Proliferation and differentiation in mammalian airway epithelium M .

Eur Respir J
REVIEW
1988, 1, 58-80
Proliferation and differentiation in mammalian
airway epithelium
M .M. Ayers, P.K. Jeffery*
CONTENTS
I.
I.L
1.2
2.
2.1
2.1.1
2.1.2
2.1.3
2.1.4
2.1.5
Introduction and techniques
Mitosis
Cell cycle
Normal proliferation and differentiation
Proliferation & normal variation
Mitotic index
Labelling index
Turnover time
Cell cycle and its phases
Growth fraction
The infrequency of mitotic figures led to dispute
amongst early workers as to the occurrence of cell
division in adult mammalian airway epithelium [30,
63, 83, 227]. It was not until 1951 that quantitative
work in the respiratory tract began to concentrate on
the distal (i.e. respiratory) portion of the lung
(reviewed by BERTALANFFY (20, 21], KAUFFMAN [132],
and MASSE et al. [159]). Studies of the lining
epithelium of conducting airways soon followed [I I,
25, 26, 27, 32, 38, 148, 208, 230]. The present review
summarizes the results of many studies of cell division
and differentiation in conducting airway epithelium.
It is divided into three major sections: I) general
principles and techniques used in assessment of cell
kinetics; 2) normal proliferation and differentiation
in conducting airways and 3) effects of irritants and
carcinogens, mechanical trauma, drugs, infection and
physiological factors on cell proliferation and differentiation. Proliferation and differentiation, which
occur during airway development, are not discussed
here: the interested reader is referred to the following
papers and reviews [24, 47, 119, 129, 132, 207, 237].
At least eight epithelial cell types are recognised in
the lining epithelium of conducting airways and three
in the epithelium lining the alveoli (for reviews see [41,
116, 117, 118]). In man the tracheobronchial surface
epithelium is pseudostratified, ciliated and columnar
• Department of Lung Pathology, Cardiothoracic Institute,
Brompton Hospital, London, England.
Correspondence: Dr. P.K. Jeffery, Department of Lung Pathol·
ogy, CTI, Brompton Hospital, Fulbam Road, London SW3 6HP,
England.
2.2 Cell differentiation
3.
Factors influencing proliferation and differentiation
3.1 Irritation
3.2 Mechanical injury
3.3 Enzymic injury (elastase)
3.4 Drugs
3.5 Infection
3.6 Neural control
3.7 Immune system
Summary
4.
reducing in thickness and degree of pseudostratification with airway generation until the epithelium is
simple columnar at the level of the smaJI bronchioles
[167]. In animals the surface epithelium of the trachea
and bronchus normally has only two layers of nuclei,
one basal and one superficial, and all cells reach the
basement membrane. In human bronchial epithelium,
obtained from grossly normal areas of resected lung,
two or more rows of basally situated cells are often
obser:ved, indicating a degree of basal cell hyperplasia
[I 54]. Figure I shows epithelial cell types distinguished according to position (basal or lumenal),
presence of cilia and secretory granules. The most
common basally situated cell, the 'basal' cell, was
described and proposed as a progenitor cell by a
number of investigators (24, 27, 63, 187, 227]. In
peripheral bronchioles, where basal cells are absent,
the Clara cell is the progenitor cell. Clara cells may
divided in response to epithelial irritation and
subsequently differentiate to form mature secretory
and ciliated cells (74, 78, 79, 81]. In the alveolus, the
type II cell is the progenitor cell [45, 70, 76] from
which the type I cell differentiates. In conducting
airways, there are three types of secretory cell,
distinguished both by the nature of their secretory
granules and the airway level at which each occurs:
serous and mucous (i.e. goblet) cells nonnally occur in
the proximal airways only [118], while Clara ce11s are
usually found distally, limited to the small bronchioles of most but not all species [171, 174]. Dividing
lumenal cells have been observed and identified as
serous and mucous cells [11, 38], but until recently the
59
PROUFERATION AND DIFFERENTIATION IN AIRWAYS
basal•
-----
Epithelial
--------
lummar•
.
/
."
'II ATt:n
----BASAL
DENSE-CORE
GRAN~
MM;
CILIATED
(DCG)b
nonciliated
~wnfuncdon
/~
SPECIAL- TYPE
C-MUCOUS -SEROUS CLARA -DCGb
BRUSH
I
I
indeterminate
Migratory
~~
GLOBULELEUCOCYT~
-LYMPHOCYTE
- MAST CELL
"'
Neural
/
- NEURO-EPITHELIAL BODIES
(NEB)
-NERVE TERMINALS
Fig. I. Mature Airway Epithelium - Cell types. a: aU cells may rest on the basal lamina but only some reach the surface (luminal); b: often
basal, sometimes luminal; e: in order to distinguish between cell types which occur in both surface epithelium and submucosal glands the terms
'surface epithelial-' and 'glandular-' should be prefixed. This is indicated where appropriate by a hyphen preceding the term. [116, 117, 155]
majority of the epithelial cells in proximal airways
have been thought to arise following the division and
differentiation of basal cells [27, 32, 52, 187). An
alternative suggestion is that the mucous cell plays the
major regenerative role in conducting airway epithelium, a hypothesis which is gaining more and more
support [153, 156].
Therefore the surface epithelial cells of the lower
respiratory tract which have a capacity to divide are
the basal, serous, mucous, and Clara of conducting
airways and the alveolar type II cells of the
respiratory portion of the lung. The number and
proportion of each, which contribute to the pool of
dividing cells, varies with airway level and species.
1. Introduction and techniques
1.1 Mitosis
Observation and simple quantitation of mitotic
figures allows three indices, which measure cell
renewal, to be determined: I) Mitotic index (MI), the
number of cells in mitosis at any one time (often
expressed as a ratio, i.e. divided by the total number
of cells counted); 2) Rate of entry into mitosis (REM)
calculated from the accumulation of mitoses during
time (t) after use of stathmokinetic (i.e. mitoticarresting) agents such as colchincine [67, 217, 221)
and the vinca alkaloids vinblastine and vincristine (48,
221]. The accumulated mitotic count is divided by (t)
to give the REM which represents a measure of the
rate at which new cells are born; 3) Turnover time
(Tf), can be determined from the REM if the total
number of cells present is known [3, 19, 145, 148, 217].
Implicit in this determination is the assumption that
all cells in the tissue are dividing, and that they are
doing so at the same rate. It should be noted,
however, that the renewal rate of a tissue depends not
only on the number of dividing cells (which in vivo is
usually only a fraction of the population) but also on
the length of time taken for each celJ to divide. When
the two variables are unknown or altered, the
calculation of Tf based on REM is inaccurate.
1.2 Cell cycle
In 1953, HOWARD and PELC [112) showed, with
radioactively labelled DNA precursors, that a finite
time existed between the end of synthesis of new
DNA (i.e. S phase, which takes time (tS)) and mitosis
60
M.M.
AYERS,
(M). They subsequently formulated the now familiar
Cell Cycle concept, proposing that dividing cells pass
through a series of complex biochemical events which
precede mitosis. The interval between mitosis and S
phase was referred to as 'gap' 1 {G1) and that between
the end of DNA synthesis and the beginning of
mitosis was called 'gap' 2 (G 2 ), (fig. 2). Any tissue is
likely to have only a fraction of its total cell number
proliferating, or cycling, at any one time and that
fraction is referred to as the Growth fraction (GF).
Cells in the GF can move into the non-proliferating
(non-cycling) fraction of the population by three
routes. They can: a) permanently leave the cycle~
perform their appropriate function and subsequently
die (i.e. the normal process of ageing), b) leave the
cycle temporarily or permanently and enter a 'resting'
state {G 0 ), yet be available to re-enter the cycle on
receiving the appropriate stimulus (e.g. liver parenchymal cells in response to partial hepatectomy), or c)
leave the cycle and die due to abnormality or
malfunction [3, 149].
When the DNA precursor thymidine (T) or
iododeoxyuridine (IUdR), labelled with tritium CH)
or carbon-14 {14C), (emitters of low-energy p particles), is made available to a population of 'cycling'
cells, those in the S phase incorporate it irreversibly
into their DNA, the extent to which this is done being
dependent on the activity of the enzyme thymidine
kinase. Thereafter, the cell is 'labelled' and can be
visualized by autoradiography [189] which enables the
proportion of cells synthesizing DNA, (i.e. the
Labelling index (LI)) to be calculated. Usually LI is
expressed as a ratio of the number of cells labelled
divided by the number of cells counted (often
expressed per 100 or 1000 cells counted). In a
population where all the cells are cycling, the number
of cells in any phase of the cycle is directly
P.K.
JEFFERY
proportional to the length of that phase. Thus as ts is
longer than tm [3, 145], the LI for a given population
will be correspondingly higher than the MI.
