsequently
respond with oedematous swelling to different degrees
(Schmiedl et al., 1990, 1993; Schnabel et al., 1991),
close attention was given to the investigation of the
relationship between parameters characterizing the
size of type I1 pneumocytes and parameters characterizing its components, with special emphasis to the surfactant-storing lamellar bodies. Hence, the study presented provides a basis for future studies of alterations
of the human surfactant system as may occur, e.g., during organ preservation and ischemia, due to experimental conditions in cell culture systems, or as a consequence of pathogenetic processes.
MATERIALS AND METHODS
In 11 cases of clinical single lung transplantations
performed a t the division of cardiothoracic and vascular surgery of Hannover Medical School, Germany
(head of the division: Prof. Dr. H. G. Borst), the contralateral donor lungs were studied by means of electron microscopy provided that they could not be made
available for a suitable recipient by The Eurotransplant Foundation Centre, Leiden, The Netherlands.
Due to distant organ procurement, donor lungs are
generally subjected to a period of ischemia, i.e., the
time period during which the organ is excluded from
circulation. To minimize the effects of ischemia and to
achieve the more favourable condition of cold ischemia,
various organ preservation procedures have been developed, which in general include a lowering of the
organ's temperature (e.g., Wahlers et al., 1986). In this
study, organ preservation of the donor double lung
block was performed according to present standard
clinical procedures as described earlier (Wahlers et al.,
1991). Following pulmonary arterial perfusion with
cold modified Euro Collins solution, the donor lungs
were stored at low temperature in the same solution
until transplantation. Left and right donor lungs were
separated immediately before transplantation. While
one donor lung was transplanted, the contralateral donor lung was fixed by instillation via the airways to
ensure rapid and uniform fixation of the whole organ
for subsequent fine structural examination. Thus, the
fine structural and morphometric data obtained are
representative of the whole organ, and differences resulting from sampling bias as may be seen in autopsy
or biopsy specimens (Bachofenand Bachofen, 1990) can
be excluded.
The period of cold ischemia of the donor lungs studied ranged between 121 and 458 minutes (mean value
s.d. of n = 11lungs: 275.5 88.2 minutes). The age
of the donors studied was in the range of 17 to 52 years
(mean value 2 s.d. of n = 11lungs: 32.3 12.6 years).
Six cases (91102; 91/05; 91/06; 91/10; 92/05; 92/06) were
characterized by excellent postoperative respiratory
function in the respective recipient as well as by absence of macroscopical contusion areas in the morphometrically examined contralateral donor lung. In 3
cases (92102; 92/03; 92/04), macroscopical inspection of
the contralateral donor lung showed areas of contusion.
However, good to excellent postoperative respiratory
function was achieved in the patient receiving the respective twin lung. In another 2 cases (91101; 92/01),
the morphometrically examined contralateral donor
lung exhibited normal macroscopical aspect, while only
mediocre postoperative respiratory function was
achieved in the respective recipient.
*
*
*
Fixation and Sampling
After separation of the double lung block in the operation theatre, the contralateral donor lung was immediately fixed, i.e., approximately at the time of
transplantation as described recently (Fehrenbach et
al., 1994). Briefly, a mixture of 1.5% glutaraldehyde
and 1.5%paraformaldehyde in 0.1 M cacodylate buffer
(pH = 7.35; buffer osmolality = 300 mOsm/kg) was
instilled via the airways at a pressure of 25 cm H,O.
Instillation was continued until the flow of the fixative
ceased automatically. After storage in the same fixative for 12.5 to 54.8 hours at 8"C, a standardized systematic sampling procedure was performed following
Cruz-Orive and Weibel(1981) and Miiller et al. (1981)
to obtain isotropic uniform random samples of each
lung for SEM and TEM examination, respectively.
Briefly, the lungs were cut into 2 cm thick slices starting apically at a random point. Then, a double-test
point system (18 A-points, d, = 12 cm; 40 B-Points, d,
= 6 cm) was superimposed at random on the lung
slices. Samples were taken at all points that hit parenchymal regions, with A-points determining samples to
be processed for SEM, and B-points determining samples to be processed for TEM, respectively.
51
MORPHOMETRY OF HUMAN TYPE I1 PNEUMOCYTES
Tissue Processing
For SEM, approximately 5 x 5 x 3 mm3 sized samples were processed according to the OTOTO method
described by Malick et al. (1975). After dehydration
through a graded series of alcohols, specimens were
critical point dried, and subsequently observed by
means of a DSM 960 (Zeiss, Oberkochen, Germany)
omitting metal-sputtering.
For TEM, approximately 1mm3 cubes of pulmonary
parenchyma were processed according to Fehrenbach
et al. (1991). Processing was standardized using a n automated tissue processor Histomat (bio-med, Theres,
Germany). Briefly, after several buffer rinses, samples
were post-osmicated, stained en bloc with uranyl acetate, dehydrated through a graded series of ethanol,
and transferred to Araldite via propylene oxide. Finally, samples were embedded in Araldite at random
orientation. Five isotropic ultrathin sections of at least
10 tissue blocks per lung were collected on Formvarcoated 1 x 2 mm slot grids and counter stained with
lead citrate by means of a n Ultrostainer (Leica, Hamburg, Germany). Qualitative and morphometric TEM
analysis was performed using a Zeiss EM 10A. To
study the fine structure of lamellar bodies a t high magnifications, ultrathin sections of approximately 40 nm
were examined by zero loss-filtering with a Zeiss CEM
902, which allows for the examination of unstained ultrathin sections at high contrast (Egle et al., 1984).
