1 Dipartimento di Cardiologia,
Angiologia e Pneumologia, U. O. di Pneumologia e Fisiopatologia
Respiratoria, Università degli Studi di Pisa, 56214 Pisa, Italy;
2 Third Department of Internal
Medicine, Cigarette smoking, the major cause of pulmonary
emphysema, is characterized by destruction of alveolar walls. Because
tissue destruction represents a balance between injury and repair, we hypothesized that cigarette smoke exposure may contribute to the development of emphysema through the inhibition of tissue contraction during the repair process. To partially evaluate this hypothesis, we
investigated the effects of cigarette smoke extract (CSE) on the
ability of cultured fibroblasts to mediate collagen gel contraction in
vitro: CSE inhibited fibroblast-mediated gel contraction in a
concentration-dependent manner (P < 0.01). Production of prostaglandin E2, a known inhibitor
of fibroblast contraction, was unchanged by CSE as was cell surface
integrin expression. In contrast, fibronectin production by fibroblasts
was inhibited (P < 0.01), and
addition of exogenous fibronectin partially restored the contractile
activity, thus suggesting at least one mechanism to explain inhibition
of gel contraction by CSE. When CSE was treated to remove volatile components, it showed less inhibitory activity on fibroblast-mediated gel contraction. Therefore, we also examined the effects of acrolein and acetaldehyde, two volatile components of cigarette smoke. Inhibition of contraction was observed at 5 µM acrolein and at 0.5 mM
acetaldehyde. In conclusion, cigarette smoke inhibited fibroblast-mediated gel contraction, and this inhibition was due, at
least in part, to the volatile components of cigarette smoke and may be
mediated, at least in part, by a decrease in fibroblast fibronectin
production. By inhibition of repair, these smoke components may
contribute to the development of pulmonary emphysema.
fibronectin; three-dimensional gel
CIGARETTE SMOKING IS THE MAJOR cause of pulmonary
emphysema (5). Current concepts suggest that emphysema results from the excessive release of destructive proteases from inflammatory cells that
overcome the anti-protease defenses of the lung (20). Excessive tissue
damage, however, represents only part of the process in the development
of emphysema. In this context, the lung has considerable capacity to
mediate repair responses. Net tissue destruction, the defining
characteristic of emphysema, must therefore represent tissue damage
with inadequate repair responses.
Tissue repair after damage is a complex process. Of particular
importance appears to be the role of fibroblasts, which are recruited
to sites of injury throughout the body. These cells can proliferate at
sites of injury, produce matrix proteins, and subsequently remodel the
newly deposited matrix through a variety of processes including that of
contraction (8). Previous studies have suggested that cigarette smoke
can impair the wound healing process by inhibiting fibroblast
recruitment and proliferation (26). An effect on inhibition of
fibroblast-mediated contraction could contribute to the enlarged air
spaces that develop in the injuries associated with pulmonary
emphysema. The current study, therefore, was designed to determine
whether cigarette smoke impairs fibroblast-mediated wound contraction.
To accomplish this, a model system using fibroblasts cultured in a
three-dimensional (3-D) native collagen gel was utilized (3). In this
model, fibroblasts are cultured in a 3-D matrix consisting of native
type I collagen fibers. This matrix culture is thought to more closely
resemble in vivo conditions than a monolayer culture. Consistent with
this, fibroblasts cultured in the 3-D gel have altered phenotype and synthetic capability (3, 21). Among the properties of fibroblasts in a
3-D gel is the ability to cause gel contraction, a phenomenon that
resembles the contraction of newly formed scar tissue (3). Our results
suggest that cigarette smoke extract (CSE) can impair this process and
thus suggest another mechanism by which cigarette smoke could
contribute to the development of pulmonary emphysema.
Cell culture.
