Collagenase secretion accompanying changes in cell shape occurs only in
the presence of a biologically active cytokine
IRENE KUTER*, BARBARA JOHNSON-WINTf, NANCY BEAUPRE and JEROME GROSS
Developmental Biology laboratory of the Medical Services, Massachusetts General Hospital, Fruit Street, Boston, MA 02114, USA
•Author for correspondence
•(•Present address: Department of Biological Sciences, Northern Illinois University, Dekalb, IL 60115, USA
Summary
We have investigated the relationship between collagenase production, cell shape and stimulatory
factors in cell culture. In a homogeneous culture of
primary rabbit corneal stromal cells, shape change
induced by a variety of agents was not effective in
stimulating collagenase secretion. Only in the presence of a biologically active cytokine or phorbol
myristate acetate was a correlation seen between
changes in cell shape (induced by a second agent)
and collagenase secretion by these primary cells.
Cell shape changes were not, however, necessary
for collagenase secretion, since certain concentrations of endotoxin or lactalbumin hydrolysate
effected secretion of the enzyme in the absence of
morphological changes. With passaged cells or
mixed cell cultures, where cell shape change did
correlate with collagenase secretion 'without the
addition of an exogenous agent, the production of
an effective cytokine (autocrine or paracrine) was
demonstrated. Thus cell shape change seems to be
neither universally necessary nor sufficient for the
stimulation of collagenase secretion. It is proposed
that the function of cytokines may be more immediately related to gene expression in this system than
is change in the shape of the cell. The hypothesis is
presented that cell shape changes may render the
target cells receptive to cytokines, perhaps by replacing the need for a natural cytokine cofactor. It
is also demonstrated here that the use of passaged
cells, mixed cell cultures containing endogenous
cytokine-secreting cells or tissue culture additives
can profoundly affect the interpretation of the effect
of various agents on collagenase secretion, and may
lead to observations that are not directly relevant to
cell function in vivo.
Introduction
as PDGF (Bauer et al. 1985), EGF (Chua et al. 1985)
and TGF/3 (Chua et al. 1985), cytochalasin B (Harris et
al. 1975), colchicine (Wahl & Winter, 1974), tumour
necrosis factor (Dayer et al. 1985), endotoxin (Wahl et al.
1974), phorbol esters (Brinckerhoff et al. 1979), urate
crystals (McMillan et al. 1981), proteases (Werb &
Aggeler, 1978), polyHEMA substrates (Aggeler et al.
1984), polyethylene glycol (Brinckerhoff & Harris, 1981),
collagen (Biswas & Dayer, 1979), latex beads (Werb &
Reynolds, 1974) and fluoride ions (Jendryczko & Drozdz,
1985). The relevance of these in vitro observations to
physiological control mechanisms is often difficult to
ascertain.
Much recent interest has been focused on the relationship of phenotypic changes of cells in culture to alterations in cell shape. Such changes have been linked to
alterations in the phenotype of chondrocytes (Zanetti &
Solursh, 1984), mammary epithelial cells (Emerman et
al. 1977), synovial cells (Aggeler et al. 1984; Werb et al.
Collagenase is a critical regulator of extracellular matrix
remodelling because of its unique ability to initiate
degradation of the native collagen fibril (Gross, 1981).
This enzyme appears to have no other natural substrate.
Its cleavage of the fibril is the rate-limiting step in
collagenolysis, and therefore the control of synthesis and
action of this enzyme are likely to be of major importance
in normal and abnormal morphogenetic processes. Study
of these processes in vivo is technically difficult, and
therefore most of our knowledge of regulation is derived
from cell culture. Stimulation or induction of collagenase
production from a variety of cell types in culture by many
agents has been reported. These include media conditioned by monocytic cells/macrophages (Dayer et al.
1979) and epithelial cells (Johnson-Wint, 1980a; Johnson-Wint & Gross, 1984), native and recombinant interleukin 1 (Postlethwaite et al. 1983), growth factors such
Journal of Cell Science 92, 473-485 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Key words: collagenase, cell shape, cytokines.
473
1986) and adipocyte precursors (Spiegelman & Ginty,
1983). In synovial cells, it has been proposed that
reorganization of polymerized actin, with loss of stress
fibres, is the common trigger for induction of the
procollagenase gene by a variety of agents (Unemori &
Werb, 1986). It has been postulated (Unemori & Werb,
1986) that cell rounding and matrix degradation would be
likely to occur together in vivo.
In contrast to most of the published studies, in which
passaged cell strains or lines, or complex heterologous
mixtures of cell types such as synovial cells or dermal
fibroblasts have been used, in this laboratory we have
been interested in regulation of collagenase secretion by
primary, homogeneous cultures. We have been particularly interested in primary cultured corneal stromal cells
(Johnson-Wint, 1980a; Johnson-Wint & Gross, 1984)
because these appear to be a homogeneous and stable
population (Conrad, 1970), which in the normal tissue
(corneal stroma in vivo) has shown no evidence of
collagenase production by immunofluorescence but will
do so when the tissue is injured (Gordon et al. 1980). We
have observed that many of the agents, including those
affecting cell shape, which stimulate collagenase secretion
by passaged or mixed cell cultures do not stimulate these
primary cultured corneal stromal cells. However, stimulation by a biologically active cytokine requires the
presence of another factor, such as cytochalasin B, which,
among its many diverse functions (Tanenbaum, 1978;
Yahara et al. 1982), affects cell shape via alterations in
the actin cytoskeleton (Cooper, 1987).
We report the results of a study of the relationship
between collagenase-stimulating cytokines, cell shape
changes induced by different agents, and their ability to
stimulate homogeneous (corneal stromal), and heterogeneous (dermal) cell populations, both in primary
culture and after passaging. The differences observed
serve to underscore the central role of cytokines in
controlling collagenase secretion, the permissive role of
cell shape changes and the complexities that can arise
when mixed populations or passaged cell targets are used.
Materials and methods
Materials
Crystalline trypsin was obtained from Cooper Biochemicals.
