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Heterogeneity of tight junction morphology in extrapulmonary and intrapulmonary airways of the rat.

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THE ANATOMICAL RECORD 198193-208 (1980)
Heterogeneity of Tight Junction Morphology in
Extrapulmonary and Intrapulmonary Airways of the Rat
EVELINE E. SCHNEEBERGER
Department of Pathology, Massachusetts General Hospital and Haruard Medical School, Boston,
Massachusetts 02114
ABSTRACT
In the present study morphology of tight junctions was related
to the various cell types lining extrapulmonary and intrapulmonary airways of
the rat. Freeze fracture replicas were prepared from extrapulmonary airway
epithelium derived from the cartilagenous and membranous sides of upper,
middle, and lower thirds of the trachea. Intrapulmonary airway epithelium was
obtained from airways < 1 mm in diameter. Tight junction fibrils on the P
fracture face were organized into three types of patterns. Type 1: parallel,
sparsely interconnected lumenal fibrils with large ablumenal fibril loops. Type 2:
richly interconnected lumenal fibrils with large ablumenal fibril loops. Type 3:
narrow network of interconnected fibrils. On the E fracture face complementary
grooves were organized in a similar pattern. Ciliated cells on both sides and all
levels of the trachea were associated with type 1 junctions. In intrapulmonary
airways, however, the junctional pattern of ciliated cells changed to type 2. Brush
cells at all levels of the airways were bounded by type 2 and occasionally by type
1 junctions. Secretory cell junctions displayed the following patterns: Mucous
cells were bounded solely by type 3, serous cells by either types 2 or 3, and Clara
cells predominantly by type 2. Cells tentatively identified as intermediate cells
displayed all three junctional patterns. The number of parallel fibrils comprising
tight junctions was higher in extrapulmonary as compared to intrapulmonary
airways. No difference was seen in the various locations sampled in the trachea.
Gap junctions were observed between secretory cells of extrapulmonary but not
intrapulmonary airways. These observations are discussed in relation to current
physiologic data.
Recent evidence suggests that cells comprising the tracheal epithelium elaborate a watery
periciliary fluid (sol)which is present beneath
the mucus (gel)layer lining the trachea (Nadel
et al., '79). One possible function for the low viscosity sol layer is to permit the cilia to beat
freely, thereby propelling the overlying mucus
layer towards the mouth. Secretion of this
watery fluid phase is a consequence of active
ion transport into the tracheal lumen (Olver et
al., '75, Mangos, '76, Al-Bazzaz and Al-Aquati,
'77), and therefore, depends on the presence of
a permeability barrier across which an osmotic
gradient can be established. Tight junctions
between cells, by virtue of their continuous
network of strands, provide such a barrier
(Staehelin, '74). Correlative studies suggest
that the number of horizontally parallel
strands comprising the junction determines, in
part, the tightness of the intercellular seal that
0003-276X/80/1982-0193$02.900 1980 ALAN R. LISS, INC.
is formed (Claude and Goodenough, '73,
Claude, '78). On the other hand, the extent to
which strands are interconnected with one
another may affect the degree to which the
junction can be stretched, either as a consequence of an increase in cell size, or of pressure
applied to the cells from within the lumen (Hull
and Staehelin, '76).
A variety of epithelial cells line the mammalian extrapulmonary and intrapulmonary airways; the relative number of each cell type,
however, varies depending on the species, and
the level of the respiratory tract from which
the tissue is taken (Jeffrey and Reid, '75,
Breeze and Wheeldon, '77). In the rat these
types include mucous, serous, intermediate,
basal, brush, ciliated, and Clara cells; the first
Received March 10. 19.30: accepted April 2, 1980.
194
E.E. SCHNEEBERGER
being sparse in number in the healthy rat and
the last being confined to the intrapulmonary
airways. While the appearance of freeze-fractured intercellular j unctions has been described
in the guinea pig (Inoue and Hogg, '77), the rat
(Marin et al., '79), and mouse (Inoue and Richardson, '79), no data are available relating the
morphology of tight junctions to either the adjacent cell type or the level of the airway from
which the junction is obtained.