Both pulse and continuous labelling with tritiumand carbon-labelled thymidine can be used to obtain
information about the length of distinct phases of
the cell cycle, the growth fraction and rates of cell
birth and loss. Four techniques will be described in
brief. Extensive discussion of procedure standardization, the problems and some of the solutions
involved in the use of these techniques, is available in
WRIGHT and ALLISON (237] in the section on
methodology. In particular, there are potential
anomalies which may be introduced in long-term
experiments, due to the re-utilization of 3 H-thymidine eH-T) following the degradation of nuclei
containing the label. If the latter is likely then an
isotope such as 3 H-IUdR (which is re-utilized to a
lesser extent) can be used.
l) A method of assessing the fraction of labelled
mitoses (FLM) was developed by QUASTLER and
SHERMAN [176] which enables cell cycle time (tC) and
the duration of each cycle phase (tG1 , tS, tG 2 , and t)
to be measured. Proliferating cells in a population are
labelled by a single pulse of 3 H-T and followed with
time as they move through G2 into mitosis to produce
a cohort of labelled mitotic figures. Consecutive
samples at short-time intervals after the pulse, and
subsequent autoradiographic analysis of the population, allows the rise and fall in the percentage of
labelled mitotic figures with time to be plotted (i.e. the
FLM curve). If all cells in the population are cycling
asynchronously and at the same rate, a well-defined
curve is generated, rising from 0 to 100% and back to
0% of the labelled mitotic figures with each complete
passage of the originally labelled cells around the
cycle. The lengths of each cycle phase and tC can be
sloughing/dying
end stage (functional)
Cycling (GF)
resting state (G0 )
---stimulus_/
\
¥
non-cycling
Fig. 2. Diagrammatic representation of cell cycle. The growth fraction (OF) consists of cycling cells each passing through a series of complex
biochemical events (e.g. S and 0 2 ) preceding mitosis (M). Thereafter cells may either continue to cycle or leave the cycle to die, terminally
differentiate or enter a 'resting' state (00 ) from which they may be re-called when required. (Modified from [3]).
PROLIFERATION AND DIFFERENTIATION IN AIRWAYS
calculated from measurements made of the FLM
curve [3, 12, 94, 108, 163, 177, 197, 211].
2) The double labelling technique enables tS and the
rate of entry into DNA synthesis to be calculated by
using both 3 H- and 14C-labelled thymidine. The
energies, and thus 'path lengths', of each isotope are
sufficiently different to enable cells, labelled with
either isotope, to be distinguished by autoradiography [3, 195, 233). A cell cohort is given a single pulse
of 3 H-T (short path length), and after a known time
interval (t), which must be shorter than tS, a single
pulse of 14C-T (long path length). The distinctly
labelled cell populations can be subsequently differentiated and counted by having two superimposed
layers of autoradiographic emulsion, one of which is
too distant to be affected by the short path length of
3
H-T. Assuming that the rates of entering and leaving
the S phase are equal (i.e. the cell population is in a
steady state), tS can be calculated [3].
3) Continuous labelling enables the growth fraction
tS, the rate of entry into DNA synthesis and tc to be
calculated [3, 93]. A cell population in vivo or in vitro
is 'continuously' exposed to a low level of 3 H-T, such
that each cell in the growth fraction, as it comes into
the S phase, becomes labelled. This can be done in
vivo either by continuous infusion [93, 162], or by
injections of low-dose 3 H-T at frequent intervals,
each shorter than tS [3]. If the proportion of labelled
cells is plotted against time, the curve first rises and
then plateaus when all of the growth fraction is
labelled. tS can then be calculated from the slope of
the curve, which measures the rate of entry into the S
phase (i.e. rS) and thus allows use of the equation:
tS=LI/rS.
The time at which the plateau occurs is equivalent
to (tG 2 +tM+G 1). If the summed time as read from
the graph is added to the calculated tS, the cell cycle
time can be derived.
4) Grain counts, i.e. the mean number of silver grains
over individual cell nuclei resulting from a single pulse
label of 3 H-T, can be plotted against time. The
halving by mitotic division occurs after a time
equivalent to tS + tG 2 + tM. If the plot of mean grain
number against time is extended, the next halving
takes place after a time equivalent to tc [87, 140, 141,
210].
Recent developments have resulted in the advent of
integrating microdensitometry and flow cytometric
techniques. Certain stains react stoichiometrically
with DNA. The Feulgen stain has been used
extensively to measure, with a microdensitomer, the
amount of light, of a specific wavelength, absorbed by
each stained nucleus. The method is laborious and the
results difficult to analyse and has in large part been
superceded. With the introduction of the fluorescence-activated cell sorter (FACS) individual (i.e.
disaggregated) cells, stained with a fluorescent dye
such as ethidium bromide or propidium iodide, are
passed in suspension through and excited by a laser
beam. The resulting fluorescence is detected and the
data expressed as a distribution of DNA content. The
61
method is quick, precise, can measure more than
one parameter at a time and, when used in conjunction with a stathmokinetic agent, allows the measurement of phase durations [100). The amount ofS phase
in tissue DNA may be achieved by incorporation of a
thymidine analogue, bromodeoxyuridine (BUdR),
which can be subsequently detected with fluoresceinlabelled monoclonal antibodies directed against
BUdR [60]. The method can be applied to cell
suspensions, originally grown in vitro or to those
derived from sections of paraffin-embedded material
[110]. Limitations of the method have been discussed
(224). Most recently a monoclonal antibody (Ki67)
has been screened, which discriminates between cells
in cycle (i.e. the growth fraction) and those in G 2
[92].
If it is assumed that the proportion of cycling cells
is randomly distributed throughout the phases of the
cell cycle (i.e. the population is asynchronous) and all
the cells are cycling independently of one another,
then population growth is dependent on the interrelationships of growth fraction size, the cell cycle time
and the ratio of cells lost through death (or migration)
and cells gained by proliferation [3). When cell loss
and cell production are in equilibrium, the population
is said to be in a 'steady state', one example of which
is the normal adult respiratory tract [43). All
determinations of kinetic parameters from the techniques described above apply to steady state populations. Of course many physiological stimuli or
exogenous irritants may alter a steady state. In
expanding populations, where cell production is
greater than cell loss, the use of complex equations
and computer simulation are necessary for adequate
descriptions [3, 145].
2. Normal proliferation and differentiation
Most cell populations are made up of both
proliferating and non-proliferating compartments.
Proliferation may be defined as an increase in cell
number by means of mitotic division. Differentiation
is the development, by a non-specialized cell, of
definable functional characteristics, a process which
may or may not be preceded by division. Differentiation is sometimes associated with a decreasing
capacity for division [148].
2.1 Proliferation
For earlier discussion on cell proliferation in the
respiratory tract the reader is referred to BoREN and
PARADISE [38), BOWDEN [40), KAUFFMAN [132), and
WRIGHT and ALLISON [237].
2.1.1 Mitotic index (MI) and rate of entry into mitosis
(REM). Dividing tracheal epithelial cells were first
found in the latter part of the 19th century [30, 44,
63]. The results of contemporary studies of MI and
REM in the respiratory tract are summarized in table
I. To enable comparison, the data have all been
recalculated and expressed as the number of cells
62
M.M .
AYERS, P.K. JEFFERY
Table I. -Rate of entry into mitosis (REM')
Airway level
Animal
Sex
Age
REM'
References
wks
Trachea
Trachea
- Basal Cell
- Mucous Cell
Bronchi
Alveolar Epithelium
a
a
~
8
12
8
5
5
3
0.16
1.7
2.6
0.9
25
26
28
32
32
hamster
hamster
a
a
9- 13
9- 13
0.25
0.18
38
38
mouse
mouse
rat
?
0
8
12
9- 12
0.3
0.15
0.4
25
26
127
guinea pig
rat
mouse
a
a
a
adult
adult
1.5
1.5
0.7
20,21
20,21
203
mouse
mouse
mouse
rat
rat
?
?
?
12
'REM: No. mitoses per 1000 total cells counted per h mitotic arrest.
entering mitosis per 1000 cells counted per colchicine
hour (i.e. REM). The REM of airway and alveolar
epithelia is indeed small, the calculated range for the
respiratory tract is 0.14- 3 mitoses per 1000 cells per
hour. By contrast REM has been estimated for rat
duodenal epithelium as 30 per 1000 cells per hour
[150] and for oesophageal epithelium of 2 month old
mice as ll per 1000 cells per hour [28].