PolyHEMA (Hydron polymer, cell culture grade) was obtained
from Interferon Sciences, Inc. Phorbol myristate acetate
(PMA), endotoxin (Escherichia coli lipopolysaccharide 026: B6
LPS), polymyxin B sulphate, monensin and latex beads (1 fim
diam.) were obtained from Sigma. Cytochalasin B (CB) was
obtained from Aldrich. Recombinant human interleukin-1/3
(rIL-1/5) was a gift from Dr Charles Dinarello. Lactalbumin
hydrolysate was obtained from Gibco, both as a 10 % solution in
distilled water and as a powder (extra soluble, tissue culture
grade). Giemsa stain was obtained from Fisher Scientific Co.
Polyclonal sheep anti-rabbit type I collagenase was a gift from
Dr C. Brinckerhoff. Horseradish peroxidase-labelled rabbit
anti-sheep IgG and rhodamine-labelled rabbit anti-sheep IgG
were obtained from Cappel. Fluorescein-labelled phalloidin was
obtained from Molecular Bioprobes. The QCL-1000 Chromogenic Limulus Amebocyte Lysate Kit was obtained from
Whittaker Bioproducts.
474
/. Kilter et al.
Cell culture
Primary nonnal rabbit conieal stromal (PoNRCS) cells. Corneas were removed from young (5 lb) New Zealand White
rabbits and processed as described (Johnson-Muller & Gross,
1978). Briefly, they were incubated overnight in cold 0'25%
trypsin, the endothelium was stripped away with forceps and
the epithelial cell layer was removed cleanly by gentle scraping
with a scalpel blade. The remaining corneal stromata were
incubated for 4h in bacterial collagenase at 37 °C to obtain a
single cell suspension. These cells were washed three times in
Dulbecco's modified Eagle's medium (DMEM) containing
penicillin and streptomycin and supplemented with 5 % foetal
bovine serum (FBS), and plated in 96-well plates at a confluent
density (6xl0 4 cells per well) or in 100cm2 dishes for future
passaging.
Primary rabbit skin fibmblasts (PoRSKF). Squares of relatively hairless skin (4 cm X 2-5 cm) were cut from the inner
surface of the ears of New Zealand White rabbits (JohnsonWint & Gross, 1984). These skin pieces were incubated
overnight in cold 0-25 % trypsin and the epithelial layer was
peeled off with forceps and discarded. The dermal patches were
cut into strips and digested with bacterial collagenase to yield a
single cell preparation. These cells were washed three times in
DMEM/5 % FBS with added penicillin and streptomycin, and
plated in 96-well plates at a confluent density (6xl0 4 cells
well" 1 ) or in 75 cm flasks for future passaging.
Passaged cells (PnRSKF, PnNRCS). Primary cultures of
corneal stromal cells and skin fibroblasts as prepared above were
subcultured by passaging with trypsin (split 1 to 3) and
propagated in DMEM/5 % FBS. At different passages, the cells
were seeded in 96-well plates at a confluent density (6x 104 cells
well" ) for use.
V2 tumour cells. A stable cell line derived from the rabbit
VX2 squamous cell carcinoma by Easty & Easty (1970) was
obtained as a gift from Dr Peter Strauli. The cells were grown in
DMEM/10% FBS in 10% CO 2 and passaged once a week
using 0-025% EDTAin Ca 2+ ,Mg 2+ -free PBS, split 1 in 5. The
cells remained stable in terms of morphology, tumorigenicity
and secretion of a cytokine with ability to stimulate collagenase
production by fibroblasts (data to be published). The cells were
free of mycoplasma contamination.
V2 tumour cytokine preparation
V2 cells were seeded in plastic 175 cm2 tissue-culture flasks
(10 7 cellsflask"') in DMEM/10% FBS. Two days later the
medium was removed, the cell layer was washed with Hank's
buffered salt solution (HBSS), and 40 ml flask"1 of serum-free
DMEM were added. After 4 days the conditioned medium was
harvested, spun, and concentrated by ammonium sulphate
precipitation (80%). The concentrated conditioned medium
was stored at — 20°C until used. No collagenase was detectable
in this conditioned medium by enzyme assay or Western
blotting. Samples of the concentrated conditioned medium
were fractionated by DEAE-Sepharose chromatography. A
preparation from the peak of stimulatory activity (/V/r17 000;
p i 4 0 ) was used as the source of epithelial cytokine in the
experiments reported.
Collagenase assay
The radioactive collagen fibril film method has been described
in detail (Johnson-Wint, 19806). Medium to be assayed for
collagenase activity was activated with 0-01 % L-(tosylamido 2phenyl) ethyl chloromethylketone (TPCK)-trypsin for 7 min at
37°C. After incubation trypsin was inactivated by the addition
of a fivefold excess of soybean trypsin inhibitor (SBTI).
Activated samples (0-2 ml well" 1 ) were incubated on I4 C-
labelled collagen films in 96-well plates at 37°C. Collagenase
activity in terms of [l4C]collagen fragments released into the
assay medium was calculated from the mean of the differences
between three replicates incubated for l h and three for 2h.
Activity is expressed in units ± standard error of the mean
(S.E.M.), where 1 unit is that amount of collagenase that
degrades 1 f.ig of collagen in 1 min. Buffer and trypsin
(lOjUgml"' final concentration) controls were always included
to ensure that the collagen substrate was in its native fibrillar
form.
Latex beads (1/im diameter, 10% aqueous suspension) were
sterilized in 70% ethanol for several hours, washed and
resuspended in DMEM/5 % FBS at a concentration of
500 ^g m l " ' . This suspension was added to confluent cells in 96well plates. Cells were exposed to the beads for 24 h. After that
time, the suspension was aspirated and the cells were washed
twice with medium and cultured for 6 days before the medium
was assayed for collagenase.