The present study on the morphology of epithelial tight junctions in extrapulmonary and
intrapulmonary airways of normal rats was designed to answer the following questions:
1. What is the morphology of the tight junctions which form between several of the combinations of the epithelial cell types enumerated above? 2. How does the morphology of the
junction differ in the extrapulmonary as compared to the intrapulmonary airways? 3. How
does the location of a cell on the cartilagenous
as opposed to the membranous side of the
trachea affect the morphology of its tight junction?
MATERIALS AND METHODS
Animals
The extrapulmonary and intrapulmonary
airways of 36 Wistar-Furth rats (Microbiological Associates, Bethesda, Md.) of both sexes,
weighing between 70 and 150 grams, were examined. The cages containing the rats were
maintained on glass enclosed shelves supplied
with filtered air. To ensure that tissue was obtained from animals free of respiratory infections, lungs from randomly selected animals
were periodically checked by light microscopy
to ascertain that there was no lymphocytic
cuffing of the airway epithelium.
Tissue preparation
The animals were anesthetized by the intraperitoneal injection of sodium pentobarbital
(Diabutal, Diamond Laboratories, Inc., Des
Moines, Iowa), 5 mgllOO gm body weight.
After exposing the trachea and lungs, and allowing the lungs to collapse, formaldehydeglutaraldehyde (FG) fixative diluted 1:2 with
0.1 M cacodylate buffer, pH 7.3 (Karnovsky,
'65) was gently instilled into the airways by
means of a 3-ml syringe and a 30-gauge needle
inserted just cephalad to the larynx. Care was
taken not to over distend the lungs with fixative. The trachea, lungs, and heart were
removed en bloc. The trachea was then divided
into three equal parts: 1. upper, 2. middle, and
3. lower trachea. These tracheal rings together
with slices of lung, 1-mm thick, were fixed in
FG fixative for an additional 20-45 minutes a t
4"C, and washed overnight in 0.15 M cacodylate buffer, pH 7.3.
Light and electron microscopy
Tracheal rings, 1-mm thick, and small cubes
(1mm3)of lung were postfixed in 1% OsO, with
15 mg/ml of potassium ferrocyanide (Karnovsky, '71) for 1 hour a t 4°C. The tissue was
stained en bloc with 1.5% uranyl acetate in
0.05 M maleate buffer, pH 6.2, dehydrated in
graded ethanols, infiltrated, and embedded in
Epon. Sections 1-pmthick from the three levels
of the trachea and the lung were stained with
toluidine blue and examined by light microscopy. For electron microscopy, thin sections
were cut with a diamond knife (BalzersUnion,
Balzers, Lichtenstein) on an LKB Ultrotome
I11 (LKB, Bromma, Sweden) picked up on
carbon-coated grids, stained with lead citrate
(Venable and Coggeshall, '69), and examined in
a Philips EM 300 electron microscope.
Freeze fracture
Small pieces of fixed tissue were taken from
lung containing airways less than 1.0 mm in
diameter (intrapulmonary airways) and from
the cartilagenous or membranous portion of
the trachea, (extrapulmonary airway) and were
infiltrated with glycerol at concentrations
increasing from 10 to 30% in 0.1 M cacodylate
buffer, pH 7.3 for 2 hours at 4°C. The tissue
was then rapidly frozen in liquid nitrogen
cooled to -210°C under vacuum, and fractured in a double replica device at -150°C,
using a Balzers high-vacuum freeze-etch unit.
The carbon-platinum replicas were washed in
5.2% sodium hypochlorite (Chlorox Co.,
Oakland, California) followed by distilled
water, picked up on Formvar-coated grids, and
examined in the electron microscope.
Measurements
In each electron micrograph of the freeze
fracture replica, the cell on which the junction
was localized was identified by morphological
criteria which are described in Results. All
measurements were made on elecron micrographs at a final magnification of x 50,000. A
grid of 0.25 pm spacings was placed on the
micrographs so that the grid lines were perpendicular to the longitudinal axis of the tight
junction network. The number of fibrils intersected by each grid line was counted and the
total length of junction examined was measured. Care was taken to count only those por-
TIGHT JUNCTIONS IN AIRWAYS OF THE RAT
tions of the junctions which appeared complete
in the fracture plane of the replica. The mean,
mode, and range of the fibril counts were determined.