MI and REM have been shown by numerous
workers to vary with the airway level, animal strain
and the sex and age of the animal studied:
a) airway level: MI decreases as the airways examined
descend from the trachea to the distal bronchiolus,
particularly in young and growing animals. In older
animals the same trend is present but it is less
pronounced [26, 32];
b) animal strain: given the same sex, age and airway
level, MJ varies between strains of mice [203] and rat
(27], although this difference may lessen with age [203];
c) sex differences: SIMNETT and HEPPLESTON (203)
suggest a trend of increasing MI with age in females
not found in male mice. In 33 day old rat airways, MI
is, however, higher in males than females: by 93 days
the difference disappears. Interestingly, the alveolar
epithelial and migratory cells of the older rats show a
higher MI for females than males and in this respect is
similar to data for mice [32];
d) age: MI shows changes with age. SIMNETT and
HEPPLESTON [203) compared young adult mice (3
months) with mice at the end of their natural lifespan
(18- 24 months of age). Whereas one strain of male
mice showed a 60- 90% reduction of their 3 month
value, another showed no significant difference.
Female mice showed an increase of MI with age. The
studies of BJORK and HARKONEN [26] and BoLDuc
and REID [32] have concentrated only on the early
phases of growth (i.e. to 3 months) where the decrease
in MI coincided with the plateau of the growth curves
for the animals.
2.1.2 Labelling index ( L/) . Since 3 H-thymidine
eH-T) is taken up during tS, which is longer than tM
[3, 50, 164], a relatively larger proportion of the
cycling population is labelled by 3 H-T (and thus is
available for quantitation) than is seen by mitotic
count (MI). However, the observations of changes in
LI with airway level, animal strain and age, parallel
those described for MI and are summarized in table
II.
a) airway level: LI decreases from proximal (central)
to distal airways [27, 32, 59, 200, 201];
b) animal strain: for the same age (or weight) and sex,
significant differences in LI in the trachea have been
reported between strains of rat [27] and, in the
alveolus, between strains of mice [203];
c) sex differences: no differences in LI have been
found at any airway level in adult rats and mice (32,
59, 201, 203], a finding confirmed in older rats (93
days old) (32]. However, in younger rats (33 days old),
males have twice the number of labelled cells than
females (32];
d) age: SIMNETT and HEPPLESTON (203) have examined
three strains of mice at ages 3, 12 and 24 months and
shown a significant decrease in LI with age. This trend
is confirmed by EVANS et a/. [73].
Extrinsic factors may also influence cell division:
e) diurnal variation: diurnal variations in mitotic and
labelling indices have been well demonstrated in
tissues such as epidermis and small bowel [4, 49] and
also in the respiratory tract. In the latter, a peak of
63
PROLIFERATION AND DIFFERENTIATION IN AIRWAYS
Table II.- Labelling index
u
Airway level
Animal
Sex
Age
wks
labelled
cells per 1000
cells counted
Trachea
rat
rat
rat
rat: conventionally derived
rat: minimal
disease
cf
adult
adult
adult
4
5- 12
0.4-0.6
164
28
240
cf
5
5
20
7
230
230
a
a
?
adult
5- 17
1~0
10
16
239
200
203
59
4
5
2
8
4
230
106
106
38
102
10
Trachea
mouse
mouse
mouse
mouse
Trachea
-basal
- mucous
-basal
- mucous
hamster
hamster
hamster
hamster
hamster
Bronchus
rat
rat
rat
hamster
Bronchus
- large
- small
- large
- small
mouse
mouse
mouse
mouse
Bronchiole
human in-vitro
Alveolar Epithelium
mouse
mouse
mouse
hamster
cf~
cf
a
a~
a~
a
8
adult
Reference
a
a
5
?
adult
adult
adult
adult
0.02-0.03
1-2
238
28
240
102
adult
adult
adult
adult
8
4-10
16
3
200
200
59
59
adult
2-7
201
7
4
4
27
27
27
102
d'~
a
a~
a~
a~
?
a
a~
either mitotic or labelling indices has been found in
the morning in rats [164, 193, 205] and the afternoon/
evening in mice and hamsters [57, 138, 140]. Studies
carried out under conditions of constant light by
BOREN (36] and BOREN and PARADISE (38] show no
significant diurnal variation in LI with time. In
normal (SPF) male rat main btoncllial epithelium,
AYERS and J EFFERY [11] found that the total number
of labelled cells in the morning (08:00) is almost
double the value found 12 hours later. Although the
labelled cell population is made up of basal, serous
and cells of indeterminate morphology, the observed
adult
adult
adult
adult
12
52
78
adult
2-4
11
change is due to a significant difference only in the
numbers of labelled serous cells;
f) vitamin A status: retinoids have been shown to play
an important role in the control of epithelial
proliferation and differentiation including that of the
respiratory tract [39, 236]. WOLBACH and HowE [234]
demonstrated excessive cellular. 'proliferation in respiratory epithelium of vitamin A-deficient animals
which subsequently underwent squamous keratinizing metaplasia. CoNDON [52], in a study of regeneration in normal and vitamin A-deficient rat tracheal
epithelia, has observed rapid epithelial proliferation: 8
64
M.M. AYERS, P.K. JEFFERY
hours after colchicine administration, 60% of epithelial cells are in mitosis compared with 'a few scattered
mitoses' present in normal epithelium. HARRIS et al.
[106] have confirmed the observations, finding large
increases in both MI and LI in vitamin A-deficient
hamster trachea. However, SHERMAN [198], in a
comparison of tracheal, corneal and epidermal epithelia from vitamin A-deficient rats, has found a
decreased MJ in all the epithelia studied. MI returns
to normal levels after local or oral application of
vitamin A acetate. LANE and GORDON [147] observe
foci of squamous metaplasia in vitamin A-deficient
rat tracheal epithelium, but report no significant
difference between the LI of rats on a normal diet and
those deficient in the vitamin. CHOPRA [46] has
examined the change in LI of basal and mucous cells
in newly established tracheal explants from normal
and deficient hamsters. Mucous cells from deficient
epithelia show only an initial transient increase in LI,
basal cells maintain a high proliferative activity
throughout the life of the explant and subsequently
give rise to squamous metaplasia.
An extensive study of vitamin A-deficiency and
restoration in hamster tracheal epithelium by
McDoWELL et a/. [156, 157] has shown that the
primary effect of deficiency is a decrease in the
proliferation rate of basal cells and (to a much greater
extent) mucous cells, with minimal morphological
change. They also described the development of
squamous lesions made up of epidermoid cells
containing PAS positive granules and where LJ was
raised above control levels. The authors conclude
that: I) vitamin A is necessary for maintenance of
normal rates of cell proliferation and that mucous
cells are particularly responsive to its absence; 2)
prolonged vitamin A-deficiency caused cell death
which resulted in a reparative response; 3) mucous
cells, which actively proliferate during repair, are
capable of expressing keratin-manufacturing or mucous secretory phenotypes, but vitamin A-deficiency
results in a predominance of the former, i.e. epidermoid lesions rather than muco-ciliary.
In a study of the effect of restoration of vitamin A
over seven days, the same authors [156] found that
basal cell proliferation remained consistently less than
that of controls, but mucous cell proliferation began
to rise two days after restoration, as did the numbers
of preciliated cells 24 hours later. Within seven days a
restored pseudostratified epithelium with normal
proportions of basal, mucous and ciliated cells was
present. The results suggest that the mucous cell plays
a primary role in normal development and repair of
tracheo-bronchial epithelium.
2.1.3 Turnover time. The turnover times (TT) of
respiratory epithelia have been calculated for a
variety of species, airway levels and ages in both sexes
(table III). Two approaches have been taken. Firstly,
in the studies of SPENCER and SHORTER (208), SHORTER
et al. [200, 201] and DrvERTIE eta/. [59] the time for
each labelled cell type to migrate (i.e. 'migration
time') to the epithelial surface and disappear
('slough') was observed and used to give an estimate
of TT for distinct cell populations. However, the
estimate was made without accounting for differences
between cell types in respect of tM, tS or their distinct
contributions to the growth fraction (see table III).
Secondly, TT bas been calculated from counts of
either the mitotic or labelling indices [31, 203). Again
no account was taken of differences either of tS or the
growth fraction (table III). BLENKINSOPP [27], assuming a value for tS of 8 hours and using LI data,
calculated that the TT for different airway levels in
two strains of rats of varying ages ranged from
between 67 and 111 days.
WELLS [230] has also calculated TT of rat tracheal
epithelium using data obtained from pulse labelling
with 3 H-T. With time, labelled basal cell:) migrate into
the superficial differentiating layer, thus the ratio of
labelled basal cells to total labelled cells in the
epithelium decreased. From the rate of decrease of the
ratio, the TT has been calculated for two 'grades' (of
microbiological status) of five week old rats, 11 - 12
days, for animals suffering from chronic respiratory
disease and 37-42 days for relatively clean animals.
TT has been shown to vary between species and
strains [27, 230], and to increase with both increasing
age and descending airway level [27]. No differences
have been reported between males and females.
2.1.4 Cell cycle and phase times. There are relatively
few studies which determine the length of the cell
cycle and its phases for distinct cell types (table IV).