Stimulator assay
SDS-gel electrophoresis
Samples of conditioned medium or preparations to be tested for
stimulator activity were dialysed against DMEM-salts solution
and then supplemented to full DMEM by the addition of
vitamins, amino acids, pyruvate and glucose as described
(Johnson-Wint, 1980a). Foetal bovine serum (5%) and cytochalasin B (CB) in ethanol carrier at a final concentration of
5/Ugml""1 were added prior to filter-sterilization. Sterile preparations of stimulatory factors or of recombinant interleukin iji
(rIL-1/3) were added to sterile, medium without filtration.
Normal primary rabbit corneal stromal cells were prepared as
described above in 96-well plates in DMEM/5 % foetal bovine
serum and incubated at 37°C for 3 or 4 days, after which the
medium was gently aspirated and the test samples added
(0'2ml well" 1 ). Each sample was run in six replicates. After 6
more days at 37°C in an atmosphere of 4% CO2 in air, the
medium was aspirated and assayed for collagenase as described
above.
DNA assay
This method has been described (Johnson-Wint & Hollis, 1982)
and was used here to determine the cell numbers in monolayers
after aspiration of the medium for collagenase assay. In brief, a
measured volume of 8% 3,5-diaminobenzoic acid (DABA) was
added to sample wells in triplicate and to standard wells
containing known numbers of diploid rabbit corneal stromal
cells. The reaction was taken to completion by heating the assay
plates at 60°C in a water bath for 45 min. The reaction products
were stabilized by the addition of 6M-HC1. Fluorescence
(excitation at 420 nm, emission at 510 nm) from the aspirated
solutions was read in a Turner model 430 spectrofluorometer,
and cell number was calculated from the standard curve.
Trypsin treatment of cells
Treatment of cells with latex beads
Gels were run by the method of Laemmli (1970). Unconcentrated culture medium was mixed 3: 1 with four times sample
buffer and lOfil were loaded, without boiling or addition of a
reducing agent, into wells of a 3 % stacking gel on top of a 10 %
running gel on a Mini Protean II Dual slab gel apparatus (BioRad). Samples were run for 40min at room temperature at
200 V. Type I collagenase was visualized on Western blots using
a polyclonal sheep anti-rabbit type I collagenase antibody
(overnight incubation), followed by addition of a horseradish
peroxidase-conjugated rabbit anti-sheep IgG for 1 h. The
substrate was developed for 10 min.
Assay for TIMP (tissue inhibitor of metalloproteinases)
activity
A partially purified preparation of type I collagenase was
activated with TPCK-trypsin as described above and samples
were added immediately to samples to be assayed for TIMP
(1: 1 mix, v/v). More TPCK-trypsin was added to activate any
latent collagenase in the samples and inactivate any serumderived inhibitors. This was followed by addition of SBTI in
fivefold excess and the collagenase was assayed as described
above. TIMP was considered present if there was a reduction in
the measurable collagenase activity.
Giemsa staining of cell monolayer
Medium was aspirated from culture wells and the cell monolayers were fixed with methanol for 3 min. After drying they
were incubated in Giemsa stain (1/25 dilution in buffered
water) for 25 min, rinsed in buffered water and allowed to air
dry prior to being photographed.
Endotoxin assay
A modification of the method of Werb & Aggeler (1978) was
used in which cells were exposed to trypsin for up to 24 h and
their subsequent secretion of collagenase over the next few days
was measured. Cells were plated in 96-well dishes at
6xlO 4 cells well" 1 in DMEM/5 % FBS. After 3 days, the
medium was aspirated and the cell layer washed four times with
serum-free medium. Trypsin at a range of concentrations in
serum-free medium was added (0-1 ml well" 1 ). After 24 h,
0-1 ml well" 1 of DMEM/10% FBS was added, bringing the
concentration of FBS to 5 %, and the plates were incubated for
5 more days at 37°C prior to harvesting the medium and
assaying the collagenase secreted.
The chromogenic endotoxin assay was used as described in the
instructions contained in the QCL-1000 assay kit. All materials
were pyrogen-free. Briefly, samples were adjusted to
pH 70-8-0 with NaOH or HC1, and endotoxin standards were
prepared in water. A 50 jt*l portion of sample, standard or blank
was dispensed into polystyrene tubes in a heating block at 37°C.
A 50 /il sample of Limulus amebocyte lysate was added to each
tube, followed 10 min later by IOOJJI of chromogenic substrate
solution. After a further 3 min, 100 j.il of 25% acetic acid were
added and the A40S was read on a spectrophotometer against a
water blank. Endotoxin concentration was calculated by linear
regression using endotoxin standards.
PolyHEMA coating of plates
Immunqfluorescent staining of cells for collagenase and
actin
A stock solution containing polyHEMA, 12% (w/v) in 95%
ethanol, was prepared with overnight rocking at 37°C. Serial
dilutions of the stock were prepared in 95 % ethanol. Samples of
each dilution were pipetted into 96-well plates, which were
placed flat in a tissue culture hood for 3 days until the ethanol
had completely evaporated, leaving polyHEMA films in the
wells. The wells were exposed to ultraviolet (u.v.) light
overnight prior to being seeded with cells.
Cells were grown in slide chambers (Lab Tek) in DMEM/5 %
FBS under a variety of experimental conditions. The coverslips
were incubated for 3h in monensin (1/UM) fixed in 4 %
formaldehyde and permeabilized in Triton X prior to staining
(Hembry et al. 1986). The monolayers were incubated in 3 %
FBS in PBS followed by anti-collagenase antibody or control
normal sheep serum (both 1/250 dilution in PBS), washed
Collagenase secretion, cell shape and cytokines
475
extensively and then exposed to rhodamine-conjugated rabbit
anti-sheep IgG (1/250 dilution in PBS). For actin staining,
fluorescein-phalloidin (15 units m l ' ) was added to the second
antibody solution. Control and anti-collagenase antibody
treated cells were photographed using identical time exposure
on a Zeiss microscope using epifluorescence optics.