RESULTS
Light microscopy
Examination of sections 1 pm thick, stained
with toludine blue, revealed neither lymphocytic cuffing beneath the tracheal epithelium
nor an inflammatory cell infiltrate in the epithelium of extrapulmonary and intrapulmonary airways of randomly selected animals. Ciliated cells, staining weakily with toluidine blue,
stood in sharp contrast to the other more darkly staining epithelial cell types. I t was possible
to distinguish mucous from serous epithelial
cells by virtue of the fact that the secretory
granules of the former were larger and stained
less intensely than those of the latter cell.
Mucous cells were sparse in number and rarely
observed caudal to the midportion of the trachea. Brush and intermediate cells could not be
identified with certainty at the light microscope level. In the intrapulmonary airways
Clara cells were easily distinguished by their
bulbous apices which projected into the lumen
of the airway above the level of the adjacent
ciliated cells. The latter cell type was more
numerous in the intrapulmonary than in the
extrapulmonary airways.
195
Secretory cells. The two secretory cell types
present in the trachea (mucous and serous epithelial cells) (Fig. l a ) are distinguished from
one another by the morphology of their secretory vacuoles: Those of the serous epithelial
cells tend to be smaller (up to 0.6 pm in diameter) and contain a homogeneous, moderately electron dense matrix; those of mucous cells
are larger (up to 0.8 pm in diameter), and
contain a rounded dense core within a weakly
electron dense matrix. The accumulation of
such vacuoles within mucous cells results in
the outward bulging of the apex and sides of
the cell, a change which is less apt to occur in
the serous cells of the normal rat. This feature
together with the larger size of the secretory
vacuoles, and the presence of mucus cells primarily in the proximal half of the trachea, are
useful in identifying these cells in freeze fracture replicas. Both these secretory cells have
on their lumenal surface a sparse array of
short, blunt microvilli, measuring 0.3-0.6 pm
in length and 0.08-0.1 pm in width. Their basolateral membranes, particularly where they are
in contact with basal cells, form interdigitations with adjacent cells. A large amount ot
rough endoplasmic reticulum is present on the
ablumenal side of the cell and moderate numbers of mitochondria are present throughout
the cytoplasm.
While the function of Clara cells remains to
be established, it is thought to be secretory in
nature (Kuhn et al., '74). In contrast to the cells
of the trachea, Clara cells have few secretory
vacuoles, moderate numbers of mitochondria,
and abundant smooth endoplasmic reticulum.
Their cell borders are smooth and show few
interdigitations. The most distinctive morphological feature of the Clara cell which is helpful
in its identification of freeze fracture is a bulbous apex which projects into the airway
lumen and has few surface microvilli (Fig. 5a).
Transmission electron microscopy
While some of the ultrastructural features of
epithelial cells lining the rat extrapulmonary
and intrapulmonary airways have recently
been described (Kuhn et al., '74, Jeffrey and
Reid, '75, Marin et al., '79), little attention has
been focused on those ultrastructural features which have been correlated with fluid secretion in other epithelia (Oschman and BerIntermediate cells usually have no distincridge, '71, DiBona and Mills, '79). These in- tive cytoplasmic features. However, occasionalclude, for example, cell shape, intercellular
ly either a small secretory vacuole or a 'fibrinojunctions, surface microvilli, and basolateral granular' accumulation indicative of ciliogenmembrane infolds. Since the present study esis may be observed, suggesting that these
deals primarily with the freeze fracture ap- cells may be precursors of either secretory or
pearance of intercellular junctions as it per- ciliated epithelial cells. The surface microvilli,
tains to the specific cell types lining the basolateral membranes, and numbers of mitoextrapulmonary and intrapulmonary airways, chondria are similar to those described for
a brief overview of the cellular morphology is secretory cells (Fig. la).
given. Comments will be made with regard to
Brush cells. These distinctive cells are few in
those structural features thought to play a role
in electrolyte and water transport, as well as number, but are distributed throughout the
those found helpful in identifying specific cell length and circumference of the extrapulmonary and intrapulmonary airways. They have a
types in freeze fracture replicas.