Morphometry
For morphometric analysis of type I1 pneumocytes, 5
random samples were studied in each case. Randomly
orientated, isotropic ultrathin sections were examined
systematically. Starting at a random point outside, the
whole section was examined along a grid of uniformly
distanced, straight lines. Micrographs were recorded of
all the cells of each section that were hit by the lines
and that were separated by at least one distance unit.
Micrographs were taken at a primary magnification of
x 4,000, to ensure that the whole cell was recorded.
The final magnification after photographic reproduction was determined by means of a calibration grid to
be x 11,450. Calibration was performed during each
photographic session.
A total of 748 profiles of type I1 pneumocytes were
analysed (mean s.d. of n = 11 lungs, 68 2 8.2 cell
profiles per lung), which due to the sampling procedure
described can be expected to meet the demands of isotropic uniform random (IUR) samples. For morphometric analysis of the cell and its components, a multipurpose coherent test system was used for point and
intersection counting (324 points, 162 test lines, length
d = 10 mm) according to classical stereological techniques (comprehensive reviews: Weibel, 1979a, 1990;
Chang et al., 1991; Bolender et al., 1993). In each case,
a minimum of 2,000 points falling on type I1 pneumocytes were counted (mean 2 s.d. of n = 11lungs, 3,855
1,497 points), so that structures accounting for 5%or
more of the total cell volume could be determined with
a standard error of less than 10% of the mean (Weibel,
1979a: p. 114).
The following morphometric parameters were recorded for each tissue sample according to the formulas
given in Table 1.
*
*
TABLE 1. Formulas used for calculation of the
morphometric parameters
Volume densities
m
C P cell,
VV(ce1litest)
i= 1
m x
pm3/pm3
Ptest
m
C
P compi
i=l
2P
pm3/pm3
cyto,
i=l
m
CPcompi
a
i=l
mx
pm3/pm3
Ptest
Surface-to-volume ratios
SVR,,)
&Xi
i=l
pm2/pm3
Volume-to-surface ratios
VsR,,,
1
pm3/pm2
S,R(x)
Cellular mean caliper diameter
H
k x vsR(cell)
Mean cell volume
V
4
3
pm3
Cell = total type I1 pneumocyte; comp = compartment (nucleus,
lamellar bodies, mitochondria, or remaining cytoplasmic components,
respectively); cyto = cytoplasm (total cell without nucleus); test =
coherent test system; P,,,, = number of points of the coherent test
system ( = 324 points); P, = number of points falling on respective
structure (x = cell, comp); I, = number of test line intersections with
respective structure (x = cell or comp); d = test line length in micrometer; a = constant, emperically determined to be 4.42; i = individual cell profile; m = number of cell profiles of a given ultrathin
section (= tissue block).
The cell
The volume density of type I1 pneumocytes was determined with reference to the test system Vv(cell/test)
A
ER
G
IC
L
M
MV
N
PC
VSR
V"
Abbreviations
Alveolar space
Endoplasmic Reticulum
Golgi apparatus
Interstitial cell
Lamellar body
Mitochondrium
Multivesicular body
Nucleus
Projection core
Volume-to-surface ratio
Volume density
52
H. FEHRENBACH ET AL.
to obtain a n estimate of the variation in cell size. Additionally, the surface-to-volume ratio S,R(cell) and
the volume-to-surface ratio VsR(cell) were determined,
parameters which are independent of alterations of the
reference space. Based on the qualitative TEM and
SEM data (see Figs. 1,2), the cells can be regarded as
oblate ellipsoids and, thus, the mean caliper diameter
H was calculated according to Weibel (1979a: Table
6.1) with the shape constant k chosen to be 9.026. The
mean caliper diameter was used-to obtain a rough estimate of the mean cell volume V according to the formula given in Table 1.
several closely adjacent cells. In cases 91/01,92/04 and
92/06, TEM showed a n overall swelling of the cisternae
of the endoplasmatic reticulum and, in places, of mitochondria which was accompanied by a clearing of the
cytoplasmic matrix (Fig. lb). The cells contained various types of lamellar bodies (Fig. 31, including immature and mature bodies both of the hemispherical as
well a s of the multilamellar type, following the terminology of Stratton (1984). While in all cases multivesicular bodies were regularly seen, homogeneous lipid
bodies were observed only in a few of the several hundreds of type I1 pneumocytes examined.
The nucleus and the cytoplasmic components
The volume densities V, of nucleus (nu), lamellar
bodies (lb), mitochondria (mi), and all remaining cytoplasmic components (cy) were determined to obtain a n
estimate of the fine structural composition of the type
I1 pneumocytes. Volume densities were determined
with reference to the whole cell as Vv(comp/cell), and
with reference to the cytoplasm, i.e., the cell without
the nucleus, as V,(comp/cyto). As one has to take into
account that these reference spaces might show considerable variations, V,(comp) were also calculated with
reference to the test point system a s V,(comp/test). In
addition, the volume-to-surface ratios V,R(comp) or in
t u r n the surface-to-volume ratios SvR(comp) were determined, parameters which are indicative of the size
of the respective structures, and which are independent
of variations of the reference space.