Results vary widely; e.g. tC of hamster tracheal basal
cells is reported as 28 h by one group and 159 ( ± SEM,
11 h) by another and that of mucous cells is 25 h or 97
( ±SEM, 4 h) respectively [37, 38]. Use of colchicineinduced metaphase block and 3 H-T labelling in mice
gives a value of 380 h for tC of tracheal basal cells
[28]. Table V shows the findings for different phases of
the cell cycle for the tracheal basal cell: tS ranges from
6-12 and tG 1 from 12- 44 h [37, 38]. Comparison of
tS of mouse alveolar wall cells in males and females of
three strains at three ages shows that: a) tS is longer in
males than females (two out of three strains); b) tS
becomes longer with age (one strain only) and c) tS
varies between strains [203]. Similar comparisons for
other species and airway generations have yet to be
made. The reported values for tG 2 are relatively
constant and lie between three and four hours for
both basal and superficial (including mucous) cells
(table V). For tM, there are only two reports for the
respiratory tract (trachea): findings for both basal and
superficial cells are similar and range from 12 to 36
min [38]. The reason for the variation is not clear. In
other tissues, tM has been reported to last between I
and 2 b in mouse forestomach epithelium [235], 2 h in
mouse ear epidermis [173], and I. 7 h in adult (8 weeks
old ) rat liver (175].
2.1.5 Growth fraction (GF) and 'resting ' cells (G0 ).
The growth fraction can be determined with either the
65
PROLIFERATION AND D IFF ERENTIATION IN AIRWAYS
Table Ill. - Migrationffurnover time
Airway Level
Animal
Trachea
mouse
mouse
adult
adult
5-7
200
200
Trachea
rat
adult
6-7
201
Bronchus
- large
-small
mouse
mouse
4
4
3 &21
7-10
208
208
Bronchus
rat
adult
7-8
201
Alveoli
mouse
mouse
4
4
7
21
208
208
Sex
?
?
?
?
Age
wks/
weight g
Migration time
days
2
Reference
Turnover Time
days
Trachea
Trachea
rat
-S.D.*
-B.H.*
-B.H.*
200 g
67
100
330 g
111
rat
-C.D.*
-M.D.*
5
11-22
5
37-42
230
230
rat
5
22-40
31
rat
7
4~0
31
rat
15
120-180
31
adult
200 g
27
27
27
73
230
330 g
126
330g
167
27
27
330 g
200
27
12
12
52
52
44
203
35
203
203
203
Trachea
hamster
5
Bronchus
-main
-small
ratB.H.*
ratB.H.*
Bronchiole
ratB.H.*
Alveolar Epithelium
mouse- NGrb
mouse- NGrb
mouse- A/Grb
mouse- NGrb
84
35
*S.D. : Sprague Dawley; *B.H. :Black Hooded; *C.D. : Conventionally Derived; *M.D. :Minimum Disease.
continuous labelling or double labelling techniques.
The only such determinations for respiratory tract
that have been reported are those of B oREN and
PARADISE [37, 38] (table VI). Their studies of hamster
trachea show that the GF consists of 2- 4% of basal
cells and 1% mucous. Studies in rats by AYERS and
(II] and DONNELLY et a/. [61] have shown
that serous and indeterminate (also referred to as
'intermediate') cells contribute to the GF in rat
tracheal epithelium.
BOREN and PARADISE (38] have observed, among the
proliferating basal and mucous cell populations, a
J EFFERY
66
M.M. AYERS, P.K. JEFFERY
Table IV.- Cell cycle times
Tracheal
cell type
Animal
Basal
mouse
Superficial
mouse
Basal
hamster
Mucous
hamster
Basal
hamster
Mucous
hamster
Age
Sex
Methods of
measurement
wks
a
a
a
a
a
a
Cell cycle
time h
Reference
8
F.L.M.
300
28
8
F.L.M.
1400
28
adult
?
28
37
adult
?
25
37
13
G.C.
159 ± 11
38
13
G.C.
97 ±4
38
F.L.M.: fraction labelled mitosis; G.C.: grain counting
Table V.- Cell cycle phase times
Cell cycle phase time
Airway Level
-cell type
Animal
Trachea
rat
Sex
Age
wks
rat: conventionally
derived
cJ
h
Reference
tM
tS
201
adult
8
5
7
3.5
230
- Basal
mouse
8
8
3.6
28
- Superficial
mouse
8
8
3.6
28
-Basal
hamster
adult
6-8
<3
106
-Mucous
hamster
adult
6-8
<3
106
-Basal
hamster
?
?
12
3.5
0.2-0.3
12
37
- Mucous
hamster
?
?
7
2.9
0.2-0.3
15
37
-Basal
hamster
9-13
11
3.2
0.6
144
38
-Mucous
hamster
9-13
10.5
2.5
0.6
87
38
very small proportion of cells that do not show grain
halving with time after a pulse label with 3 H-T. This
suggests the presence of small numbers of both basal
(0.3%) an6i mucous (0.1 %) cells which were either
resting (i.e. in G 0 ) or, at least, proliferating mo~e
slowly than other labelled cells.
To summarize proliferative activity in the respiratory tract: I) normally very few cells are dividing,
resulting in a 'long-lived' epithelial s.urface (i.e. slow
turnover); 2) the numbers of dividing cells vary
between species and strains: they decrease from
proximal to distal airway levels and they vary with
increasing age; 3) there ·is di\irnal variation in the
number of proliferating cells; 4) vitamin A is an
important influencing dietary factor; 5) there is very
little information about cell kinetics m airway
epithelium, with most from animal data and virtually
nothing known about human lungs.
67
PROLIF ERA T ION AND DIFFERENTI ATION IN AlRWA YS
Table VI. - Growth fraction
Method of
measurement
Growth
fraction%
Reference
Tracheal
cell type
Animal
Sex
Age
wks
Basal
hamster
?
?
?
1.8
37
Mucous
hamster
?
'!
?
1.4
37
Basal
hamster
a
9-13
C.L.
3.9
37
Mucous
hamster
a
9-13
C.L.
1.3
37
C.L. : Continuous labelling.
2.2 Cell differentiation
The process of epithelial cell renewal is seen as the
passage of cells through proliferative, maturation and
functional compartments in response to death or
sloughing of previously existing cells. In 1881,
DRASCH (63] suggested that dividing respiratory
epithelial basal cells differentiated first into mucous
cells and then, in time, these became ciliated, without
an intervening division. On the other hand, the
suggestion that daughters of dividing basal cells
matured first into ciliated columnar and then into
mucous cells arose from observations of respiratory
epithelia from astJ1matic patients and experimental
injury and repair in ra bbit and ra t tracheas [52, I ll ].
The scq ttcnce of mo rphological changes (i. e. ' pathways') during differentiation has been studied by
pulse labelling a cohort of cells synthesizing DNA
with 3 H-T a nd observing changes in the pattern of
labeWng, with lime. Using this method BI NDREIITER
era/. (24] has studied proliferati on a nd diflerentiation
in young normal rats a nd suggests tha t differentia tion
proceeds from basal to mucous a nd then to ciliated
cells. A continuous labelling study of rat airway
epithelium has led BLENKINSOPP [27] to suggest that
basal cell division produces one basal and one
superficial ccJI, i.e. there is an asymmetric division
where one daughter cell is committed to differentiation and the other remains in the basal compartment.
The former then produces two daughter cells, one of
which is lost by sloughing.
AYERS and JEFFERY [II] have studied differentiation
in main bronchial epi thelium of normal specific
pathogen free rats fo llowing a single pulse of 3 H-T.
Wi tb Lime. a sigrtifica nt decrease in lhe pro portion of
cells contnining la bel occurs. suggesting a gradual
continuous loss of cells from surface epithelium.
Examination of the change in percentage label in each
cell type suggests that basal cells differentiate to both
mucous and ciliated cells, and that the pathway
includes an 'intermediate' cell type, which acts as an
uncommitted cell stage through which differentiating
cells pass. Detailed quantitative light and electron
microscopic studies of hamster tracheal epithelium
[155] have, however, established that true intermedi-
ate cells are very rare and cells, so designated at the
light microscope level, are a heterogenous population
including secretory cells and 'tall' basal cells which are
difficult to classify (i.e. indeterminate) [177, 155].
A cell kinetic study of hamster tracheal epithelium,
(38] has suggested a 3-compartment model of cell
renewal: the first comprises a self-renewing one of
proliferating basal cells which gives rise to a compar tment consisting of mucous cells. Some mucous cells
retain the ability to divide while others lose it and
become fully differentiated . The third compartment
consists of fully differentiated cells only, which do not
divide, and have a finite life span.
A different proposal has been suggested by DoNNELLY et al. [6 1], who observed changes in both the
numbers of each labelled cell type and the combinations of labelled 'adjacent cell pairs' which, in theory,
represent the recent progeny of a single cell's division.