196
E.E. SCHNEEBERGER
Fig. 1.a. Epithelium obtained from the upper third of the rat trachea. A serous cell (SC). ciliated cell (CC). basal cell (BC),
intermediate cell (IC).and a mucous cell (MCI are indicated. Mag. x 9,560. b. Portion of a brush cell present in the upper third of
the trachea. Bundles of microfilaments (asterisk)extend upward into the surface microvilli. The tight junctions are indicated
(arrow heads). Mag. x 15.120. c,d,e.f. Tight junctions between ciliated and intermediate cells (c). mucous and mucus cells (d),
ciliated and mucous cells (e),serous and serous cells (f). Areas of close membrane apposition are indicated by short lines. Mag. x
99,000
TIGHT JUNCTIONS I N AIRWAYS OF THE RAT
characteristic array of straight, stiff-appearing
surface microvilli, measuring approximately 2
pm in length and 0.2 pm in width, which are the
most useful feature in their identification in
freeze fracture replicas (Fig. lb). Dense bundles of microfilaments extend from the underlying cytoplasm into each microvillus. A moderate number of mitochondria are present, and
the basolateral cell membranes show frequent
areas of interdigitation with adjacent cells.
Ciliated cells. The cells increase in number
towards the intrapulmonary airways, and are
readily identified by their numerous apical
cilia which contain the characteristic array of
nine outer doublet and two inner microtubules.
Surrounding each cilium are numerous microvilli measuring up to 1.1 pm in length and 0.08O.lpm in width. The basolateral membranes
are relatively straight. While the number of
mitochondria is similar to that of the cell types
described above, the electron density of the
cytoplasm is consistently less (Fig. la).
Junctional complexes. All the above cell
types are joined by junctional complexes
which, by transmission electron microscopy,
are similar in appearance, but vary in the width
of the tight junction band, and the prevalence
of desmosomes. The junctional complexes consist of an apical tight junction which varies in
width from 0.12-0.52 pm, and displays varying
numbers of membrane contacts in which the
extracellular space is apparently obliterated
(Figs. lc,d,e,f). Distal to the tight junction an
intermediate junction is often present. Desmosomes are infrequent in the lumenal junctional
complexes, except for those adjacent to brush
cells where several may be observed in a single
plane of section.
Freeze fracture
Technical considerations. Fixation of airway
epithelium from 20-45 minutes did not affect
the length of tight junctional areas exposed by
the freeze fracturingprocess. However, shorter
fixation times resulted in the tight junction
fibrils being particulate and caused them to
partition onto both P and E fracture faces.
Similar observations have been made in fetal
lamb lungs (Schneeberger e t al., '78).
Conversely, a longer fixation times gave rise to
continuous fibrils on the P face and empty,
complementary grooves on the E face. These
observations support the findings of Van
Deurs and Luft ('79), and emphasize the
importance of examining complementary
replicas of the same junction before drawing
197
any conclusions as to its integrity. Approximately 108 double replicas were examined and
these yielded a total of 116 freeze-fractured
junctional areas which could be catergorized as
to the cell types with which they were associated (Table 1).
Classification of tight junctions. In both the
extra (trachea)pulmonary and intrapulmonary
airways (bronchioles)essentially three geometrical patterns of tight junction networks are
observed (Fig. 2). The first (type 1)consists of
sparsely interconnected, but fairly closely
spaced parallel fibrils (P face) along the
lumenal portion of the junction. This arrangement of junctional elements forms oblong, interfibril compartments, the long axes of which
are parallel to the lumenal surface of the cell.
On the ablumenal side of the junction, the
fibrils form large, irregular loops with the ends
of some of the fibrils not connected to the junctional network (Fig. 2). Mirror-image replicas
show complementary grooves containing a
variable number of junctional particles (E face)
(Figs. 3a,b). The second (type 2) consists of a
richly interconnected network of fibrils (Pface)
on the lumenal side of the junction and irregular large loops of fibrils on the ablumenal side
(Figs. 2,3c).As in the type 1junctions, some of
the ablumenal fibrils end blindly, and mirrorimage replicas show complementary grooves
containing a variable number of junctional
particles (E face). The third (type 3) consists of
a narrow band of richly interconnected fibrils
(P face) and complementary grooves (E face)
with no large ablumenal loops (Figs. 2,4,a,b).
In both types 2 and 3 junctions the interfibril
compartments are smaller and more irregular
than in type 1,and they do not show the parallel alignment with the lumenal surface.