Human fetal lung fibroblasts (HFL1; lung, diploid, human) were obtained
from the American Type Culture Collection (Rockville, MD). The cells
were cultured in DMEM (GIBCO) with
10% FCS and refed three times weekly in 100-mm tissue culture dishes
(Becton Dickinson Labware, Lincoln Park, NJ). Cells were trypsinized
(Trypsin-EDTA, GIBCO; 0.05%
trypsin-0.53 mM EDTA-4Na) for use in contraction assays
when "subconfluent," i.e., ~3 × 106 cells/dish.
Collagen.
Collagen gels were prepared as described by Strom and Michalopoulos
(33). Type I collagen was extracted by stirring ethanol-washed adult
rat tail tendons for 48 h at 4°C in sterile 4 mM acetic acid. After
centrifugation (3,000 rpm for 20 min at 4°C), the supernatant was
stored. An aliquot was lyophilized to determine collagen concentration.
CSE.
CSE was prepared by a modification of the method of Carp and Janoff
(7). Briefly, two cigarettes without filters were combusted with a
modified syringe-driven apparatus. The smoke was bubbled through 50 ml
of serum-free DMEM. The resulting suspension was adjusted to pH 7.4 with concentrated NaOH and then filtered through a 0.20-µm pore
filter (Lida Manufacturing, Kenosha, WI) to remove bacteria and large
particles. CSE was applied to fibroblast cultures within 30 min of
preparation. To examine the effect of volatilization, CSE was
lyophilized and reconstituted to the initial volume with distilled
water. A separate aliquot was bubbled with a stream of
N2 gas at room temperature for 30 min.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Contraction assay. Effects of CSE on fibroblast-mediated collagen gel contraction were examined by the method of Bell et al. (3). Briefly, the fibroblasts prepared as described in Cell culture were trypsinized and resuspended in DMEM-1% FCS (Biofluids, Rockville, MD). The cells were gently mixed with a solution containing rat tail tendon collagen, 4× concentrated DMEM, FCS, and distilled water, all prepared at 4°C. Volumes were adjusted so that the final collagen concentration was 0.75 mg/ml, DMEM was 1×, FCS was 1%, and cell concentration was 105 cells/ml. Fibroblasts were added after other components had been mixed. Three milliliters of cell suspension containing 3 × 105 cells were dispensed into six-well tissue culture plates, with each well being 35 mm in diameter (Becton Dickinson Labware). The plates were then incubated for 20 min at 37°C to allow the collagen solution to gel. After this, 3 ml of DMEM with 1% FCS containing different concentrations of CSE were added on top of the gel. The gels were then gently detached from the walls and the bottom of the dishes using a sterile metal spatula to prepare a floating gel. The resulting cultures were incubated at 37°C and 5% CO2 and were refed every day with fresh medium containing CSE. Gel contraction was quantified by measuring the area of the gel after 24, 48, and 72 h using an image-analysis system (Optomax V, Burlington, MA) to quantify an image of the plate prepared on a Xerox copier and expressed as a percentage of the original area. All cultures were performed in triplicate.
Cytotoxicity. To assess the cytotoxicity of CSE for human lung fibroblasts, lactate dehydrogenase (LDH) release into gels and into supernatant was measured using a commercially available kit (LDH-20; Sigma). This method is able to detect LDH release from cells incubated with cytotoxic levels of acrolein and acetaldehyde (26).
DNA assay. To estimate cell number in 3-D gels, DNA was assayed fluorometrically with Hoechst dye no. 33258 (Sigma) by a modification of a previously published method (17). Collagen gels were solubilized with collagenase (22), and cell suspensions were collected by centrifugation at 500 g for 10 min and resuspended in 1 ml of distilled water. After freezing and thawing twice, the suspensions were mixed with 2 ml of TNE buffer (3 M NaCl, 10 mM Tris, and 1.5 mM EDTA, pH 7.4) containing 2 µg/ml of Hoechst no. 33258. Fluorescence intensities were measured with a fluorescence spectrometer (LS-5, Perkin-Elmer) with excitation at 356 nm and emission at 458 nm.