At the magnification used for morphometric analysis, the type I1 pneumocyte profiles account for only a
fraction of the test system. To facilitate comparison and
graphic presentation, the discrete values of V,(comp/
test) obtained from a n individual ultrathin section
were multiplied with a constant a, which was empirically determined to be a = XPtest/XPcell= 4.42. Thus,
values of dimensions comparable to the values of
Vv(comp/cell) were obtained.
Statistics
Data referring to individual tissue samples are given
as discrete values obtained according to the formulas
given in Table 1. Data referring to individual lungs are
given a s mean values f SEM of five tissue samples
unless stated otherwise. Data referring to several
lungs are given a s mean values f S.D. unless stated
otherwise. Correlation between variables was performed according to standard least-squares linear regression. The levels of significance P of the correlation
coefficients r are given according to Sachs (1992: Table
193). The condition of P c0.05 is considered to be significant.
RESULTS
Qualitative Scanning and Transmission
Electron Microscopy
The type I1 pneumocytes of the eleven human donor
lungs studied exhibit the normal fine structural appearance, as seen both by TEM (Fig. 1)and SEM (Fig.
2). In detail, however, inter- as well as intra-individual
variations were observed. While most frequently, type
I1 pneumocytes were single epithelial cells widely separated from one another, in some cases (91/01, 91/10,
92/01) they could be seen to constitute groups or rows of
Morphometry
The cell (Fig. 4)
The volume density of the type I1 pneumocytes referred to the test system V,(cell/test) varied between
the tissue samples of a n individual lung, and considerable differences were noted from one case to another.
As the data histogram of all 55 tissue samples analysed
largely resembles a gaussian distribution (Fig. 4A), the
type I1 pneumocytes studied were considered to constitute a normally distributed population of cells. Correspondingly, the volume-to-surface ratio V,R(cell) as
well a s the respective surface-to-volume ratio SvR(cell)
showed normally distributed variations. Both parameters were significantly correlated with V,(cell/test). As
was shown for V,R(cell), the correlations were significant not only at the level of the tissue samples (Fig. 4B)
but also at the level of the organs (Fig. 4C). Notably,
highest values of V,R(cell) were observed in both cases
showing macroscopical contusions (92/02; 92/04), as
well a s in case 91/01, one of the 2 cases (91/01; 92/01)
characterized by mediocre postoperative respiratory
function in the corresponding recipient.
On the basis of V,R(cell), the mean caliper diameter
was calculated according to Weibel(1979a) to be 9.4 to
12.6 pm (mean ? s.d. of n = 11lungs: 10.8 f 1.0 pm).
The estimated mean volumes of the t pe I1 pneumocytes were in a range between 457 pm2and 1,160 pm3
(mean f SEM of n = 11 lungs: 763 f 64 pm3), which
is similar to the mean cell volumes of 613 pm3 to 1,483
pm3 (mean t SEM: 889 +- 101 pm3) reported for 8
normal human lungs (Crapo et al., 1982: Table 2).
The nucleus and the cytoplasmic components
Due to this considerable variation in the size of the
cells, the volume densities of nucleus, lamellar bodies,
mitochondria, and remaining cytoplasmic components
were given not only with reference to the cell and to the
cytoplasm, as V,(comp/cell) and Vv(comp/cyto) (Table
11, respectively but also with reference to the constant
test system, a s V,(comp/test).
Nucleus (Fig. 5 )
The volume density V,(nu/cell) of the nucleus of the
lungs studied ranged between 18.0 and 24.8% of the
respective cell volume (mean 2 s.d. of n = 11 lungs:
21.9 t 2.2%). Linear regression analysis did not indicate significant correlations between V,(nu/cell) or
V,(nu/cyto) and VsR(cell), respectively (Fig. 5A). However, if V,(nu/test), the nuclear volume density calculated with reference to the test system, or the volumeto-surface ratio VsR(nu), which is independent of the
MORPHOMETRY OF HUMAN TYPE I1 PNEUMOCYTES
Fig. 1. Fine structural appearance of human type I1 pneumocytes as
seen by transmission electron microscopy. The apical surface, facing
the gas containing alveolar space, bears numerous microvilli. Characteristic features of the basal surface are cytoplasmic processes
which may (arrows) or may not (arrowheads) perforate the basal lamina, and may then be in close association with interstitial cells. At the
lateral face, the cell membrane forms junctional complexes with adjacent type I or type I1 pneumocytes. Variations in fine structure
53
are most obvious in the appearance of the cytoplasmic components. a:
(case 91/05): nucleus, lamellar bodies, multivesicular bodies, and cisternae of the endoplasmic reticulum as well as the cytoplasmic matrix
are of normal appearance, while mitochondria may show partial loss
of matrix ( 0 ) . b: (case 91/01):Cisternae of the endoplasmic reticulum
are dilated, the basal part of the cell shows a clearing of the cytoplasmic matrix, and disintegration of cristae.
H. FEHRENBACH ET AL.
54
Fig. 2. Fine structural appearance of human type I1 pneumocytes as seen by scanning electron microscopy. While in some cells, the apical surface is characterized by the microvilli being restricted to a
marginal zone entouring a bald-like central patch, it may be completely studded with microvilli in others
(inset). In places, globular protrusions (arrowheads) may be seen, which probably are lamellar bodies
ready to be excreted.
reference space, were considered, strong positive correlations with V,R(cell) were observed (Fig. 5B, 5C). The
relationship between nuclear and cellular parameters
was apparent not only a t the level of the tissue samples
(not shown) but also a t the level of the organs.