Distribution of types of tight junctions i n
pulmonary airways. Ciliated cells on both the
cartilagenous and membranous side, at all
levels of the trachea are bound by type 1 tight
junctions- that is, junctions with poorly connected parallel lumenal fibrils and large
ablumenal loops (Fig. 3a,b, Table 1).While the
mean number of fibrils is slightly less on the
cartilagenous than on the membranous side of
the trachea, the difference is not statistically
significant (Table 2). The mode and range of
fibril numbers of ciliated cells in both locations
of the trachea are also similar. In the intrapulmonary portions of the airways, at the level of
the bronchioles, however, the geometrical
pattern of the tight junctions between ciliated
E.E. SCHNEEBERGER
198
1
2
Fig, 2. 'Racings of the three junctional patterns observed. Type 1, present on tracheal ciliated cells. consists of parallel,
sparsely interconnected lumenal fibrils and large ablumenal loops. Q p e 2, present on serous, intermediate, and brush cells in
the trachea and on ciliated, Clara, and brush cells in bronchioles, consists of a richly interconnected network of lumenal
fibrils and large ablumenal loops. Q p e 3, present on tracheal mucous and serous cells, consists of a narrow interconnected
network of fibrils.
Fig.S.a,b.c. Complementary double replica (a,b)of a tight junction between ciliated cells in the mid-portion of the trachea. The
junction consists on the P face of parallel, sparsely interconnected,lumenal fibrils which form large hops on the ablumenal side
(b), and complementary grooves on the E. face (a). Some of the ablumenal fibrilsigrooves end blindly (small arrow). Mag. x
50.000. c. Freeze fracture replica of a tight junction between ciliated cells in a bronchiole. In contrast to the tight junctions in the
trachea, the lumenal fibrils form a richly interconnected network. The ablumenal fibrils form large loops, and some end blindly
(arrows).Mag. x 50,000.
TIGHT JUNCTIONS IN AIRWAYS OF THE RAT
Figure 3
199
200
E.E. SCHNEEBERGER
cells changes to that of type 2 (Fig. 3c). Rarely
a type 3 pattern may be observed (Table 1).
Moreover, the mean number of fibrils is slightly reduced (0.02 < P < 0.05) and the lower
limit of the range of fibril numbers is smaller
than in the trachea (Table 2).
In both extrapulmonary and intrapulmonary airways the tight junctions of brush cells
are most frequently of the type 2 variety (Table
1, Fig. 4d). Unlike the ciliated cells, the mean
number of fibrils is not significantly reduced
(Table 2) in the intrapulmonary airways and in
none of the brush cells examined was a type 3
pattern observed. Brush cells were observed in
replicas from both the membranous and carti-
TABLE 1. Frequency distribution
Level in
airway
Trachea3
Bronchioles
~~
~
lagenous side of the trachea; however, only
those from the cartilagenous side yielded fracture planes through the tight junction areas
(Table 2).
Among the secretory cells (mucous, serous,
Clara cells) of the extrapulmonary and intrapulmonary airways, essentially two patterns
of tight junctions are present: type 2 and type
3 (Table 1).Except for some serous cells in the
membraneous portion of the trachea, the type 1
pattern is not observed. Serous cells at all
levels of the trachea may have either a type 2
or type 3 pattern with the latter predominating
(Table 1,Fig. 4b). Mucous cells areconsistently
bounded by type 3 junctions having relatively
of the three types of tight junction patterns in extrapulmonary
and intrapulmonary airways
Percent of tight junction type2
5Pe2
5Pe3
Cell type'
5pe 1
Nonsecretory: ciliated, brush cells (29)
Secretory: serous, mucous cells (25)
Nonciliated: intermedate cells (15)
90
4
7
104
21
40
Nonsecretory: ciliated, brush cells (34)
Secretary: Clara cells (11)
Nonciliated: intermediate cells (3)
6
-
a5
73
1005
-
75
53
9
27
-
~
'The information in this table is derived from the same cells a s are listed in Table 2. The numbers in parentheses indicate the number of
tight junctions examined in each cell lype.
"@pe 1 junction: parallel poorly interconnected lumenal fibrils and ablumenal loops.
Type 2 junction: interconnected luinenal fibril network and ablumenal loops.
Q p e 3 junction: interconnected network of fibrils, no ablumcnal loops.