Prostaglandin production. Prostaglandin E2 (PGE2) produced by fibroblasts is known to inhibit collagen gel contraction and could function as an autocrine or paracrine mediator. Therefore, we quantified PGE2 in both supernatant media and solubilized gels (22) by RIA (9, 27) (Advantage Magnetics, Cambridge, MA).
Inhibition of cyclooxygenase. To further explore the role of cyclooxygenase and prostaglandin activity in regulating gel contraction, monolayer cell cultures were incubated with 1 µM indomethacin for 45 min before exposure to different concentrations of CSE. After pretreatment, cells were trypsinized and cast into gels that also contained 1 µM indomethacin. To determine whether indomethacin treatment could directly alter fibroblast-mediated gel retraction, control experiments were performed in which indomethacin was added to the cell suspension and mixed with other components.
Integrin expression.
Cell surface integrins, in particular
2
1-integrin,
are thought to be required for fibroblast-mediated collagen gel
contraction (16, 30). To evaluate cell surface integrin expression,
subconfluent fibroblast monolayer cultures were incubated overnight in
serum-free DMEM containing different concentrations of CSE. Cell
surface expression of integrins was evaluated using epifluorescence
flow cytometry. Briefly, cells were suspended in PBS containing 3% BSA
and incubated with control mouse ascites or mouse monoclonal anti-integrin antibodies (1:200 dilution), followed by counterstaining with FITC-conjugated anti-mouse IgG (1:200 dilution, Sigma). The stained cells were fixed in 1% paraformaldehyde, and the relative fluorescence intensity per cell was measured with fluorescence flow
cytometry (FACS II, Becton Dickinson, Sunnyvale, CA). Data are
expressed as the mean channel fluorescence intensity of 5,000 cells. To
compare fluorescence intensities, the mean channel fluorescence intensity for 5,000 cells was converted to linear fluorescence intensity units (25) so that background fluorescence with control antibodies could be subtracted (32).
Measurement of fibronectin by ELISA. For quantification of fibronectin production, the media were harvested and the gels were solubilized with collagenase as described in DNA assay. Fibronectin in the gels and the media was assayed by an ELISA that is specific for human fibronectin and does not detect bovine fibronectin (29).
Effect of plasma and cellular fibronectin. To evaluate modulation of gel contraction by fibronectin, in separate experiments, 50 µg/ml of human plasma fibronectin (GIBCO BRL, Grand Island, NY) or human cellular fibronectin (Upstate Biotechnology, Lake Placid, NY) were added to the collagen solution before gels were made. Gels were then cast and cultured, and the areas of the gels were measured as described in Contraction assay.
Statistical evaluation. The data presented are means ± SE. Experimental values were compared using a one-way ANOVA for repeated measures and an unpaired two-tailed Student's t-test for single comparisons. Comparisons were considered statistically significant at P < 0.05.
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RESULTS |
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The effect of CSE on fibroblast-mediated gel contraction. Control gels consisting of native type I collagen in which fibroblasts were embedded contracted rapidly during the first 24 h of incubation, reaching 35.8 ± 3.4% of their original area, and further decreased their area during the next 72 h (Fig. 1). CSE-exposed gels also contracted but significantly less so. After 24 h of incubation, the area of the gels incubated in media containing 4% CSE was still 57.9 ± 1.5% of the original area, and gels incubated in media containing 6% CSE were 90 ± 2.0% of the original area (P < 0.01, both comparisons with control by ANOVA). In contrast, the area of the gels incubated with 2% CSE did not differ from control. When media were changed every 24 h to media containing fresh smoke extract, the differences between control and 4 or 6% CSE persisted for up to 72 h (Fig. 1).
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Cytotoxicity of CSE. Cytotoxicity of CSE was assessed by quantification of LDH release into gels and supernatants. No release of LDH was induced at any concentration used (Fig. 3). In contrast, the supernatant of fibroblasts incubated with 1% sodium azide, used as positive control, showed a significant increase in LDH release. As a separate means of evaluating fibroblast viability, fibroblast number was estimated by DNA quantification. No significant differences were observed over 72 h after exposure to CSE (data not shown).