No significant changes occur in labelling indices over
a ten day period , following a pulse of 3 H-T. F1owever,
changes in la belled pairs of adjacent cells suggest that:
a) ciliated cells ca n develop from cells of in termediate
(indeterminate) mo rp hology wit hin 24 h and b) that
both superfic ial mucous (goblet) and ciliated cell
development is preceded by two divisions, a basal cell
division foll owed by an intermediate cell division. The
cell type produced depends on the balance of
environmental influences. The studies of McDOWELL
and TRUMP [153] on human tissue and parallel studies
of epiLhelial injury in animals. indica te that secretory
cell hyperp lasia. sta tifi cation, epidermoid metaplasia
and 'carcinoma in situ· are closely related histogenetically. The authors believe that the lesions arise mainly
fro m hyperplastic proliferation of mucus-secreting
cells associated with pheno typic changes. Cell quantification and la belling e;>:periments with 3 1-I-T indicate that 'pre-ciliated' cells are the progeny of
secretory cell divisions and t hat many of the former
still contain secretory granules carried over from
parent cells following their division. The authors
propose tha t secretory cells play the major role in
genesis of ciliated cells during foetal and neonatal
development, during adult epithelial cell turnover and
during epithelial regeneration.
In summary, several studies have shown that in
68
M.M.
AYERS,
tracheo-bronchial epithelium, mucus-secreting and
indeterminate cells comprise the proliferating fraction
and that there is basal cell differentiation to both
mucous and ciliated cells. Mucous cells not only
proliferate but can differentiate into ciliated cells with
the latter a poorly dividing end stage but highly
functional cell. 'Compartments' serving different
kinetic and functional roles have been suggested but
their exact components and quantitative relationships
and the factors which influence their functional
relationships are as yet unknown.
3. Factors influencing proliferation and differentiation
Numerous extrinsic agents, shown to affect cell
division and thought to alter patterns of differentiation in airway epithelium, have been implicated in the
aetiology of respiratory disease. Foremost are airborne irritants such as tobacco smoke and industrial
or domestic environmental pollutants (e.g. oxides of
sulphur, nitrogen, ozone and vehicle exhaust fumes),
chemical carcinogens and infective agents. Studies of
the sequence of reparative changes after mechanical
trauma, and administration of certain therapeutic
drugs which may change proliferative activity provide
further insights into the extent to which respiratory
tract epithelial cells can divide and differentiate.
3.1 Irritation
Tobacco smoke is a complex organic mixture and
may damage each airway level in distinct ways,
whereas simpler inorganic substances often have a
predilection for one particular airway level or
another, depending on their physical characteristics,
such as solubility in water [104].
i) Tobacco smoke (TS)
Tobacco smoke comprises the combustion products
of well over 1500 components [17]. Morphological
and proliferative changes may be induced by whole
TS and both its particulate and gaseous phases
contribute [5, 18, 22, 51, 188]. Tobacco substitutes
may also be harmful [18].
Whole tobacco smoke, when given daily to experimental animals for short periods of up to twelve
weeks, causes an increase in epithelial thickness, and
mucous (goblet) cell hyperplasia in proximal airways
[22, 109, 115, 121, 125, 127, 144, 160,225, 226). There
is also an increase in the number and size of alveolar
macrophages [225, 226]. Continued exposure may
lead to loss of ciliated and goblet cells, basal cell
proliferation, and focal squamous cell metaplasia
which, with cell atypia, may be regarded as 'carcinoma in situ' [8, 9, 10, 18, 55, 58, 62, 105, 139, 151,
182]. Similar findings are described in man [7].
a) Proliferation- The effects ofTS on MI and Ll have
been studied only after short-term exposure (table
VII). In all species studied, MI increases in response
to TS in both extrapulmonary and intrapulmonary
airways, but mainly in the former [31, 34, 127). Nearly
all studies have utilized male animals and although
P.K.
JEFFERY
sex differences have been reported in the mucous cell
(hyperplastic) response [I 09, 113], no comparison of
MI or LI has been made. Both LAM» and REID (144)
and WELLS and LAMERTON [229) have found a doserelated response to TS, the highest number of
cigarettes smoked producing the greatest number of
mitotic divisions. The peak of the mitotic rise is at 24
h which, in spite of continued exposure, falls to
control levels by about two days following commencement of exposure [229). If, however, there is a
break in TS exposure, a second peak is generated on
recommencement [34, 121].
The effect of TS on LI has been studied by BoREN
[36] and AYERS and JEFFERY [11]. In the former study
on hamsters there is a significant increase in LI (up to
four-fold) in the airways of 400 ~tm or less, after only 8
h of TS. A second peak in LI is found at 40 h of
exposure, in the alveolus but not in the intrapulmonary conducting airways. Trachea and main bronchi
were not examined in the study. AYERS and JEFFERY
[11] have examined the LI in rat main bronchus at
selected time intervals of up to 14 days of TS exposure
and find a significant rise at 24 h, which remains at 3
days, falls to control levels by 7 days and is
unchanged at 14 days. Labelled basal cells produce
the increase seen at 24 h, whereas at 3 days both
labelled indeterminate (intermediate) and secretory
cells, containing a mixture of serous and mucous
granules (i.e. transitional), are the main dividing cells.
Although epithelial LI returned to control levels by
seven days, the distribution of dividing cells is altered.
In control animals, 72% oflabelled cells are basal and
17% serous; no mucous cell is labelled. In contrast,
after seven days ofTS exposure, 37% of labelled cells
are basal and 40% mucous. This finding suggests that
the population of mucous cells can respond to
irritants by proliferation, and indicates one important
mechanism by which the TS-induced increase in
mucous cell number may be brought about.
b) Differentiation - JEFFERY and REID [I 15, 121]
suggest that the increase in mucous cell number seen
in the rat is initially due to transformation of existing
serous cells as cells with transitional features are
found. AYERS and JEFFERY [I I] have labelled cohorts
of proliferating cells with a single pulse of 3 H-T and
monitored differentiation by recording changes in the
proportions of each cell type labelled with time of TS
exposure. Compared with unexposed controls, there
is no marked change in the pathways of differentiation; the ciliated cell still forms the major end product
of differentiating (labelled) basal cells. However,
newly occurring mucous cells do not arise as a
consequence of basal cell differentiation, but rather
from the transformation of pre-existing serous cells
and subsequent division of newly-formed mucous
cells.
ii) Sulphur dioxide (S0 2 )
Sulphur dioxide is associated with environmental
pollution (both domestic and industrial) where it may
act synergistically with other particulate pollutants
[14, 114). It is highly water soluble and ionisable and
69
PROUFERATION AND DIFFERENTI ATION I N AIRWAYS
Table VII. - Effect of tobacco smoke on proliferation
Animal
Exposure time
cigs/exposure
Airway level
Rat
2 hours
(48)
tracheobronchial
epithelium
Rat
48 hours
(32)
bronchi
bronchioli
alveoli
Rat
4 days/week
for6 weeks
(25)
trachea
Rat
14days
(25)
main bronchus
24 hours
3 days (25)
7 days
14 days
tracheobronchial
epithelium
lOOpuffs
tracheal basal
cells
Rat
Guinea Pig
MI
LI
max. change
max. change
Reference
229
t 10 X
(24h)
t3 X
t4 X (8 hr)
t4 X
36
34, 127
bx
(6 weeks)
h
X
(24 h)
11. 121,122
f 12 x (7 days)
after short-term exposure appears to affect central
(large) more than distal, airways (56, 120, 124, 143).
Studies of the morphological changes induced by S02
are plentiful [6, 114, 160, 168, 209]. Only two studies,
however, have reported quantitative data about S02 induced changes in respiratory epithelial cell kinetics
(table VIII). The first of these by REm [178] has used
S0 2 to 'model' human bronchitic changes. Specific
pathogen free rats are exposed to 400 parts per
million (ppm) of S02 three hours daily for up to six
weeks, sufficient to induce mucous cell hyperplasia. In
the trachea and central intrapulmonary airways, Ml
twx
)
)
f9x
px
)
t 14 X (24 h)
241
) 229
increases from 0.15% in controls to about 1.4% at
four days of exposure. T hereafter, it falls slightly by
three weeks but is maintained, for up to six weeks, at
a level significantly higher than that of controls. In
central airways, the early rise in MI is followed by a
rise in mucous cell number. In distal bronchioli, MI is
not significantly raised, yet a rise ~n mucous cell
number also occurs.
OKUYANA et al. [170] have examined the response
in chickens to doses of S02 given daily for fourteen
days. There is mucosal hypertrophy and a 2- I 0 fold
increase in MI, an increase in the numbers of mucous
Table VIII. - Effect of sulphur dioxide on proliferation
Animal
Exposure time
Airway level
Rat
5 days/week
trachea
h2x*
143
Rat
5 days/week
45 days
hilar airway
hilar airway
t
t
)
)168
4 hours/week
trachea
t
)
5 months
bronchi
14 days
trachea
Dog
Chicken
* max.
seen at 4 days.