'The cells examined are derived from both the membranous and cartilagenous portions of the trachea a t levels 1, 2. 3.
'The junctions of three out of the four brush cells examined were of the type 2 pattern. All the junctions of the ciliated cells were of type 1.
'Too few junctional areas in nonciliated cells were exposed, in the replicas examined, t o state with assurance whether any of the other types
of junctions were present.
TABLE 2. Tight junctions in extrapulmonary and intrapulmonary airways of the rat
~
Fibril number
Area in
Airway
Cell type2
Mean f S.E.
Membranous
portion of
trachea a t
levels 1, 2. 3
Ciliated cells (11)
Serous cells ( 12)
Nonciliated cells (lo)*
Mucous cells (3)
7.56
6.88
6.07
4.80
f 0.24
f 0.29
Cartilagenous
portion of
trachea a t
levels 1, 2, 3
Ciliated cells (14)
Serous cells (9)
Nonciliated cells (5)
Brush cells (4)3
Bronchioles
Cliliated cells (31)
Clara cells (11)
Nonciliated cells (3)
Brush cells 13)
6.50 0.11
5.96 f 0.23
6.00 0.44
6.43 f 0.44
Mode
Range
Length of
junction
measured (pm)
i 0.35
6
6.7
6
4,5
5-12
4-10
3-9
3-8
7.30 f 0.13
6.28 0.25
7.10 f 0.26
6.88 f 0.30
7
6
6
6.7
5-10
4-9
5-11
5-9
22.8
9.7
6
5
4
56
3-12
2-11
4-10
3-11
52.1
20.5
4.0
5.6
f 0.18
*
*
*
16.3
1.7
13.6
4.8
7.4
4.0
'The absence of d a t a from a particular cell type in either the membranous or cartilagenous portion of the trachea indicates t h a t no tight
junction of that cell type was present in the replicas examined. However. all five cell types were present The number in parenthesis
indicates the number of junctions measured in each cell type.
'Nonciliated cells are those in whirh the fracture plane passed solely through the lateral membrane. They could, therefore. represent either
serous. niucous. or intermediate cells.
'llrush cells were also present in the replicas from the inenibranous portion of the trachea. hut unlike those of the cartilagenous portion of
thc trachea. none nf the fracture planes passed through the Light junctinn region.
TIGHT JUNCTIONS IN AIRWAYS OF THE RAT
few fibrils and no ablumenal loops (Table 2,
Fig. 4a). Clara cells, on the other hand, are
bounded primarily by type 2 junctions (Fig.
5c,d). As in the case of ciliated cells in extrapulmonary and intrapulmonary locations,
the mean number of fibrils comprising the
tight junctions of serous cells is somewhat
greater than Clara cells (0.02 < p < 0.05).
Cells designated as nonciliated include those
in which the fracture plane passes along the
lateral cell membrane without entering the underlying cytoplasm. This makes it impossible
to determine whether these cells are serous,
intermediate, or possibly mucous cells which
have recently discharged their secretory vacuoles. In the trachea all three types of junctional
patterns are observed in these nonciliated
cells; however, types 2 and 3 predominate (Fig.
4c, Table 1). Too few nonciliated cells were
available in the replicas from intrapulmonary
airways to say with assurance whether types
other than 2 are present.
Distribution of gap junctions and rectilinear
arrays. While the primary purpose of this
study is an analysis of the morphology of epithelial tight junctions in the airways of the rat,
observations are also presented with regard to
the presence and location of gap junctions in
the freeze fracture replicas examined.
The gap junctions observed are present in
replicas derived from the extrapulmonary airways and are associated with serous, mucus,
and possibly intermediate cells (Fig. 5b). They
are usually present towards the basal portion
of the cell, although an occasional gap junction
is observed on the lumenal half of the lateral
cell membrane. They vary in maximum diameter from 0.30 to 0.91 pm, and more than one
gap junction per cell may be observed. The location of gap junctions near the base of the secretory cells suggests that some of these junctions may serve to connect secretory to basal
cells. Although they were not present in any of
the ciliated cells examined, rare gap junctions
have been reported to be present near the
lumenal half of ciliated cells in the rat (Marinet
al., '79). Insufficient numbers of Clara cells
were examined to make a definitive statement
as to the presence of gap junctions in these
cells; however, none were observed in the
replicas obtained of these cells. Rectilinear arrays of intramembranous particules, such as
those described in the cells of the guinea pig
trachea (Inoue and Hogg, '77), were seen in the
lateral membranes of only a single Clara cell.