CM-cellulose absorption of collagenase
In order to remove collagenase from samples to be assayed for
stimulator, media were dialysed against lOmM-Tris-HCl,
pH7-2, containing l-OniM-CaC^, and mixed with a sample of
carboxymethyl-cellulose that had been equilibrated with the
same buffer (1 ml resin per 5 ml sample). After gentle stirring
for 30min the mixture was spun and the supernatant was
removed and dialysed against DMEM salts in preparation for
testing.
Results
Effect on collagenase secretion of agents affecting cell
shape
Cytochalasin B, a potent disrupter of actin microfilaments, and phorbol myristate acetate (PMA), an activator of protein kinase C and pleiotypic effector, produced changes in shape in all cells tested (Fig. 1). The
effect of CB was quite dramatic, producing a characteristic dendritic morphological transformation equally obvious in all cell types whether primary (P o ) or passaged
(P,,) or of corneal or skin origin. The effect of PMA was
more subtle, but nevertheless there was a dose-dependent
increase in refractility of the cells exposed to it, and the
development of a 'transformed' appearance to the cell
monolayer, with loss of the normal 'cobblestone' appearance in the case of the primary corneal stromal cells.
Despite similar shape changes in the different cell
types exposed to each of these two agents, there was
diversity in the resultant production of collagenase.
Table 1 presents the cumulative collagenase secreted by
cells continuously exposed to either CB or PMA. Passaged skin or corneal fibroblasts were markedly stimulated by either CB or PMA. Similarly, primary skin
fibroblasts, which constitutively secrete collagenase, were
strongly stimulated by either agent. In contrast, primary
corneal stromal cells were not stimulated by either PMA
or CB to secrete any detectable collagenase.
The effects of other agents that perturb cell shape were
also examined. Proteases presumably affect cell shape by
destroying macromolecules mediating cell-substratum
attachment. In serum-free medium, trypsin had similar
effects on the morphology of primary and passaged
stromal cells and skin fibroblasts. Cells began to round up
at a trypsin concentration of 0-5-l-0^gml ; at
5-20 fig ml" nearly all the cells were rounded and
aggregated. They remained viable after exposure to
trypsin for 24 h, as demonstrated by their ability to
spread out and re-form a monolayer after the addition of
FBS. The morphological changes were blocked by
adding 5 % FBS during exposure to trypsin. PolyHEMA
coating of tissue culture dishes apparently interferes with
the ability of cells to form attachments to the plastic, thus
inhibiting them from spreading (Folkman & Moscona,
1978). We observed a transition from flattened to
rounded cell morphology with polyHEMA films of approximately 500;itgcm- . There was no obvious difference in the morphological response of the various cell
types.
Whereas P 0 RSKF, P,,RSKF and P,,NRCS cells were
stimulated by both trypsin and polyHEMA in a dosedependent fashion, P Q N R C S cells were not stimulated to
secrete collagenase by either of these agents. Table 1
summarizes the data on collagenase secretion by the
primary and passaged cell types in response to the various
Table 1. Effect of various agents affecting cell shape on collagenase secretion by primaiy and passaged conieal
and skin fibroblasts
Collagenase (units per 106 cells ±S.E.M.)
Baseline, unstimulated
PoNRCS
P,,NRCS
PQRSKF
P,,RSKF
0-02 ±0-03
0-00 ±0-03
6-16±0-32
0-10 ±0-05
0-03 ±0-02*
5-47 ±0-23*
30-70 ±1-08*
13-72 ±0-82*
0-00 + 0-02*
25-45 ± 1-10*
26-70 ±0-77*
8-07 ±0-77*
0-00 ±0-02*
0-00 ±0-04*
ND
0-28 ±0-02*
9-93 ±0-87*
0-06 ±0-06
14-55 ± 1-28*
0-19 ±0-03
0-10 ±0-06
0-08 ±0-01*
0-20 ±0-06*
002 ±0-01t
0-30 ±0-00
2-01 ±0-03
3-08 ±0-10
6-50±0-25t
CB
(5 fig ml"')
PMA
(20ngmr')
Trypsin
(lfigmr 1 )
(5 fig ml-')
(5 figmP' + FBS)
(20 fig mP')
(20 fig ml"' + FBS)
PolyHEMA
(313 fig cm"2)
(1250 fig cm-2)
(5000 fig cm"2)
Latex beads
* Cell shape change.
•f Endocytosis of beads.
| N D , not done.
§ Trypsin was toxic to PQRSKF cells.
476
/. Kuter et al.
ND
0-00 ±0-09*
NDf§
ND§
ND§
ND§
ND§
2-06 ±0-28*
11-34 ±0-44*
0-00 ±0-08
13-92 ±0-56*
0-02 ±0-04
7-S6± 0-12
9-10 ±0-35*
7-21 ±0-31*
0-08 ±0-01
1-15 ±0-20*
6-81 ±0-17*
ND
ND
Control
PMA
mmwm*^ ^^m* *-*W%W&
>;.\*Ji.V/':/^i^vi*,-Av-.i ^ . * ^ y / ^ ^ k w ^i? r i^-«=s«^i\.^
Fig. 1. Effects of CB and PMA on morphology. Primary and passaged cells in 96-well plates were treated with CB (5 ;Ugml )
or PMA (20ngml~ ). Cells were incubated for 6 days before the medium was harvested and assayed for collagenase (results in
Table 1). The cell monolayer was photographed under phase-contrast microscopy to demonstrate the morphological response to
these agents. A-C. P 0 NRCS; D - F , P n N R C S ; G - I , P 0 RSKF; J - L , P 9 RSKF. A,D,G,J, control; B,E,H,K, +CB;
C,F,I,L, +PMA. Bar, 50 fim.
agents affecting cell shape. Despite a similar morphological response, the primary corneal stromal cells lack the
collagenase response elicited from the other cell types
exposed to these agents. DNA assays showed no evidence
for significant cell loss in the P Q N R C S cultures exposed
to any of these agents, with the exception of some
detachment from the dish at the highest polyHEMA
concentrations.