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PGE2. Fibroblasts are known to produce PGE2 (19), and PGE2 has been reported to inhibit fibroblast-mediated collagen gel contraction (27). Augmented PGE2 production in response to CSE could be one mechanism to explain reduced gel retraction after exposure to CSE. To evaluate this possibility, the supernatant media from collagen gel cultures were harvested, and the gels were dissolved with collagenase. When the gels were completely dissolved, the resulting solutions were centrifuged to remove cells, and the supernatants were harvested. Release of PGE2 by fibroblasts into the cultures was then quantified in both gels and supernatants. CSE induced a slight but not statistically significant increase in PGE2 release (Fig. 4; P > 0.3, ANOVA). To further explore the role of endogenously produced prostaglandins in modulating gel contraction, fibroblasts were pretreated with or without indomethacin, an inhibitor of prostaglandin synthesis. Indomethacin was able to inhibit PGE2 production by fibroblasts (data not shown) but did not block the ability of CSE to inhibit fibroblast-mediated collagen gel contraction (Fig. 5).
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2
1-Integrin
expression.
Fibroblast contraction of collagen gels is thought to be mediated by
cell surface integrins, specifically
2
1-integrin.
Inhibition of integrin expression, therefore, could be a possible
mechanism to explain CSE-mediated inhibition of fibroblast contraction
of collagen gels. All fibroblasts, however, were well stained with antibody to both
2- and
1-integrin. CSE exposure did
not alter integrin expression assessed as mean fluorescence (data not
shown).
Fibronectin. Potential mechanisms to explain CSE-mediated inhibition of fibroblast contraction of collagen gels also include inhibition of fibronectin production. CSE exposure resulted in a significant decrease in fibronectin production by fibroblasts in gel culture at all conditions used (Fig. 6; P < 0.01). Thus a decrease in fibronectin production may contribute to CSE inhibition of contraction. To further evaluate this possibility, the effect of exogenously added plasma and cellular fibronectin was also investigated. Fibroblasts were cast into gels with or without cellular or plasma fibronectin (50 µg/ml) and then exposed to 0 or 6% CSE. Both cellular fibronectin and plasma fibronectin were able to partially restore the ability of fibroblasts to mediate contraction after exposure to CSE (P < 0.01; Fig. 7).
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Inhibition of fibroblast-mediated gel contraction
and fibronectin production by volatile components of cigarette
smoke.
To help determine which components of CSE contributed to the inhibition
of fibroblast-mediated collagen gel contraction, we assessed the effect
of CSE after lyophilization and after "volatilization" to remove
volatile components. CSE lost most of the inhibitory activity for
fibroblast-mediated collagen gel contraction after removal of volatile
components (Fig. 8). We next assessed the effects of two volatile components present in high concentration in
cigarette smoke, acrolein and acetaldehyde. Both volatile components of
CSE inhibited fibroblast-mediated gel contraction. This inhibition was
observed at 106 M acrolein
and at 5 × 10
4 M
acetaldehyde (P < 0.01; Fig.
9, A and
B). Acrolein and acetaldehyde were
not cytotoxic at these concentrations, as assessed by LDH release (data
not shown), but both significantly reduced the amount of fibronectin
recovered from fibroblast-containing gels (Fig. 10).