ChangeinMI
Reference
) 242
)
t
170
70
M.M. AYERS, P.K. JEFFERY
and peptide-secreting cells (epithelial and glandular),
macrophages, and recruitment of inflammatory cells.
iii) Nitrogen dioxide (N0 2 )
Extensive studies by Evans and his colleagues have
shown that the primary site of damage by N0 2 is at
the level of terminal and respiratory bronchioli and
adjacent alveoli (see (42]). Short-term exposure (e.g.
daily for 40 days), induces bronchial and bronchiolar
hyperplasia with tall columnar epithelium and frequent mitotic figures. Longer exposure (i.e. for the
animals' lifetime) induces emphysema.
Various studies [53, 70, 75, 77, 79, 85, 86, 102, 137]
have each used 3 H-T labelling to elucidate which
airway level and epithelial cell is most profoundly
affected by N0 2 and which cells take part in the
proliferative response. Ciliated cells of the terminal
bronchioli and type I cells of adjacent alveoli are
destroyed by N0 2 [212]. Subsequently, nonciliated
bronchiolar (Clara) cells and alveolar type II cells
proliferate within 24 h, reaching a maximum at 24-28
h [102, 2 12]. They return to control levels by 2-4 days
[82, 215], (table IX). The proliferating Clara and type
Table IX. -Effect of nitrogen dioxide on proliferation
Animal
Hamster
Exposure time
h
6
6
Airway level
trachea
main bronchus
Change in LI
Time after
N0 2 days
t12x
f 49 X
Reference
)
)
1
)
)
6
Hamster
small bronchi
b6x
6
bronchioli
6
alveoli
t 23 X
t 2x
24
trachea
h.4x
1
1
lh
) 137
)
)
)
)
)
)
24
bronchus
fi43x
(0.03% 4.3%)
1h
)
) 102
)
24
small airway
24
alveoli
Rat
48
alveoli - type
llcell
Rat
24
terminal
bronchiole
24
terminal
bronchiole
24
24
terminal
bronchiole
b3x
lh
4x
lh
)
)
)
1
170
t 50x
lh
t 35
)
)
)
1
X
1 60 X
)
)
)
3
) 75,79
)
)
terminal
bronchiole
45 X
10
24
terminal
bronchiole
45 X
15
)
Rat -1 month
old
24
terminal
bronchiole
28 X
1
)
)
Rat - 25 months
old
24
terminal
bronchiole
14 X
2
)
)
)
)
)73
)
PROLIFERATION AND DIFFERENTIATION IN AIRWAYS
II cells differentiate into ciliated and type r cells
respectively [70, 76, 79, 81]. Older animals show the
same pattern of response, but the onset of proliferation is slower, more tissue damage occurs, and the
magnitude of the proliferation is greater than that
observed in younger animals [73]. EvANS et a/. [82]
have shown that the proliferative response is dose
related, and that once the LI has returned to control
levels, it remains so despite continued exposure,
irrespective of age. Thus it appears that the newly
repaired epithelial cell populations become resistant
to the toxic effect of the irritant [73, 82].
iv) Ozone (0 3 )
Ozone is a highly toxic component of photochemical
smog: its sources and effects have been reviewed by
STOKINGER (218), DUNGWORTR et a/. (64) and Bn..s
and CHRISTIE (23). It causes injury in proportion to
the exposure dose [75, 78, 196], the trachea is severely
affected, and terminal and respiratory bronchioli as
well as adjacent alveoli are subject to more damage
than more distal alveoli [86, 213, 214, 216]. Exposure
conditions vary and the doses used in a number of
species range from 0.1 to 3.5 ppm. Quantitative
observations ofMI have been made by SCHWARTZ et
al. [196], quantitative studies of LI reported by EvANS
and co-workers [75, 78, 80] and CASTLEMAN eta/. [45],
and studies of differentiation have been made by
STEPHENS et a/. (215) and CASTLEMAN et a/. (45] (table
X). The prime targets for damage are the ciliated cells
of conducting airways and type I cells of the alveolus
71
[29, 35, 45, 75, 78, 152, 161, 172, 194, 196, 213, 214,
215]. The nature of the proliferative response and the
resultant sequelae appear to be the same whether
exposure is continuous or not [215]. PENHA and
WORTHHEIMER [172] have reported a mitotic response
in tracheal basal cells with subsequent hyperplasia
and squamous metaplasia, after daily 2 hour exposure
of young mice to 2.5 ppm for 45 days. In rats and
monkeys proliferation of Clara cells reach a peak
after 2-3 days [45, 75, 78, 152, 213, 2 14, 215]. High
doses (15 ppm) produce an increase in LI in all
epithelial cells, which peaks at 2- 3 days [ 199]. When
exposure is prolonged beyond 3 days, LI returns to
control levels by about 4 days in spite of continuing
exposure [75, 78], although LuM et a/. [152] have
observed a raised LI throughout the 168 h of
exposure. Associated with the increase in Ll is a
change in the relative proportions of bronchiolar
epithelial cells. Clara cells increase in number and
ciliated cells decrease [45, 75, 78, 152]. EVANS and
colleagues [75, 78] have found that Clara cells
constitute all of the originally labelled population, but
by 4 days, 33% of labelled cells are ciliated and 68%
are Clara cells, and the authors suggest that the
proliferating Clara cells differentiate into ciliated.
Grain counts indicate that Clara cells divide at least
once before their subsequent differentiation. Following type I cell necrosis, type II cell proliferation results
in alveolar repair within 48 h [45, 75, 78, 215],
indicating that the latter is the alveolar stem cell.
Table X.- Effect of ozone on proliferation
Animal
Exposure time
[03 ppm]
Change in
Airway level
Reference
LI
MI
time after 0 3
Rat
24h
t 9 x non-ciliated
(0.7)
terminal
bronchiole
Rat
168 h
(0.8)
terminal
bronchiole
Rat
48h
(0.5, 0.9)
alveoli - type
II cells
2 h/day
trachea
hasal cells
120 days
(2.5)
bronchi alveoli
ttype II cells
)
) 172
)
Mouse
8 days
(0.5-0.8)
bronchioli
tClara cells
223
Mouse
6h
(0.5-3.5)
alveoli
Monkey
50h
respiratory
bronchiole
Mouse
(0.8)
secretory (24 h)
t 30
X (72h)
78
152
214
(24-30 h)
t (24h)
80
tcuboidal bronchiolar
45
t(72 h)
cells (50 h)
72
M.M.
AYERS,
P.K.
Kultchitsky cells have also been estimated to have an
LI of 0.5% in control animals and show a response to
ozone, with an increase in LI to 9% after 36 h of
exposure [45].
There is also evidence of the development of
tolerance to ozone [35, 152, 213, 2 15]. After 48 h
exposure, the epithelial proliferative response in the
terminal bronchioli stabilizes and repair begins to
return epithelial morphology to normal [214]. LI
decreases to near control levels within 4 days and in
spite of continued exposure (maintained at 0.4 and 0. 5
ppm), does not show a further rise [78]. Tf the dose is
increased, there is a second rise and subsequent fall in
LI, with a corresponding new level of tolerance [75,
78]. STEPHENS et al. [215] have shown that, prior to
weaning, bronchial epithelium of rats is resistant to
oxidant-induced damage. Oxidants produce little or
no injury until 35 days of infancy.
EvANS et. a/. [80] have also shown, in a study using
ageing rats ( 18- 20 months), that LI is initially
lowered in response to a single 6 h exposure (0.5- 3.5
ppm), then increases to control levels or above by 72
h. T hus age influences the proliferative response to
ozone.
v) Oxygen (0 2 )
Oxygen in high concentrations (40- 100%) is toxic
to lung tissue, and induces: I) an exudative phase
comprising alveolar oedema, haemorrhage, fibrinous
exudate, hyaline membrane formation and alveolar
necrosis; 2) a subacute proliferative phase leading to
alveolar epithelial (type II) hyperplasia, interstitial
fibrosis and partial resolution [23]. Experimental
studies using rats, mice and monkeys have shown that
the severity of the damage 'to proximal airways and
alveoli depends on concentration [169, 223]. Most
experimental studies have concentrated on the alveolus where oxygen induces type I cell necrosis follow~d
by type II cell proliferation. I n response to 90- 100%
0 2 , EvANS and colleagues [71, 72] and ADAMSON and
BOWDEN [I] found an initial decrease in alveolar cell
proliferation followed by hyperplasia, resulting in a
JEFFERY
cuboidal alveolar lining (see table XI). LuM eta/. [152]
have compared the effects of ozone (0.8 ppm) and
80% 0 2 on the LI of terminal bronchioli and found
that the 0 2 -induced proliferative response is delayed
and is smaller than that initiated by 0 3 . Differential
cell counts show that the nonciliated (Clara) secretory
cell is the most important proliferating cell in the
response to both oxidants. Oxygen also appears to
affect the proliferative component of repair. Following initial damage by ozone, exposure to oxygen at
concentrations higher than 60% inhibits the proliferation by which repair to ozone normally proceeds
[103].
vi) Chemical carcinogens
Of the aromatic hydrocarbons, benz(a)pyrene (BP)
and dimethyl-benz-anthracene (DMBA) have been
widely studied, although few papers have examined the
early proliferative changes induced in the respiratory
tract. Binding of tritiated BP occurs with DNA of
ciliated, mucous and basal cells, and is dependent on
enzyme activation [107, 219]. During Vitamin A
deficiency, which has been linked to increased risk of
developing carcinoma [ 13], binding is increased and
appears to be greatest in areas of squamous metaplasia
(HARRIS eta/., unpublished observation quoted in [91]).