20 1
freeze fracture replicas prepared for the present study, the epithelial tight junctions at the
point of transition between respiratory bronchioles and alveolar ducts were also examined.
I t is in this region that Macklin ('55) has
suggested that pulmonary sumps are located
beneath the epithelium. Furthermore, it has
been postulated that these sumps may be anatomical sites into which excess alveolar fluid
can drain (Macklin, '55) or from which alveolar
flooding occurs in a retrograde fashion (Staub,
'79). I t is, therefore, of interest to know what
the structure of the tight junctions is in this
region of the pulmonary epithelium. Only a
single such tight junction (Fig. 6) was apparent
in the freeze fracture replicas examined, and its
structure resembles that of the junctions observed between type 1 pneumocytes (Schneeberger and Karnovsky, '76) rather than that of
junctions present between epithelial cells lining the respiratory bronchioles.
DISCUSSION
The present study shows that in the epithelium lining extrapulmonary and intrapulmonary airways, the morphology of tight junctions
varies depending on the cell type involved and
its level in the tracheobronchial tree, but not
on its location on the membranous or cartilagenous side of the trachea. The junctions are
organized into three geometrical patterns.
w p e 1, consisting of the P fracture face of
poorly interconnected parallel fibrils and large
ablumenal loops, is primarily limited to ciliated cells and occasionally to some brush cells
at all levels and on all sides of the extrapulmonary airway (trachea).Q p e 2, consisting on the
P fracture face of an interconnected network of
lumenal fibrils and large ablumenal loops, is
the predominant pattern for ciliated, Clara,
and brush cells in intrapulmonary airways
(bronchioles), but is also seen on some serous
and nonciliated (intermediate ?) cells in the
trachea. Finally, type 3, consisting of a narrow
band of interconnected fibrils on the P face
with no ablumenal loops, is present primarily
on mucous and serous cells in the trachea. The
factors governing the morphological organization of tight junctions are at present unclear. I t
is of interest however, that the greatest uniformity of tight junction pattern is observed
among ciliated cells which are the most common cell type in the airways (Breeze and
Wheeldon, '77) and are, therefore, the most
likely to form tight junctions with each other.
A quantitative assessment of the impermeTight junctions of transition between respir- ability of tight junctions to solutes is obtained
atory bronchioles and alveolar ducts. In the by the measurement of the electrical resistance
202
E.E. SCHNEEBERGER
Figure 4
TIGHT JUNCTIONS I N AIRWAYS OF THE RAT
203
Fig, 4. a. Tight junction (P face) on the surface of a mucous cell. The junction consists of a relatively narrow network of
interconnected fibrils. Rarely a fibril ends blindly on the ablumenal side. Mag. x 62.000. b. Tight junction (Pface) on the surface
of a serous cell. The junction consists of a network of interconnected fibrils with some slightly larger ablumenal compartments.
Mag x 64.000. c. Tight junction (E face)from a presumptive intermediate cell. The junction consists of lumenal parallel grooves
which show relatively few interconnections and large ablumenal loops. Mag. x 63,000. d. Tight junction (mostly E face) of a
brush cell. The junction consists of an interconnected network of lumenal grooves with some larger ablumenal loops. The closely
spaced surface microvilli are seen on the left. Mag. x 42.000.
204
E.E. SCHNEEBERGER
Figure 5
TIGHT JUNCTIONS I N AIRWAYS OF THE RAT
205
Fig. 5.a. Clara cell (Cl)and ciliated cells (CC)from a respiratory bronchiole. The Clara cell is characterized by it bulbous apex
which often contains a moderate number of secretory vacuoles. Mag. x 4,600. b. Two gap junctions [G)present on the basolateral membrane of a secretory cell in the midportion of the trachea. Mag. x 67,000. c. Freeze fracture replica (Pand E faces) of
the apical portion of a Clara cell in a bronchiole. The apical, bulbous portion of the cell extends into the lumen (L)above the level
of the tight junction. A portion of the junction on the right hand side (within rectangle) is enlarged in Figure 5d. Mag. x 16,400.
d. Enlargement of a portion of the Clara cell tight junction shown in Figure 5c. The P face (P)is on the left and the E face (El is on
the right. Mag. x 44.000.