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DISCUSSION |
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The current study demonstrates that cigarette smoke is capable of
inhibiting fibroblast-mediated collagen gel contraction. This effect
depends on the concentration of smoke extract to which collagen gels
are exposed. The effect develops over 24 h and persists for up to 72 h
with continual exposure. The effect is, however, at least partially
reversible. Removal of smoke after 24 h of exposure results in cells
resuming their contractile activity. This reversibility suggests that
the effect of smoke is not due to a cytotoxic effect, and, consistent
with this, cigarette smoke was not associated with LDH release or with
any significant change in cell number in the collagen gels as assessed
by DNA content. Several mechanisms could explain the inhibitory effect
of CSE on fibroblast-mediated collagen gel contraction (1, 10, 24). An
increase in endogenous PGE2
production does not appear to account for the majority of the
inhibitory effect, however, because cigarette smoke was not associated
with significantly augmented PGE2
production nor was indomethacin associated with a blockade of the smoke
effect. Similarly, loss of the collagen-binding
2
1-integrin
would be expected to be associated with a loss of contractile activity (16, 30), but no changes in fibroblast expression of these integrin
chains were observed when monolayer cultures were exposed to CSE.
Fibronectin has also been reported to augment fibroblast-mediated gel
contraction (2, 11). CSE was observed to inhibit release of
fibronectin, thus providing a potential mechanism to account for the
effect of CSE on contraction. In support of this mechanism, exogenous
fibronectin could partially restore the ability of fibroblasts to
support contraction of collagen gels. The inhibitory activity present
in cigarette smoke appears to be a volatile component. Moreover,
multiple components present in the volatile phase of cigarette smoke
are likely to have activity because two active volatile components,
acrolein and acetaldehyde, each demonstrated the ability to inhibit
both fibroblast-mediated collagen gel contraction and fibronectin
production. Together, the current study supports the concept that
cigarette smoke may impair wound healing processes by altering
fibroblast contraction.
Contraction of collagen gels by fibroblasts is a complex process, and
cigarette smoke could be acting at several levels. This process appears
to involve both the collagen-binding
2
1-integrin and fibronectin-mediated processes (2, 11, 30). The current study
suggests that no significant changes in
2
1-integrin
expression resulted from cigarette smoke exposure. On the other hand,
integrin affinity for substrate could be altered, as could the
mechanisms by which integrins interact with the cytoskeleton (15). Such an integrin-mediated mechanism is not excluded by the results of the
present study.
In the present study, increased endogenous PGE2 production does not appear to account for the inhibition of fibroblast-mediated collagen gel contraction caused by cigarette smoke. This is interesting, particularly when compared with the effects of gamma irradiation. Such radiation can also impair fibroblast-mediated collagen gel contraction in a reversible manner (6). The radiation effect, however, appears to be largely mediated through the production of endogenous PGE2, as evidenced by measurement of PGE2 production and reversibility of the effect in the presence of indomethacin. In contrast, cigarette smoke did not increase PGE2 release measured after 72 h. An effect on early PGE2 release is not excluded but seems unlikely because indomethacin added both before and together with smoke extract to ensure complete inhibition of PGE synthesis did not reverse the effect of smoke. Thus cigarette smoke appears to be inhibiting fibroblast-mediated gel contraction by a mechanism different from that of irradiation.
Some investigators have suggested that fibronectin plays a role in fibroblast-mediated gel contraction (2, 11). Our studies demonstrated that fibronectin production was significantly reduced by CSE as well as by acetaldehyde and acrolein. Reduced contraction due to reduction of fibronectin could account for the reduced contraction of cells exposed to CSE. This is supported by the observation that plasma and cellular fibronectin were capable of reversing, in part, the inhibitory effect of CSE. Similar results were observed with acetaldehyde and acrolein (data not shown). These results also suggest that the effect of CSE was not due to a nonspecific toxic effect.
Variant forms of fibronectin result from differential splicing of mRNA derived from a single gene (31). Using skin fibroblasts, Asaga et al. (2) suggested that only the "cellular" form was capable of supporting fibroblast-mediated gel contraction. In contrast, also working with skin fibroblasts, Gillery et al. (11) suggested that both forms could support this activity. The results from the current study suggest that lung fibroblasts can utilize either form of fibronectin to augment contraction that has been inhibited by CSE.