In an in vitro study, using neonatal rat tracheas,
CRocKER eta!. [54] have found that exposure to BP,
DMBA or methyl-cholanthracene (MCA) at a range
of doses and for a maximum of 14 days, causes loss of
differentiated (superficial) cells and early increases in
both MI and LI in the remaining basal cells. The
increases in MI and LI are not always proportional,
nor are they alike for each agent. Continuation of
exposure with the higher doses of each agent results in
the prqduction of squamous metaplasia. Intratracheal
administration of BP, repeated weekly for up to
twenty weeks, gives rise to early cell proliferation of
either basal [179, 180] or mucous cells [16]. In either
case, these changes precede the development of
squamous metaplasia, keratin formation and premalignant changes.
Table XI. -Effect of oxygen on proliferation
ArUmal
Exposure time
[02 %)
Airway level
Change in
MI
Reference
LI
time of max.. change
Mouse
Mouse
4days
(20- 95%)
terminal
bron chiole
60 days
(90%)
alveoli
Mouse
Rat
hx
bx
type II
(36 h)
alveoli
168 h
(80%)
103
(48 h)
terminal
bronchiole
72
t 15x
Clara
(168 h)
152
PROLIFERATION AND DIFFERENTIATION IN AIRWAYS
The action o f nitrosamines, (in particular diethyl
nitrosamine (DEN)) on airway epithelium has been
s tudied by REZNIK-SC'HULLF.R a nd co-workers [1 8 1,
183- 186]. Jn hamster trachea, DEN induces basal cell
proliferation, mucous cell diffe rentia tio n a nd epithelial cell hyperplasia, followed by d evelopment of
papillary po lyps made up
mucous and 'intermediate' cells. Tn hamster bronchi, severalnitrosami nes arc
found to specifical ly affect C l<lra and APUD (Kultchitsky-like) cells, inducing their proliferalion and the
subsequent production sq uamous metaplasia [ 186].
A single exposure to urethane can elicit bronchiolar
nonciliated (Clara) cell ~1.nd type rl alveolar cell
hyperplasia with multiple adenoma formation i11 mice
[1 3 1. 132). DYSON and HEPPJ.ESTONE [66] have given
urethane as a single injection, wb.ich ca uses a rise in
LI of alveolar cells, peaking at two weeks after
urethane and returning to control levels after two
months. Subsequent tumour formation is first observed four weeks later. In another study by KAUFFMAN [131], mjce were continually exposed to urethane
in drinking wa ter, resulting in an early d ecrease in Ll
and type ll cell necrosis. This is followed by a n
increase in LJ which peaks at six weeks, together with
a doubling of the number of type IT cells. Over the
next sixteen weeks, LI returns to control levels, type II
and type I cell numbers decrease and tumours
develop.
These observations are in accord with the multistage concept of carcinogenesis, i.e. two distinct phases
following a carcinogenic stimulus: 1) a nonspecific
response in which there is cell proliferation and 2)
later development of atypic and malignant changes
(often in the absence of continuous carcinogen
exposure).
or
or
3.2 Mechanical injury
The healing following mechanical trauma o f tracheal epithelium has been studied as a n experimental
model of airway cell proliferation and differentiation.
Two techniques bave been used: I) gentle stroking
with cotton swabs, which removes superficial cells
only, leaving basal cells essentially intact (95- 99, 146,
147] and 2) curettage, which removes all epithelial
cells and aims to leave a denuded basement membrane [ 15, 52, L33- 136. 158, 23 1].
I) Stroking - LANB and GoRDON [146, 1471 have used
the gentle stroking technique on the ventral (i.l!.
anterior) surface of rat trachea and have given
colchjc ine a nd 3 H-T LO visualize the proliferative
response. The a uthors found that immediately after
injury. there is a small early 'vave of dividing cells
which peaks at seven ho u rs post-injury and comprises
4% of rhe epithelia l cell population: the 4% represerus mostly s uperficial mucous cells [96, 97]. Subsequently, there is a larger peak of Ll at 22 h, made up
of 42% of the to tal cell populatio n. A large peak in
Ml then occurs at 32 h (also comprising 42% of cells),
which is followed by a smaller peak in LI at 39 h,
made up of 7% of the population [98].
73
The authors used double labelling and grain
counting to distinguish between differe nt epithe lia l
cell populations. Do uble labelling has shown that the
early minor wave of dividing cells (7 h, Ml peak) i a
different population fro m that which is synlhesi7j ng
DNA at 22 b [97]. Grain counts show that the cells
synthesizing DNA at 22 h go o n to e nter mitosis at 32
h. forming the ~econd a nd major mitotic peak. T he
authors suggest tha t the last mentio ned population of
cells is no rmally at ·resr in the G 1 phase of the cell
cycle. Grain counts also show that the cells synthesising DNA at 39 h do not enter mitosis thereafter, and
thus come to rest in the 0 2 phase of the cell cycle.
The results suggest, therefore, three separate epithelial cell populations. one cycl ing and two ' resting',
one in G 1 and the other in G 2 • The response to mild
injury is a single cycle of synchro nous cell division in
the two 'resting' cell populations. The G 1 population
is thought to be basal and the G 2 population mucous
[97, 147].
Study of the subsequent differentiation and reconstruction of a functional mucociliary epithelium [146]
reveals that the percentage of labelled basal cells
decreases and that of labelled mucous cells increases,
without changes in grain counts, indicating that
during this process mucous cells differentiate from
basal cells without prior division [98]. Cell cycle phase
times have been determined by stimulating another
wave of DNA synthesis witli a second injury at the
original site: tS for basal cells is 8-9 h, tG 2 is 2.53.5 h and tC is 28 h. tG 1 is then calculated as 14.516.5 h (96, 97].
2) Curettage - l ni tia lly, curettage induces cell migration (52, 23 1] followed by mitosis at 24-48 h postinjury in both basal and mucous cells [ 158]. The result
is a sq uamous epithelium which covers the wound
area. Mitosis continues among the newly-migrateu
cells, producing immature 'indifferent' daughter cells
which subsequently ditferenLiute over 72- 96 h postinjury into functional ciliated a nd mucous cells (158].
KEENAN and colleagues [133- 136] have quan 1ified
the kinetic responses in hamsters. Control values of
MI and LI in the trachea of untreated animals were
found to be 0.19 and 0.63% respectively, made up of
dividing mucous a nd basal cells in the raUo of
approximately 2: I respectively. The autho rs emphasize the impo rta nce o f the secretory cell in the regenerati ve process. As evidence they cite the fo llowing
observa Lio ns: I) secretory ceUs form the grealcst
proportion of the dividing cell population in control
epithelium and 2) in response ro the stimulus of
injury, a greater percentage by far of secretory cells
than basal cells, proliferate. When labelled cells were
each taken as discrete populations, a larger proportion of labelled secretory cells than basal cells were
found in metaphase (arrested by colchicine), at a
given time interval after wounding, suggesting that
the movement of the former through the cycle was
more rapid. In addition, epidermoid cells covering the
wound area contain PAS-positive material. Therefore, the authors consider that the resulting epider-
74
M.M . AYERS, P.K. JEFFERY
moid epithelium is mainly the result of dividing
secretory cells. By continuous infusion, nearly all the
epidermoid cells become labelled as they continue to
proliferate. The proliferation rate returns to normal
between 60- 120 h and epidermoid metaplasia gives
way to a nearly normal mucociliary epithelium.
CONDON (52], WILHELM (232) and LANE and
GoRDON [147] have examined proliferation and repair
in airway epithelium of vitamin A-deficient animals.
Deficient epithelia develop multiple foci of keratinizing stratified squamous metaplasia. CONDON [52] has
found that many more epithelial cells than normal
divide, whereas LANE and GORDON [147] find no
difference between LI in deficient and normal epithelium. In vitamin A-deficient animals, curettage results
in the same sequence of cellular events, as in normal
repair, but the wound area is restored with a
keratinizing squamous metaplasia whether the original surface is squamous or columnar and mucociliary
in type. The repair time is also shorter (i.e. 6- 8 days)
than in normal animals (i.e. 12- 14 days) [232].