Endocytosis of latex beads has been reported to stimulate collagenase secretion from synovial fibroblasts (Werb
Collagenase secretion, cell shape and cytokines
477
8
Mrx10~3
92-5—
66-2•53-0
45-0 —
21-5Fig. 2. Effects of CB and PMA on collagenase secretion. Primary and passaged cells in 96-well plates were treated with CB or
PMA at a range of concentrations. Cells were incubated for 6 days before the medium was harvested and analysed by SDS-gel
electrophoresis. A Western blot of the SDS-gel was developed with sheep anti-rabbit type I collagenase and horseradish
peroxidase-coupled rabbit anti-sheep IgG. Lanes 1-4, medium from PoNRCS cells; lanes 5-8, medium from P 2 6NRCS cells;
lanes 1, 5, control DMEM; lanes 2, 6, DMEM + CB ( 5 ^ g m l " ' ) ; lanes 3, 7, DMEM + PMA (lOngmP 1 ); l a n e s 4, 8,
DMEM + PMA (lOOngmP 1 ). The position of molecular weight standards is indicated. Arrow identifies collagenase band at
53 X 103. The high molecular weight band present in all lanes is due to cross-reaction of the rabbit anti-sheep IgG with a
component of FBS.
& Reynolds, 1974). Although changes in cell shape have
not been reported for phagocytotic stimuli, it has been
suggested that the perturbation of membrane activity and
cytoskeletal reorganization that accompany endocytosis
might be responsible for inducing collagenase secretion
(Aggeler et al. 1984). We exposed primary and passaged
corneal stromal cells to latex beads. Both cell types
extensively phagocytosed the beads, yet collagenase secretion was detected only in the medium from the
passaged cells (6-5 units per 10 cells); none was secreted
by the primary cells (0-02 unit per 106 cells).
Lack of response of primary corneal stromal cells is not
due to the presence of TIMP
Because the measurement of total collagenase (active plus
latent) secreted by cells in the experiments described
above depends on a functional assay, there was a concern
that the PoNRCS cells might actually be secreting the
enzyme, but its presence was not being detected because
of endogenous tissue inhibitor(s) of metalloproteinases
(TIMP) (Reynolds, 1986), which, in theory, might be
binding to the enzyme upon activation by trypsin (Herron et al. 1986). To investigate this possibility, media
from P 0 NRCS cells (control and CB-stimulated) were
screened for the ability to inhibit the enzymic activity of
type I collagenase prepared from passaged corneal
stromal cells stimulated by CB and activated by trypsin
478
/. Kuter et al.
(see Materials and methods). No significant inhibition in
any of these culture media was found (data not shown).
Western blot analysis confirmed the presence of imraunoreactive type I collagenase in the media from stimulated
passaged cells and its absence in the media of PoNRCS
cells exposed to CB or PMA (Fig. 2).
Further evidence that the primary corneal stromal cells
exposed to CB are not making collagenase that is masked
by TIMP was provided by indirect immunofluorescence
staining for collagenase. PoNRCS cells exposed to CB
did not stain with anti-collagenase antibody, whereas
passaged NRCS cells stain brightly under the same
conditions (data not shown).
Primary corneal stromal cells will respond to shape
changes if exogenous cytokine or an endogenous source
of cytokine is present in the culture
Since the above experiments suggested that cell shape
change alone is not enough to initiate collagenase secretion by PoNRCS cells, and since we have reported
(Johnson-Wint, 1980a) the observation that CB appears
to permit PoNRCS cells to respond to stimulatory
epithelial cytokines, we asked if shape changes and
cytokine stimulation were both required for collagenase
secretion to occur in the primary corneal cells. A partially
purified cytokine from a V2 rabbit carcinoma cell line
(Kuter, Johnson-Wint & Gross, unpublished) was used
-o- — V2 cytokine
••- +V2 cytokine
2000
4000
6000
PolyHEMA (/(gem" 2 )
8000
12
V2 (cell number per well x 10
H 10-
Fig. 4. Collagenase secretion by PoNRCS cells cocultured
with cytokine-producing cells. PoNRCS cells were seeded
into 96-well plates (6xlO + cells well" 1 ) together with V2 cells
at a range of added cell density; 3 days after plating, the
medium was changed to regular DMEM ± CB and the plates
were incubated for a further 6 days before the medium was
harvested for collagenase assay. Error bars show the S.E.M.
—V2 cytokine
+ V2 cytokine
64-
10
u
J
12
-o- —V2 cytokine
•*• +V2 cytokine
2
" 4
D.
H 3-
40
' 60
-80
PMAfngmP 1 )
100
120
Fig. 3. Effects of changes in cell shape on collagenase
secretion by PoNRCS cells in the presence of exogenous
cytokine. PoNRCS cells were plated either in regular 96-well
plates (B,C) or plates coated with polyHEMA at a range of
concentrations (A). After 3 days the medium was changed to
control DMEM or DM EM containing active V2 cytokine,
with or without added CB (B) or PMA (C) at a range of
concentrations. After 6 days of incubation the medium was
harvested and assayed for collagenase. Error bars show the
S.E.M.
to stimulate PoNRCS cells in the presence of increasing
concentrations of polyHEMA, CB or PMA (Fig. 3). In
the presence of the V2 cytokine, polyHEMA (Fig. 3A)
and CB (Fig. 3B) each stimulated the P 0 NRCS cells to
secrete collagenase. A dose-response relationship between the amounts of these agents and the quantity of
collagenase secreted was apparent. In contrast, PMA
(Fig. 3C) showed limited ability to stimulate the
PoNRCS cells in the presence of the V2 cytokine. In all
three cases no stimulation occurred in the absence of
cytokine.
When P 0 NRCS cells were cocultured with cells that
produce cytokines capable of stimulating collagenase
secretion, the degree of collagenase secretion was
influenced by the type of cell supplying the cytokine, by
the density of this cell type in the coculture, and by the
presence or absence of CB. In the case of V2
cell/P 0 NRCS cell cocultures (Fig. 4), minimal collagenase secretion was noted in the absence of CB, and in its
presence marked stimulation was seen but was limited to
cultures containing between 1X104 and 3xlO 4 V2cells
well" . We have demonstrated (unpublished data) that
this is the cell density at which the V2 cells are secreting
maximal amounts of an active cytokine. Secretion of the
V2 cytokine is not affected by CB. Similar stimulation of
collagenase secretion was seen in stromal cells cocultured
with macrophages (data not shown), although in this case
(submaximal) stimulation was seen even without the
addition of CB.