Lamellar bodies (Figs. 6, 7)
The volume density of the lamellar bodies varied
considerably from lung to lung (Fig. 6). In 9 cases,
Vv(lb/cell) ranged between 5.3 and 10.3% of the respecs.d. of n = 9 lungs: 8.4 k
tive cell volume (mean
l.8%), and Vv(lb/cyto) was in the range of 7.0 to 13.6%
of the respective cytoplasmic volume (mean 2 s.d. of n
= 9 lungs: 10.8
2.3%). In 2 cases, the volume densities reached considerably higher values with Vv(lb/
cell) of 16.5% and Vv(lb/cyto) of 20.8% in case 91/06,
and V,(lb/cell) of 16.2% and V,(lb/cyto) of 19.9% in
case 92/05. Similar degrees of variation were seen as
concerns Vv(lb/test) (range of n = 9 lungs: 6.3 to
10.7%, mean s.d. 9.0 -C 1.4%; case 91/06: 17.5%; case
91/05: 17.4%).
Due to this considerable difference as compared to
*
*
*
the other cases, which awaits further investigation,
case 91/06 and 92/05 were excluded from the correlation analysis concerning lamellar body related parameters. V,(lb/cell) (Fig. 7A) as well as V,(lb/cyto)
showed a strong negative correlation with VsR(cell),
i.e., the bigger the cells or their respective cytoplasmic
volumes were, i.e., the more pronounced they were
swollen, the smaller was the lamellar body volume
fraction. However, neither V,(lb/test) nor VsR(lb) were
significantly correlated with VsR(cell) (Fig. 7B,C).
Mitochondria (Fig. 8)
The volume density V,(mi/cell) of the mitochondria
of the lungs studied ranged between 4.2 and 8.4% of the
respective cell volume (mean k s.d. of n = 11lungs: 6.2
1.3%). At the level of the tissue samples, V,(mi/cell)
showed a wider range of 2.4 to 10.2% (mean s.d. of all
55 tissue samples: 5.8 1.6%).Linear regression analysis revealed no correlation between V,(mi/cell) or
Vv(mi/cyto) and VsR(cell), respectively (Fig. 8A). In
contrast, the volume density referred to the test system
Vv(mi/test) (Fig. 8B) a s well as the volume-to-surface
*
*
MORPHOMETRY O F HUMAN TYPE I1 PNEUMOCYTES
55
Fig. 3.Lamellar body sub-structure of human type I1 pneumocyte as seen in the zero loss-mode of an
energy-filtering transmission electron microscope (Zeiss CEM 902). The ultrathin section was not
counter-stained with lead citrate. The lamellar body shown is of the hemispherical type according to
Stratton (1984).The concentric lamellae end on the sub-structured projection core material. The limiting
membrane (arrows) of the body forms a projection (open arrowhead), which is in close association with
cisternae of the endoplasmic reticulum.
ber of studies (Campiche et al., 1963; Weibel, 1963;
Balis and Conen, 1964; McDougall and Smith, 1975;
Gehr et al., 1978; Stratton, 1978; Weibel and Taylor,
Remaining cytoplasmic components (Fig. 9)
1988). Morphometric studies of human lungs, however,
At the level of the organs, the volume density of the have focussed on the composition of the pulmonary paremaining cytoplasmic components V,(cy/cell), i.e., of renchyma, and special attention has been given to the
all the cellular components other than nucleus, lamel- relationship of structure and function at the level of the
lar bodies, and mitochondria, ranged between 56.6 and air-blood barrier (Weibel, 1963, 1979b; Cassan et al.,
66.7% of the respective cell volume (mean 5 s.d. of n = 1974; Gehr et al., 1978; Crapo et al., 1982; Zeltner et
11 lungs: 62.4 2.9%). At the level of the tissue sam- al., 1987; Stone et al., 1992a). Until now, however, a
ples, Vv(cy/cell)ranged between 49.4 and 76.6% (mean detailed morphometric study of the human type I1
t s.d. of all 55 tissue samples: 62.4 k 5.6%).While only pneumocyte and its fine structural components has not
a very weak positive correlation of Vv(cy/cell) with yet been presented.
The lungs examined in this study were obtained durVsR(cell) (Fig. 9A) was observed, V,(cy/test) showed a
strong positive correlation with VsR(cell) (Fig. 9B). ing clinical single lung transplantations from human
This relationship was observed not only at the level of donors, whose contralateral lung could not be made
the tissue samples (Fig. 9B) but also a t the level of the available for a suitable recipient. Clinical organ preservation of the donor double lung block was performed
organs (Fig. 9C).
according to standard procedures with Euro Collins soDISCUSSION
lution (Wahlers et al., 1991), which has recently been
The normal human type I1 pneumocyte has qualita- shown to yield an adequate preservation of the parentively been described in detail by transmission (TEM) chymal fine structure in lungs subjected to 3.5 to 5
andlor scanning electron microscopy (SEMI in a num- hours of cold ischemia (Fehrenbach et al., 1994). In the
ratio V,R(mi) (Fig. 8C) were positively correlated with
VsR(cell).