Interestingly, the wave of mucous cell proliferation
reported in normal repair after gentle stroking [98]
does not occur in vitamin A-deficient animals [147].
3.3 Enzymic injury
Elastase given as a single intratracheal injection
increases MI and LI in three cell types, e.g. the LI of
I) non-ciliated, non-secretory bronchiolar cells increases from I to 8% at 24 h, 2) type II alveolar cells
increases from 0.5 to 15% at 2 days and 3) endothelial
cells increases from 0.2 to I 0% at 4 days [228].
3.4 Drugs
A variety of pharmacologically active chemicals,
either in use clinically or experimentally, affect
proliferation in the respiratory tract. Isoprenaline
sulphate, a potent stimulator of DNA synthesis,
increases M I at three extra- and two intrapulmonary
airway levels when given daily at high dose [33, 11 5].
The effect is seen in male but not female rats. More
cells are found in division in the superficial than as
normal in the basal zone of the epithelium. Pilocarpine nitrate (also daily at a high dose) causes less of an
increase than isoprenaline, significant only in the
trachea and proximal intrapulmonary airways. Daily
administration of either drug increases the number of
airway mucous cells [128, 220]. Oestrogen (ethanyl
oestradiol) given by mouth to guinea pigs, produces
an initial increase of mucous cell numbers, which is
later replaced by foci of squamous epithelia with a
high MI [68, 69].
Conversely, several nonsteroidal and steroidal
drugs have been shown to inhibit the increase in
mucous cell numbers due to irritation by cigarette
smoke [101 , 122, 126, 190, 191). As it is known that
one component of the cigarette smoke-induced increase in secretory cell number is cell division [I I,
122], it is possible that these dr ugs produce their effect
by inhibition of proliferation. Supportive evidence
comes from other organ systems which show that the
nonsteroidal anti-inflammatory drug, Indomethacin,
inhibits increases in the LI of rat epidermis induced
by promoting agents such as 12-0-tetradeca-inoyl"
phorbal-13-acetate (TPA) and also of kaolin-induced
granulomata and methylcholanthrene-induced tumours [84, 89]. Interestingly, the proliferative response to TPA is restored by addition of prostaglandin (PG) E 2 but not PGF 2 .. [84, 88- 90]. In the
respiratory tract, the anti-tussive agent, Phenylmethyl-oxadiazole (PMO), present as 2% by weight of
tobacco, partiaJiy inhibits increases in MJ seen at two
weeks of exposure to cigarette smoke (121, 127). PMO
delays (by I day) the mitotic response seen d uring the
first 24 h after exposure, but, paradoxically, the
delayed response is almost twice the amplitude of the
earlier response without drug (34}. Apart from the
inhibitory effect of Dexamethazone on L1 of foetal
(16- 18 day) mouse lung tissue [130], little is known of
the effects of steroids on pulmonary cell division.
Early administration of Methyl-prednisolone to adult
mice inhibits butylated bydroxytoluene-induced type
II cell proliferation (as measured by LI), providing
the degree of lung injury is 'mild' [206].
Colchicine and the vinca alkaloids, vinblastine and
vincristine, are classic inhibitors of cell division
arresting mitosis at metaphase [48, 67]. In this context
it is interesting that vinblastine sulphate (given
subcutaneously at 0.05 mg · kg- • daily for 21 days)
prevents cigarette smoke-induced mucous cell proliferation in the upper trachea and proximal airways
of the lung. However, at the dose used, vinblastine
does not appear to inhibit cell division as there is no
accumulation of metaphase arrests with time (EVANS
et al., unpublished; [126]) suggesting an alternative
mechanism of action such as inhibition of mucous
synthesis [2).
Lastly, t here are some findings regarding a novel
action of a mucolytic drug N-acetylcysteine (NAC).
NAC has now been shown both to inhibit cigarette
smoke-induced secretory cell increase [123, 191] and
to attenuate, but prolong, the early proliferative
response [123]. The early proliferative response to
tobacco smoke is formed by both basal and mucous
cell division and NAC particularly inhibits the
former. However, orally administered NAC also
causes degeneration and sloughing of rabbit tracheal
goblet cells after 20 min [142]. Nonsteroidal antiinflammatory agents, including NAC, also appear to
speed recovery after experimental cessation of cigarette smoke [192).
3.5 Infection
The MI, LI, tS and turnover time have been
determined in rats with minimal disease and compared with those of conventionally derived rats, many
with chronic respiratory disease [229, 230]. MI and LI
are highest and turnover time shortest in the small
number of diseased animals, while the values for tS
PROLIFERATION AND DIFFERENTIATION IN AIRWAYS
are similar in both [230]. An anecdotal finding is the
report of one infected coutrol animal in u study by
Bowuc and REID [33], which showed that boLh
mucous cell numbers and MJ are increased a t each
airway level examjned. ln another study by SHORTER
et a!. [201]. two rats which showed evidence of
purulent tracheo-bronchitis bad the Ll at three levels
greatly increased with respect to the cont rols (without
disease).
3.6 Neural control
As certain neuromimetic drugs alter the rate of cell
proliferation, it would seem reasonable to hypothesize that the nervous system is one component which
may control or, at least, affect cell division. There is
some evidence from other body systems, e.g. in the
intestine it has been suggested that autonomic nerves
may have the capacity to respond to changes in the
rate of cell loss and, in a highly localized manner,
contribute to the balance of maintaining ceil production a nd loss (222]. (n addition, SILEN et al. [202] have
shown that vagotomy is invariably associated with an
increase in the fraction of cells in the S phase in the
dog duodenum and jejumtm six weeks postoperatively. lmmunosympathectomy in rats also greatly
increases Ml (i.e . decreases turnover time) of jejunal
epilhelium [65). Since gut and lung epithelia share a
common origin. it mjght be of interest to look for a
neural component in the COJltrol of airway epithelial
proliferation. Indeed, the reqtLirement of both intact
motor and sensory nerves for effective epithelial
repair and regeneration (albeit in amphibia) has
already been well described [204].
3.7 Immune system
The mucosal immune system (gut and bronchusassociated lymphoid tissue) has recently received
much attention as a first line of defence against
invading micro-organisms. In this context MILLER
and NAWA [165] and MrLLf:R eta/. [166] have studied
the instestinal epithelial response to infec tion by
Nippostrongylus brasielien.tes. There is an increase in
the mucous cell number, a thymus-dependent phenomenon in which thoracic duct lymphocytes, either
directly or indirectly, appear to regulate the 'djfferentiation' of intestinal mucous cells. Thus, the regulatory role of the lymphocytes, present in bronchusassociated lymphoid tissue, on cell division and
differentiation may well be a fruitful area for future
investigation.
4. Summary
The proliferative potential of the various cell types
present in airway epithelium have been described. The
techniques used to assess their proliferative activity
(MI and Ll), growth fraction and length of each
phase of the cell cycle have been given in outline. By
use of a variety of techniques, there is ample capacity
75
to recruit non-cycling cells rapidly into the cell cycle.
Under certain circumstances (e.g. cigarette smoke
exposure and mechanical trauma) the mucous (goblet) cell is proliferative and its role in epithelial repair
has been clearly underestimated. Factors contributing
to the variation seen in baseline proliferation include
species, sex, hormonal status, airway level, age,
diurnal rhythm and pathogen-free status. A variety of
stimuJi increase proliferation as part of a reparative
process: I) irritation by oxidants, chemical carcinogens or enzymes, 2) mechanical i11jury, 3) infection
and 4) certain drugs, some of which stimulate division
whjlst others inhjbit the proliferative response to
irritation and may be useful in controlling the
response.
lncreases in the numbers of some cell types may
be due to differentiation rather than proliferation and
the 3-compartment model can be applied to airway
epithelium comprising: I) the self renewing/
proliferating compartment. made up of basal and
secretory cells in the large airways, nonciliated
broncruolar (Clara) cells in the small airways, a nd
type II cells in the alveolus; 2) the differentiation
compartment containing mature functional cells,
some of which retain the capacity to divide, e.g.
serous, mucous (goblet), Clara, type II cells; 3) the
fully-mature end stage cells (e.g. ciliated cells) which
do not normally divide.
The nervous system and lymphocytes present in
epithelium and bronchus-associated lymphoid tissue
may also have a part Lo play in controlling cell
division and their roles should be investigated furlher.
However, the lung remains relatively unexplored with
regard to the factors which control the proliferation
and differentiation of its many distinct cell types. both
in health and disease.
AcknoH'led~:emtmts: We wannly acknowledge the
s upport given to M .M. Ayers by the Cystic Fibrosis
Research Trust (England) and the patience shown by
J. Billingham for help in preparing the manuscript.
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