PMA can mimic cytokine action
The observation that the V2 tumour cytokine (unpublished data) and normal epithelial cytokines (JohnsonWint, 1980a; Johnson-Wint & Gross, 1984) need the
presence of CB to stimulate P 0 NRCS cells to secrete
collagenase prompted us to ask whether PMA, ineffective
by itself, could also stimulate P 0 NRCS cells in the
presence of CB. Fig. 5 compares the concentrationdependent stimulation of P 0 NRCS cells by the V2
cytokine (Fig. 5A), rIL-1/3 (Fig. SB) and PMA
(Fig. 5C): in each case there is no stimulation of collagenase secretion in the absence of CB but a clear dose-response curve in its presence. In essence, then PMA
mimics the effects of the V2 cytokine and rIL-1/? on the
Collagenase secretion, cell shape and cytokines
479
-O--CB
•*- -CB + PMX
• +CB
— +CB + PMX
64 32 16 8
4
2
V2 cytokine (reciprocal of dilution)
100
150
200
250
Endotoxin (/igmP1)
•o- LH alone
— LH + PMX
• LH + CB
— LH + CB + PMX
0-2
0-4
0-6
0-8
Lactalbumin hydrolysate (%)
Fig. 6. Stimulation of collagenase secretion from primary
corneal stromal cells by endotoxin and lactalbumin
hydrolysate. PoNRCS cells were plated in 96-well plates as
described, and 3 days after plating endotoxin or lactalbumin
hydrolysate (LH) at a range of concentrations in DMEM/5 %
FBS was added in the presence or absence of CB and/or
polymyxin B (PMX). After 6 days of incubation, the medium
was harvested and assayed for collagenase. Cell monolayers
were fixed and Giemsa stained to demonstrate morphology
(see Fig. 7). A. Collagenase secretion in response to
endotoxin. B. Collagenase secretion in response to
lactalbumin hydrolysate. Error bars show the S.E.M.
PMA (ngmP 1 )
Fig. S. Effect of cytokines and PMA on collagenase secretion
by primary corneal stromal cells in the presence and absence
of CB. P 0 NRCS cells were plated in 96-well plates as before.
Three days after plating, V2 cytokine (A), rIL-l/J (B) or
PMA (C) at a range of concentrations was added, in the
presence or absence of CB. The V2 cytokine preparation was
initially diluted l/lOO, then further diluted as shown. After 6
more days of incubation, the medium was harvested and
assayed for collagenase. Error bars show the S.E.M.
primary corneal stromal cells in that it is stimulatory only
in the presence of a cofactor such as CB.
Collagenase secretion can occur in the absence of shape
changes
The effect of bacterial lipopolysaccharide (endotoxin)
was tested, since this agent has been reported to stimulate
480
/. Kuter et al.
collagenase production by macrophages (Wahl et al.
1974). Fig. 6A shows the results of exposing P 0 NRCS
cells to endotoxin at a range of concentrations. Endotoxin
at 30/xgmP 1 triggered considerable collagenase secretion. When CB was present a similar stimulation was
observed at 3 /igml" 1 and the peak response was greater.
Polymyxin B, commonly used to inhibit other biological
effects of endotoxin (Jacobs & Morrison, 1977), was
noted to inhibit the endotoxin effect on collagenase
secretion except at the highest endotoxin concentrations
on the dose-response curve. The inability of ZO^gmP 1
of polymyxin to inhibit the higher concentrations of
endotoxin is in keeping with a stoichiometric endotoxin/
polymyxin interaction (Jacobs & Morrison, 1977). The
polymyxin alone was not stimulatory. The P Q N R C S cells
secreting collagenase in response to endotoxin in the
absence of CB underwent no detectable change in morphology compared with the control, unexposed cultures
Endotoxin
LH
• #
J >v'
%
V
o
o
although they did develop vacuoles (Fig. 7A-D). In the
presence of endotoxin and CB they underwent the
characteristic changes effected by CB alone.
A second example of collagenase secretion by PgNRCS
Fig. 7. Morphological effect of
endotoxin and lactalbumin
hydrolysate on P0NRCS cells.
Cells were treated with endotoxin
or LH and then Giemsa stained as
described in the legend to Fig. 6.
A-D. Endotoxin at 0 (A), 1 (B),
10 (C) and 100 (D) ^gml~';
E-H, LH at 0-01 (E), 0-1 (F), 0-5
(G)and 1-0 (H)%.
cells in the absence of changes in cell shape was observed
when, in an attempt to maximize the viability of the cells
when grown for several days in serum-free medium,
lactalbumin hydrolysate (LH) was added to the cultures
Collagenase secretion, cell shape and cytokines
481
in lieu of serum (Werb & Aggeler, 1978). Because this
change of conditions altered the outcome of some experiments, the effect of LH alone on collagenase secretion
was investigated. Whether or not 5 % serum was present
in the cultures, LH was able to stimulate collagenase
secretion by PoNRCS cells in the absence of CB
(Fig. 6B). There was considerable variation from experiment to experiment in the amount of LH required to
stimulate the cells, but in at least some experiments the
threshold for the effect was about 0-2%, which is the
concentration commonly used as a serum substitute.
Similar effects were observed with both the LH purchased as a 10% solution and with the tissue-culture
grade powder product, although the latter gave a lesspronounced stimulation. The addition of CB increased
the sensitivity of the PoNRCS cells to stimulation by LH.
Passaged cells were even more strongly stimulated by LH
in the absence of CB (data not shown). As with the
endotoxin, the stimulation of collagenase secretion by
LH in the absence of CB was not accompanied by
perceptible alterations in cell morphology (Fig. 7E-H).