*
56
H. FEHRENBACH E T AL.
present study, very similar conditions were noted, i.e.,
2 to 5.5 hours of cold ischemia. In only one lung, a
period of 7.5 hours of cold ischemia following ECS preservation was achieved. However, there were no indications that the prolonged duration of ischemia had any
detrimental effect to the fine structure of the tvpe I1
" _
pneumocytes. This is in line with recent experimental
data from rat lung transplantations that showed surfactant function being impaired only after onset of reperfusion but not after ischemia alone (Erasmus et al.,
1994).
A rough estimation of the mean volume of the cells,
calculated from the volume-to-surface ratio of the cell;
yielded a range of 457 pm3 to 1,160 pm3 (mean SEM
of n = 11 lungs: 763 ? 64 pm3). Similar mean cell
volumes of type I1 pneumocytes, 613 pm3 to 1,483 pm3
(mean SEM: 889 k 101 pm3) have been given for 8
normal human lungs (Crapo et al., 1982). A mean volume of 815 pm3 ( 2 SEM of 144 pm3)has been reported
from the morphometric analysis of 4 resected human
lung lobes (Stone et al., 1992b). Thus, a gross oedematous swelling of the cells analysed in this study can be
excluded. Similar variations in the values reported for
the mean type I1 pneumocyte volume are seen comparing different studies of normal adult Spra ue Dawley
rats: 440 pm3 (Young et al., 1981), 525 pm8 (Young et
al., 19851, 385 pm3 (Randell et al., 19911, and 443 pm3
(Stone et al., 1992b),respectively. Moreover, a comparative study of lungs of 10 mammalian species including
man revealed that there was no significant change in
the size of type I1 pneumocytes with increasing body
weight (Stone et al., 199213). Hence, inter-species differences in the size of type I1 pneumocyte appear to be
relatively small as was also indicated by the volume
densitv of the cvtodasm referred to the test svstem
(Massaro et al., "1975). However, substantial inter-individual differences may be seen. This in line with our
study, as indicated by the inter-individual variations
in the volume-to-surface ratio of the cells and the volume density of the cells referred to the test system.
Apart from numerous reports of alterations during
ontogenetic processes or due to specific pathogenetic
conditions, the detailed consideration of which is beyond the scope of this paper, comparative studies of the
fine structure of type I1 pneumocytes of various mammalian species have revealed qualitative and quantitative inter-species differences as concerns the cytoplasmic components. Qualitative inter-species differences
have been reported for example, to be present with respect to the sub-structure of lamellar bodies (Kikkawa
and Spitzer, 1969;Pattle et al., 1974; Stratton, 1984).In
agreement with Stratton (19781, the lamellar bodies of
the human donor lungs studied are of the hemispherical
*
CELJ LAR PARAM ETERS
10
9
8
7
6
5
4
3
2
1
4
2.0 - r
n
E
a.
0.8
-
0.4
1
1.6
Y
1.2
m
>
-
12 20
28
y
I
0.0
0
1.6
I
I
10
I
-
+ 0.427
0.032~
I
20
52
36 44
I
I
30
I
I
I
50
40
r = 0.824. p<O.OOl
n
E
a.
*
/o!
U
U
m
>
+
y = 0.024~
0.0.i.P
0
I
'
20
I
25
'
I
30
0.825
'
I
35
Vv ( c e I I/t e st) [%]
Fig. 4
f
Fig. 4. Morphometric data. A Histogram showing the distribution
of the discrete values of the volume density of the cell referred to the
test system V,(cell/test) of all the 55 tissue samples analyzed. Each
count represents the result of the analysis of all type I1 pneumocyte
profiles collected from a n individual ultrathin section, which corresponds to an individual tissue block. The distribution of the discrete
values of V,(cell/test), which is a n indicator of the volume of the cells,
largely follows the corresponding Gaussian distribution curve. B,C:
There is a strong positive correlation of the volume-to-surface ratio of
the cell VsR(cell) with Vv(cell/test) not only at the level of tissue
samples (B) but also at the level of the organs (C). Values of the lungs
of those cases that showed contusion areas (92/02; 92/04) or that were
characterized by mediocre postoperative transplant function (91101;
92/01) are indicated.
As in Figs 5, 7-9, solid lines represent the linear regression curve,
the mathematical description of which is shown in the lower right
corner. Dashed lines represent the corresponding 95% confidence interval. The regression coefficients and levels of significance are displayed in the upper left corner.