The current study demonstrates that the volatile components of cigarette smoke were particularly important in inhibiting fibroblast-mediated gel contraction. Cigarette smoke contains in excess of 6,000 components, approximately one-half of which are relatively volatile (14). Only a minority of these components have been studied in detail for their toxicities. Among the more toxic species present in high concentration, however, are reactive aldehydes, of which acrolein and acetaldehyde are particularly prominent (13, 14). These moieties are capable of binding to and interacting with various cellular components including contractile proteins (23). Such a mechanism could contribute to the inhibition of contraction by smoke. It was for this reason that these specific species were selected for testing in the current study.
It has been estimated that one cigarette can yield 980 µg of
acetaldehyde and 85 µg of acrolein. Measured concentrations of acetaldehyde in our smoke extract were ~50% of this theoretical yield. Such a concentration, when diluted according to the protocols used in the current study, would have been just below the
concentration-response range tested for acetaldehyde alone. With
allowance for the 70-80% recovery we observed for acetaldehyde in
standard samples, it is possible that acetaldehyde acting alone could
have accounted for some of the observed effect. Recovery of acrolein
was much less efficient (10% or less), perhaps because of its greater
lability, and the concentration of acrolein was below detectability in
all our extracts. However, had acrolein been present in the ratio expected to acetaldehyde in fresh smoke extract, it would have been
present at 1.77 µg/ml (2.16 × 105 M), well within the
range at which toxic effects on fibroblast contraction were observed.
In addition to acetaldehyde and acrolein, cigarette smoke contains many
other components that may also contribute to its toxicity. Because
these components of cigarette smoke may interact with multiple targets
within the cell, there is the potential that these various toxins could
interact in an additive or even synergistic way to impair fibroblast
function. Thus, although we believe that we have demonstrated that
acetaldehyde and acrolein could contribute to the toxic effects of CSE,
we believe that the effects of the extract likely represent a combined
effect of these and other toxins.
The ability of fibroblasts to mediate contraction of a collagen gel is thought to be a model of part of the wound-repair response (12, 28). This response is also thought to involve fibroblast recruitment and proliferation, both of which are also inhibited by CSE (26). These in vitro findings are consistent with the impaired healing noted in cigarette smokers in a variety of settings. One example of this impaired healing is pulmonary emphysema. In this disease, cigarette smoke results in destruction of alveolar walls and enlargement of air spaces (5, 20). Undoubtedly, the release of potent proteases in excess of anti-protease defenses plays an important role in the damage of the alveolar structures. Emphysema results when this damage is not balanced by an appropriate repair response. The ability of cigarette smoke to impair fibroblast-mediated repair mechanisms could, therefore, be another mechanism by which cigarette smoke contributes to the development of diseases such as pulmonary emphysema. In this regard, it is of interest that emphysema develops variably among smokers (5). Fibrosis-like changes, moreover, may be present at selected sites within emphysematous lungs (18, 34), suggesting that repair processes are active in emphysema and that the final lesion represents a balance among several processes. It is conceivable, therefore, that individuals differ in their susceptibility to cigarette smoke alteration of repair responses, and this could represent an important variable in determining who is at risk for the development of emphysema.
In conclusion, we have shown that CSE can inhibit fibroblast-mediated collagen gel contraction. This effect is not likely to be due simply to an effect on cell growth or to cytotoxicity. The effect appears to be dependent on volatile components of cigarette smoke and may be mediated by a decrease in fibroblast production of fibronectin. Impaired wound healing is a feature of cigarette smokers and may be a particularly important feature in the development of some diseases such as pulmonary emphysema. The ability of cigarette smoke to inhibit fibroblast-mediated healing may play a particular role in this process.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Borgerding for assistance in quantification of acetaldehyde and acrolein and Lillian Richards for administrative assistance.
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FOOTNOTES |
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This work was supported in part by the National Research Council of Italy, the State of Nebraska, and the Larson Endowment of the Univ. of Nebraska Medical Center.
Address for reprint requests: S. I. Rennard, Univ. of Nebraska Medical Center, 600 South 42nd St., Omaha, NE 68198-5300.
Received 31 July 1996; accepted in final form 8 January 1998.
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