Polymyxin B was observed to inhibit the LH effect
most effectively at low concentrations of LH and the
inhibition was seen in both the presence and absence of
CB. An attempt to quantify endotoxin levels in the
lactalbumin hydrolysate using the chromogenic assay
revealed a level of several ngml" in the 0 - 2% LH
preparation; however, there was also evidence for inhibition of endotoxin activity in the chromogenic assay
(non-quantitative recovery of activity of endotoxin standard added to samples), so that its reliability in quantifying the true endotoxin concentration in this setting is
questionable.
Collagenase-secreting cells produce a biologically active
cytokine
The ability of the passaged cells to respond to CB and
polyHEMA in contrast with P 0 NRCS cells, which require a source of biologically active cytokine for collagenase secretion, raised the possibility that the passaged cells
were producing their own cytokine, i.e. an autocrine
factor. We had already observed that the EVX2-135 cell
line, a fibroblastoid type isolated from the V2 carcinoma
(provided by Dr E. Voelkel), secreted such a factor
constitutively, and that this cell line responded to CB by
secreting collagenase (Kuter et al. 1984). Johnson-Wint
(1987) has reported that passaged corneal stromal cells
secrete an autocrine factor that stimulates collagenase
production. We determined that a stimulatory cytokine
was secreted by passaged stromal cells that were actively
producing collagenase in response to other agents. Media
conditioned by passaged corneal stromal cells
(P26NRCS) in the presence or absence of CB, or by cells
exposed to latex beads, was adsorbed with CM-cellulose
to remove any collagenase present. The enzyme-depleted
medium was tested on PoNRCS cells for its ability to
stimulate collagenase secretion (Table 2). Collagenasedepleted medium from the passaged NRCS cells exposed
to CB or to latex particles induced collagenase secretion
by the primary NRCS cells in the presence of CB. It is
unlikely that the activity was due to endotoxin introduced
482
/. Kuter et al.
Table 2. Stimulation of collagenase secretion by
PoNRCS cells by conditioned medium from passaged,
collagenase-secreting ATRCS cells
Collagenase
(units ml" 1 in P26NRCS
conditioned medium)
Fresh conditioned medium
Conditioned medium after
CM-cellulose absorption
Absorbed conditioned medium
after incubation with
PoNRCS cells*
Control
CB
Latex
0-03
0-02
2-65
0-19
2-44
0-01
2-11
2-85
018
*PlusCB.
Table 3. Effect of lactalbumin hydrolysate on
collagenase secretion by PoNRCS cells in response to
PMA
Collagenase
(units per 106 cells ± S.E.M.)
0-1% LH alone
PMA alone (ngmP 1 )
0
20
100
0-1% LH + PMA (ngml" 1 )
20
100
0-07 ±0-03
0-08 ±0-01
0-07 ±0-01
0-08 ±0-02
2-98 ± 1-33
3-54 ±0-55
from processing with the resin, since stimulation was not
observed by the conditioned medium from the passaged
cells not exposed to CB or latex particles.
Responsiveness of collagenase-secreting cells to
stimulators is influenced by the presence of other active
agents
Since both endotoxin, a ubiquitous contaminant, and
LH, which is commonly used to supplement cell culture
media, were able to stimulate collagenase secretion in the
absence of alterations in morphology, we asked whether
these agents might, at levels below the threshold of their
activity, affect the responses of target cells to stimulation
by other agents. Table 3 shows that this is indeed the
case. Although neither 0-1% LH alone nor PMA alone
(20-200ngml" 1 ) were stimulatory on the P 0 NRCS cell
target, the combination of the two agents at these
concentrations was effective.
Discussion
Alterations in cytoskeletal architecture, reflected in cell
shape changes, are well known to accompany changes in
gene expression in such diverse cell types as mammary
epithelium (Emerman et al. 1977), chondrocytes (Zanetti
& Solursh, 1984), adipocyte precursors (Spiegelman &
Ginty, 1983) and cells from synovial tissues (Aggeler et
al. 1984; Werb et al. 1986). The recent report (Unemori
& Werb, 1986) that disruption of the actin cytoskeleton is
correlated with stimulation of procollagenase and stromelysin secretion by rabbit synovial fibroblasts has led to
speculation that cell rounding, more particularly perturbation of the actin microfilaments, might be linked to the
expression of genes involved in the initiation of extracellular matrix degradation. We have confirmed that
some agents that alter cell shape can also modulate the
production of collagenase by passaged rabbit corneal or
skin fibroblasts and primary skin fibroblasts in vitro. Our
findings, especially the polyHEMA experiments, suggest
that changes in cell shape may influence the response of
the cells to other biological stimulators of collagenase
secretion.
We have demonstrated that there is not a consistent
cause and effect relationship between alterations in the
shape of cells that are capable of collagenase synthesis and
their production of the enzyme. Treatment of primary
corneal stromal cells with trypsin, polyHEMA, PMA,
CB or latex beads alone results in the same morphological
changes that they induce in passaged cells, yet the former
do not secrete collagenase in response to these agents,
while the latter do. We have ruled out the possibility that
the response of these primary corneal stromal cells may
be masked by the presence of tissue inhibitors of metalloproteases (TIMP), and the correlation between detection
of immunoreactive collagenase in the cells and the
enzymically active species in the medium confirms that
we are not simply dealing with a block in secretion of the
enzyme by PoNRCS cells.
We have also observed the reverse situation: collagenase secretion can be stimulated without a concomitant
change in cell shape. With either primary or passaged
corneal stromal cells as targets, this occurs on exposure to
certain levels of endotoxin or lactalbumin hydrolysate.
Whether the LH effect is due to endotoxin is not clear;
we are attempting to quantify more accurately the
amount of endotoxin in the LH preparations in order to
answer this question. At the light-microscopic level, not
only do the collagenase-secreting cells appear flat and well
spread, but their actin cytoskeleton does not appear to be
obviously altered when the cells are stained with fluorescein-phalloidin.