57
MORPHOMETRY OF HUMAN TYPE I1 PNEUMOCYTES
TABLE 2. Volume densities and volume-to-surfaceratios of components of type I1 pneumocytes of various
mammalian species (mean values f s.d. unless indicated otherwise)
V,(nuicell)
VJmiicell)
V,(mi/cyto)
V,(lbicell)
VJlbicyto)
(%I
(%I
(%I
(%)
(%I
5.8 f 0.9
7.5 f 1.1
9.8 f 3.6
12.6 f 4.4
Species
Man
21.9
Baboon
28.9 f 2.4'
f 2.2
6.99 f 0.75'
Sheep
13.4 f 1.3
9.3 f 1.1
Macaque, old
32
f 3.1'
6.7 i- 0.7'
8.9 t 1.3
9.37 f 1.54'
Mongrel dog
22.7 i- 1.5'
Mongrel dog
12.4-16.5,
Phospholipid
stabilisation'
OsO,; UAc
oso,
oso,
oso,
oso,
8.9 t 3.4'
11.1 f 2.1
Macaque, young
VsR(lb)
(nm)
179 f 21
242
f
52,3
OsO,
Mongrel dog
9.1 f 1.5'
20.5 f 0.3'
oso,
oso,
Mongrel dog
9.4 t 0.Ol2
33.1 f .05'
050,
Rabbit
Rabbit
Rat. CFN-COBS
strain
Rat, Wistar
Rat, Sprague
Dawley
Rat, Sprague
Dawley
Rat, Sprague
Dawley
Rat, Sprague
Dawley
Rat, Fischer
Rat, Fischer
24.4 2 2.3' 236 i- 14.5'~~OsO,
13.6 f 1.7'
oso,
10.9 f 1.4'
4.9
f 0.1'
21.9 2 0.P
7.6 f 0.4'
8.9 f 0.2'
8.2
f 0.3'
12.9 f 0.3'
12.7 f 0.7'
26.9 t 0.6'
145 f 0.5'
OsO,; UAc
OsO,; UAc
oso,;
12.8 i- 1.5'
24 t 3
8.4 2 1
1 2 -t 1
OsO,; UAc
25
f 0.8
8.8 2 0.4
9.8 t 0.6
OsO,; UAc
25
Ifr
3
8 f 1
12 t 1
OsO,; UAc
9 f 1
8.4 f 0.33
15 f 1
15.9 0B3
21 2 2
25.9 t 1.63
oso,
*
Rat, Long-Evans
Mouse, C57BLi6J
strain
OsO,; UAc
18.6 f 0.6'
22.7
f
235 t 92,3
1.6' 229 f
Oso,
OsO.
References
This study
Fracica et al.,
1994
Shimura et al.,
1986
Shimura et al.,
1986
Lakritz et al.,
1992
Massaro and
Massaro, 1975
Shepard et al.,
1982
DeFouw and
Chinard, 1983
DeFouw and
Berendsen,
1977
Massaro and
Massaro, 1975
Gail et al., 1975
VidiC and Burri,
1981
Ishii et al., 1989
Massaro and
Massaro. 1973
Young et al.,
1985
Young et al.,
1991
Kliewer et al..
1985
Pinkerton et al.,
1990
Balis et al., 1988
Massaro and
Massaro, 1975
Massaro and
Massaro. 1975
'Postfixation with osmium tetroxide (OsO,) only, or additional en bloc staining with Uranyl acetate (UAc).
'Mean values t SEM.
3Calculated from original values.
,Range of values from n = 5 normal canine lungs.
and multilamellar type, and may contain the typical,
sub-structured projection core material (see Fig. 3). As
described by Shimura et al. (19831,lamellar bodies were
seen in close association with the cisternae of the endoplasmic reticulum, the membranes of which have recently been discussed to directly contribute to lamellar
body growth (Risco et al., 1994). We also regularly observed multivesicular bodies, which comprise several
structurally different types (Williams, 1987),and which
may play a role not only in the de novo formation of
lamellar bodies (Chevalier and Collet, 1972)but also in
the recycling andlor degradation of alveolar surfactant
(Young et al., 1993). Neutral lipid bodies, however,
which in cells isolated from human lungs were shown to
be sites of cyclooxygenaseactivity (Dvorak et al., 1992),
were only very rarely seen in our preparations, while
they have regularly been observed in canine lungs (Fehrenbach et al., 1991), and were abundantly present in
human type I1 pneumocytes cultured on EnglebrethHolm-Swarm tumour matrix (Edelson et al., 1989). An
increase in neutral lipid bodies has been reported with
age in monkeys (Shimura et al., 1986), and after haemorrhagic shock in dogs (Barkett et al., 1969).
Quantitative inter-species differences with respect to
lamellar bodies have been reported to be quite small as
indicated by the volume densities referred to the cytoplasm and by the surface-to-volume ratios (Massaro
and Massaro, 1975). However, there are differences in
the morphometric data reported by different authors
studying type I1 pneumocytes of the same species (see
Table 2). This may at least partly be due to the fact that
lamellar bodies contain high amounts of stored phospholipids (Hawgood, 1991: Van Golde et al., 19941, and
thus, are highly susceptible to the particular fixation
and tissue processing procedure applied (for review see,
e.g., Stratton, 1984). The application of en bloc staining
with uranyl acetate prior to dehydration and embedment, as was performed in our study, has recently been
shown to preserve the sub-structure of lamellar bodies
in conventionally processed samples in a way that is
close to the results obtained by special lipid-carbohydrate retention techniques (Fehrenbach et al., 1991).
Quantitative inter-species differences have additionally been reported with respect to mitochondria of type
I1 pneumocytes. Mitochondria1volume density referred
to cytoplasm for example has been reported to be pos-
58
H. FEHRENBACH ET AL.
NUCLEUS
LAMELLAR BODIES
25
bQ
Y
30
cI
cI
n
w
I
U
-<
n
n
-
t
0
t'
y
-
5.34x
+
15.43
I
I
I
I
1.0
1.2
1.4
1.6
mVr(lb/toot)
mVr(lb/odf)
aVv(lb/oyto]
20
15
10
5
0
Case
y
1.0
3 8 . 0 1 ~-20.29
1.2
1.4
Fig. 6. Morphometric data. Volume densities of lamellar bodies are
given with reference to the test point system VJlbitest), the whole
cell V,(lb/cell), and the cytoplasm V,(lb/cyto), respectively. Differences between individual lungs may vary considerably depending on
the reference space chosen for calculation of the lamellar body volume
density (compare, e.g., case 91/01and 91/02).While in most cases, the
values of V,(lb/test) and V,(lb/cell) are very similar and lower than
the values of V,(lb/cyto), in case 91\01 V,(lb/test) reaches a value
nearly twice the value of V,(lb/cell).