206
E.E. SCHNEEBERGER
Fig. 6. Tight junction between epithelial cells at the bronchoalveolar junction. A type I (I)cell is on the right and a cuboidal
epithelial cell (C) lining the bronchiole is on the left. The tight junction forms on interconnected network of fibrils (arrow)
which resembles the tight junctions usually observed between type I cells. The air space (AS) and capillary Iumen (CL) are
shown. Mag. x 51.300.
across the epithelium (Fromter and Diamond,
'72), and a rough correlation appears to exist
between the electrical resistance and the number of sealing fibirls comprising the junction
(Claude and Goodenough, '73, Claude, '78). In
airway epithelium of several species an electrical resistance of about 300 Q cm2 has been
measured (Nadel et al., '79), putting it in the
category of a moderately tight epithelium
(Schultz, '77). While it is recognized that junctional permeability may be governed by factors other than fibril numbers (MartinezPaloma and Erlij, '75, Dziegielewska et al., '79,
Hainau et al., '79), the present morphological
study suggest that the airway epithelium of
the rat may be expected to be moderately tight
as well. A consistent reduction in the mean
number of fibrils comprising all tight junctions
was observed in the intrapulmonary as compared to the extrapulmonary airway epithelium, suggesting the possibility that the latter
may be less permeable than the former.
Similarly, the number of fibrils constituting
the junctions of secretory cells is consistently
less than of ciliated cells.
Recent studies have shown that tracheal epithelium actively transports C1- towards the
lumen and Na' towards the submucosa. While
the Na-K-ATPase has been localized to the
basolateral membranes of tracheal epithelial
cells (Widdicombe et al., '79), the anatomic
location from which C1- is secreted has not
been established. Furthermore, it is not known
whether C1- secretion is a property of all cells
comprising the tracheal epithelium. The present study shows that most of the cells lining
the extrapulmonary and intrapulmonary airways are endowed with some surface microvilli
of varying dimensions, and their basolateral
membranes display a moderate degree of interdigitation with adjacent cells thereby forming
tortuous intercellular spaces; these are all
features that have been observed in epithelia
involved in isotonic fluid secretion (DiBona
207
TIGHT JUNCTIONS IN AIRWAYS O F THE RAT
and Mills, '79). Of the tight junctions associated with the various cells lining the extrapulmonary and intrapulmonary airways, those associated with the mucous cells have the fewest
fibrils and might be expected to provide the
least resistance to the passage of small water
soluble molecules. However, in the normal rat
these cells are few in number and are limited to
the proximal portions of the trachea. Since the
secretion of the watery periciliary fluid appears to occur throughout the airways, it is
likely that C1- transport is not limited to a
single cell type.
In their studies of the tight junction morphology in the alimentary tract of Xenopus
laevis, Hull and Staehelin ('76) suggested that
tight junctions with parallel, poorly interconnected networks of fibrils are flexible and can
be stretched more readily than those with
richly interconnected networks. Interestingly
in Xenopus laevis the flexible type of junctions
are associated with mucus cells and the junctions rich in interconnections are seen on
ciliated and absorptive cells. The reverse
appears to be the case for cells in the mammalian airways. Not only are the poorly interconnected tight junctions limited largely to ciliated cells in the trachea, but the fibrils of these
junctions become more richly interconnected
in the distal airways, where physiological evidence suggests that the airways are more distensible (Martin and Proctor, '58, Burnard et
al., '65). Furthermore, it might be expected
that the membranous part of the trachea is
more likely to be subjected to stretching than
the cartilagenous side, and yet the tight junctions show similar, poorly interconnected fibrils in both locations. I t remains to be
established to what extent the degree of fibril
interconnection plays a role either in the
permeability properties or distensibility of
tight junctions.
ACKNOWLEDGMENTS
The expert technical help of Mrs. Joanne McCormack and Mssrs. Bruce Kaynor and William Flaherty and the critical review of the
manuscript by Dr. R.D. Lynch are gratefully
acknowledged. This study was supported by
USPHS grant HL-25822.
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