Furthermore, in those instances where cell shape
changes were concomitant with enzyme secretion, the
correlation was less than perfect. Stimulation of primary
skin fibroblasts (constitutively 'turned on') by polyHEMA could be detected at concentrations at which
there were no observable morphological changes, and at
higher concentrations where cell shape change was most
affected, collagenase secretion declined.
When shape change and collagenase secretion occurred
together in these studies, a biologically active cytokine
was also present. The source of the cytokine was either
exogenous (as when V2 conditioned medium was added
to the P 0 NRCS cells on polyHEMA), a paracrine factor
(from cocultured tumour, epithelial or mononuclear
cells) or an autocrine factor (from the cells that secreted
the collagenase). Thus, it is not apparent that a causal
relationship exists between cell shape change and the
induction of collagenase secretion. Others (Baker et al.
1983; Dayer et al. 1984) have shown that shape changes
in synovial tissue cells can be dissociated from collagenase
secretion stimulated by mononuclear cell factor (IL-1).
We propose that biologically active cytokines, which
may be derived from epithelial cells, mononuclear cells,
tumour cells (paracrine) or fibroblasts (autocrine), are
the key regulatory factors in collagenase secretion. Shape
changes may affect the response of the target cells to
cytokines, and in the case of passaged cells may also
stimulate secretion of an autocrine factor. Cell shape
change may be mimicking the effect of a naturally
occurring cofactor of cytokine action. Shape change is not
observed in primary stromal cells stimulated by macrophage-conditioned medium, or by cocultured macrophages (Kuter & Gross, unpublished) or normal rabbit
skin epithelial cells in the absence of CB (Johnson-Wint,
1980a). This is consistent with the possibility that a
cofactor for the epithelial or macrophage cytokines,
analogous to CB, may be produced by these cell types.
A number of models explaining the cofactor requirement could accommodate the possiblity that this function
may be mediated by changes in the cytoskeleton. Endotoxin appears to replace the requirement for cytokine and
at higher doses both the cytokine and the cell shape
change. This probably reflects the ability of endotoxin to
mimic multiple biological effectors (Bradley, 1979; Morrison & Rudbach, 1981); in this case the cytokine and its
cofactor. There are several instances where endotoxin has
been reported to initiate the secretion of biologically
active mediators from a variety of cells (Colucci et al.
1985; Bradley, 1979; Libby et al. 1986), and such a
mechanism may be involved in its action on the PoNRCS
cells, stimulating release of an autocrine stimulatory
factor.
It is likely that collagenase secretion is not the only
differentiated function that depends on the action of both
a cytokine and a second factor that can be simulated by a
cytochalasin-induced mechanism. It was recently
reported (Brown & Benya, 1988) that dihydrocytochalasin B (DHCB)-mediated disruption of actin microfilaments correlated with resumption of synthesis of type II
collagen by cultured chondrocytes that had lost their
chondrocyte-specific function. However, the DHCB effect alone was not sufficient for stimulation of type II
collagen synthesis: 10% FBS, which presumably contains an active factor, was also necessary. Furthermore, it
is known that cartilage-inducing factor A (Zanetti &
Solursh, 1984; Rosen et al. 1986), which has recently
been shown to be TGF/3 (Seyedin et al. 1986), commits
mesenchymal cells to chondrogenesis, but the differentiated phenotype is not expressed without the action of
DHCB. It has also been noted that multiple factors acting
together may bypass the need for the cell shape change in
the induction of chondrogenesis (Koskinen et al. 1985);
this would support the notion that in such situations the
shape change is substituting for the biological effect of a
cofactor.
Primary cultures of cells such as dermal or synovial
fibroblasts that consist of mixed cell populations can give
misleading information on regulated processes because
Collagenase secretion, cell shape and cytokines
483
these cells may not interact in vivo in unrestricted
fashion. For example, epithelial cells derived from hair
follicles may secrete stimulators of fibroblast secretion of
collagenase that are effective in vitro, but in vivo may be
prevented from interacting with their potential target,
perhaps by the barrier function of basement membrane
or by secretion of an inhibitor of enzyme production
(Johnson-Wint, 1980a). In addition, agents that appear
to be stimulatory may be acting indirectly on a noncollagenase-producing cell, causing release of stimulatory
cytokines, which then secondarily activate the collagenase-producing cells. Lastly, the interpretation of results
obtained in vitro is subject to the criticism that certain
culture additives (advertent or inadvertent) may themselves influence the response of the target cells to the
regulators of interest. This may well be the case for
lactalbumin hydrolysate used as a serum substitute, and
endotoxin present in some serum batches. This effect,
shown here, is apparent when subthreshold levels of each
of two agents added together result in a response.
Finally, the data presented here re-emphasize a caveat
often heard (Bissel, 1981) but seldom heeded: passaging
of cells in culture results in disruption of normal regulatory processes. In this case, passaging corneal stromal
cells results in their (inducible) secretion of an autocrine
factor, which allows them to be stimulated to secrete
collagenase by any agent that mimics the cofactor. We
have evidence that at early passages of the corneal stromal
cells CB is required for the secretion of the autocrine
factor, whereas at later passages this cytokine is constitutively secreted (Johnson-Wint, unpublished data). Primary corneal stromal cells, on the other hand, depend on
the presence of an exogenous cytokine for stimulation,
and this translates into a paracrine model for control of
collagenase secretion, with regulation lying, at least in
part, with a heterologous cell type. We predict that the
primary cell cultures more accurately reflect the regulatory mechanisms in operation in vivo, and have evidence
(Wagoner, Gross & Johnson-Wint, unpublished) that
collagenase secretion by stromal cells in intact corneas is
regulated in a similar fashion to that observed in primary
corneal stromal cells in culture.
This is publication no. 1047 of the Robert W. Lovett
Memorial Group for the Study of Diseases causing Deformity.
The work was supported by National Institutes of Health grants
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(Received 29 August J 988 -Accepted, in revised form,
2 December 1988)
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