1.6
mitochondrial inner membrane and cristae has been
reported to be negatively correlated with the lung's
oxygen consumption (Massaro and Massaro, 1977). NoE
tably, the mean mitochondrial volume density V,(mi/
Y
cyto) of 7.5 -+ 1.1% as determined in our study for hu0.8
man type I1 pneumocytes, is very close to the value of
n
3
8.3% calculated according to the equation V,(mi/cyto)
c
= 7.1 + 0.1 x respiratory rate given by Massaro et al.
U
(1975: Fig. 21, on the basis of a value of 12 breaths per
0.4
Q)
minute given for the respiratory rate of a normal hu>
man being (Fishman, 1988: Appendix B).
Discrepancies in the morphometric data reported by
y
0 . 6 1 ~+ 0.07
I
I
I
different authors for a particular species, may also
0.0
arise from differences in the age of the individuals
1.0
1.2
1.4
1.6
0.0
analysed (Shimura et al., 1986), from factors related to
circadian rhythms (Ishii et al., 1989), or due to differVsR(cel1) [ p m ]
ences in the sampling procedure performed, which may
be particularly true if only a fraction of the whole orFig. 5. Morphometric data. Relationships of volume-to-surface ratio
gan has been available as, e.g., in studies of autopsy or
of the cell V,R(cell) and volume density of the nucleus referred to (A) biopsy specimens (Bachofen and Bachofen, 1990).
the whole cell V,(ndcell), (B) the test point system V,(nuitest), and
In most studies investigating the fine structure by
(C) the volume-to-surface ratio of the nucleus V,R(nu), respectively
means of morphometry, the volume densities of the nu(mean values t- SEM of 5 tissue samples per lung). While V,(nu/cell)
i s not correlated with V,R(cell) (A), significant positive correlations
cleus and the cytoplasmic components are given with
are seen between V,R(cell) and V,(nu/test) (B) and V,R(nu) (C) re- reference to the whole cell (VidiC and Burri, 1981;
spectively.
Kliewer et al., 1985;Young et al., 1985,1991;Pinkerton
et al., 1990; Lakritz et al., 1992) and/or with reference
itively correlated with the respiratory rate and the to the cytoplasm (VidiC and Burri, 1981; Massaro and
lung's oxygen consumption of a given species (Massaro Massaro, 1975; DeFouw and Berendsen, 1977; DeFouw
et al., 1975), while the surface-to-volume ratio of the and Chinard, 1983; Shimura et al., 1986; Snyder and
n
1.2
a
I
-
59
MORPHOMETRY OF HUMAN TYPE I1 PNEUMOCYTES
LAMELLAR BODIES
r
r
ae
n
15
10
M ITOCH 0 N D R IA
-
0571. p<O.OOl
z
’
<.-E
1OI-
0
5 ’
U
00
5 ’
>
-13.97~
y
0
0.0
1.0
I
I
1.2
1.4
>
+ 25.26
I
y
0
0.0
1.6
0
8
Q)
0.8
-
+
0.002~
I
I
1.2
1.6
5.82
I
2.0
I
’
1
t
l5
r
0.552, p<O.OOl
n
10
<
n
Q)
v
s
5
-5.38~
0
0.0
+
15.48
y
0
1.0
1.2
1.4
1.6
0.0
0.20
=t
U
0.20
5
U
m
>
1.2
- 0.18
5.28~
I
1.6
1
2.0
r = 0.361, pq0.01
a
0.18
0
0.12
0.16
0.14
0.16
U
n
v
0.8
-
n
n
E
I
IL/
0.00
0.0
v
1
5
+ 0.17
>
0.08
0
y = 0.001~
I
I
I
1
1.0
1.2
1.4
1.6
VsR(cel1) [ p m ]
0.00
L
0.0
0
y
0.8
1.2
0 . 0 2 9 ~ + 0.079
1.6
2.0
VsR(cel1) [ p m ]
Fig. 7. Morphometric data. Relationships of volume-to-surface ratio
of the cell V,R(cell) and volume density of lamellar bodies referred to
(A) the whole cell V,(lb/cell), (B) the test point system V,(lb/test), and
(C) the volume-to-surface ratio of the lamellar bodies VsR(lb), respectively (mean values ? SEM of 5 tissue samples per lung). V,R(cell)
shows a strong negative correlation with V,(lb/cell), while V,(lb/test)
and V,R(lb) are not correlated with V,R(cell).
Fig. 8. Morphometric data. Relationships of volume-to-surface ratio
of the cell V,R(cell) and volume density of the mitochondria referred
to (A) the whole cell V,(mi/cell), (B) the test point system V,(mi/test),
and (C) the volume-to-surface ratio of the mitochondria V,R(mi), respectively (each point represents a n individual tissue block). While
V,(mi/cell) is not correlated with V,R(cell) (A), weak but significant
positive correlations are observed between V,R(cell) and V,(mi/test)
(B) and VsR(mi) (C), respectively.
Magliato, 1991). However, as with each “density,” i.e.,
a ratio of two compartments, the observation of alterations is only meaningful if the reference space is
largely constant throughout the individual or the experimental groups studied as was pointed out, e.g., by
Massaro and Massaro (1983)and Bolender et al. (1993).
H. FEHRENBACH ET AL.
60
Variations in the size of cells or their cytoplasm, a s may
result from pathogenetic processes (e.g., Balis e t al.,
1988; Fracica et al., 19941, will be of considerable influence on the morphometric data obtained. This is
clearly illustrated by our study of the relationship of
cellular parameters and the volume densities of nucleus, lamellar bodies, mitochondria, and the remaining
cytoplasmic components, which were determined with
0
0
reference to the whole cell, the cytoplasm, and the test
point system, respectively. On the basis of data referring to these different reference spaces, opposite correlations of the respective volume densities to the volumeto-surface ratio of the cells VsR(cell) were obtained.
While, the lamellar body volume densities referred to
40
the cell V,(lb/cell) or to the cytoplasm Vv(lb/cyto) decreased with increasing VsR(cell), the volume density
y
8 . 2 7 ~+ 52.48
referred to the test system remained constant. The test
I
1
I
0
'
system was the only reference space with constant size,
0.0
0.8
1.2
1.6
2.0
while the cell and the cytoplasm showed considerable
variations. Therefore, the decrease in V,(lb/cell) and in
0
V,(lb/cyto) has to be interpreted as a n indication that
the lamellar bodies were diluted in a n increased cellular
120
or cytoplasmic volume, respectively, rather than as a n
indication of a decrease in stored surfactant.
100
On the other hand, the volume densities of nucleus
and mitochondria referred to the cell or to the cyto80
plasm remained more or less constant with increasing
60
VsR(cell), while the volume densities referred to the
test system showed a positive correlation with VsR40
(cell), a n index of cell size (V,(nu>: r = 0.633, P <0.001;
r = 0.552, P <0.001). Hence, we may conclude
Vv(mi):
20
that the larger the cells were, the bigger the nucleus
y
7 6 . 0 1 ~ 24.761
and the mitochondria have been. This was also reflected by the positive correlation of VsR(cell) with vs0.0
0.8
1.2
1.6
2.0
R(nu) (r = 0.468, P <0.001) and VsR(mi) (r = 0.361,
P
<0.01), respectively. As concerns all components of
120
the cell other than nucleus, mitochondria, and lamellar
n
bodies, subsumed in the remaining cytoplasmic compo100
nents, the volume density referred to the whole cell
showed a very weak positive correlation with VsR(cell)
n
+
80
(r = 0.382, P <0.01), while a strong positive correlacn
tion (r = 0.870, P <0.001) was observed as regards the
60
volume density referred to the test system.
Based on the correlations of VsR(cell) with the vol40
ume densities referred to the test system of the respecW
tive organelles (correlation coefficients: rcy > r,, >
20
rmJ,we may conclude that a n oedematous increase in
Y
8 6 . 2 0 ~- 37.03
the size of the type I1 pneumocytes is mainly determined by a n increase in the volume of cytoplasmic com0
ponents other than mitochondria and lamellar bodies,
0.0
1.0
1.2
1.4
1.6
and only secondly by a n increase in nuclear size. Mitochondrial contribution is only of a minor extent, and
VsR(cel1) [pm]
lamellar bodies do virtually not contribute to the increase in cell size related to a n oedematous swelling of
Fig. 9. Morphometric data. Relationships of volume-to-surfaceratio
the type I1 pneumocyte.
of the cell V,R(cell) and volume density of the remaining cytoplasmic
Therefore, if considerable variations in the size of the
components (i.e., the whole cell except nucleus, mitochondria, and
lamellar bodies) referred to (A) the whole cell V,(cy/cell), and (B, C) cells to be studied have to be expected, a reference
space must be chosen, which is independent of these
the test point system V,(cy/test). While V,(cy/cell) and V,R(cell)
show only a very weak positive correlation, a strong positive correla- variations. This demand is perfectly met by the multition is observed between V,R(cell) and Vv(cy/test). The correlation of
purpose test system itself. In addition, parameters
V,R(cell) and V.,(cy/test) is significant not only a t the level of the
which are independent of variations in the reference
tissue samples (values obtained from the analysis of all cell profiles
space as, e.g., the volume-to-surface ratio or in t u r n the
sub-sampled from an individual ultrathin section) (B), but also at the
level of organs (mean values * SEM of 5 tissue samples per lung) (C). surface-to-volume ratio (Schmiedl et al., 1990, 1993),
may be used in quantitative descriptions.
Although one has to take into account the lungs' spe-
REMAl N IN G CYTOPLASM
t
2oL, -
-
-
-
-
-
m
.
.
I
.
;c
2
g
MORPHOMMETRY O F HIJMAN TYPE I1 PNEUMOCYTES
cific donor history, the study presented provides a basis
for future morphometrical analysis of alterations of the
fine structure of human type I1 pneumocytes as may
occur, e.g., during extended ischemia, due to differences in organ preservation procedures, due to experimental conditions in cell culture systems, or as a consequence of pathogenetic processes.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the expert technical assistance of S. Freese, H. Huhn, and A. Gerken.
Many thanks are owed to M. Ochs and B. Will for assistance in fixation and sample preparation, and for
fruitful discussions. This study was supported in part
by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 330 Organprotektion, Teilprojekt B 12).
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