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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Contraction of three-dimensional collagen gels is a model of
the contraction that characterizes normal healing and remodeling after injury. In the current study, we evaluated the hypothesis that a number of inflammatory factors, including tumor necrosis factor (TNF)-
, interleukin (IL)-1
, and interferon
(IFN)-
, modulate this process by induction of prostaglandin
(PG) E2 and nitric oxide (NO) production and that these secondary mediators function in an autocrine or paracrine manner to modulate contraction. Human fetal lung fibroblasts
(HFL) were cultured in type I collagen gels and floated in medium containing TNF-, IL-1, or IFN- alone or in combination (cytomix). All cytokines inhibited the contraction significantly. The potency order was IL-1, TNF-, IFN-. The cytomix was no more potent than was IL-1 alone. PGE2 production was increased by TNF- (5.0 versus 0.16 ng/ml, P < 0.01), IL-1 (5.3 versus 0.16 ng/ml, P < 0.01), and cytomix
(5.9 versus 0.16 ng/ml, P < 0.01), and was completely inhibited by indomethacin. Indomethacin (P < 0.05) and L-NG-monomethyl arginine citrate (L-NMMA) (P < 0.05) alone both
partially attenuated the inhibition of contraction caused by
cytokines alone or by cytomix. Indomethacin and L-NMMA together attenuated inhibition more than either alone (P < 0.05). Exogenous PGE2 and exogenous NO donors (DETA nononate and 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride) inhibited the contraction significantly. The protein kinase
A inhibitor KT5270 and the protein kinase G inhibitor Rp-pCPT-cGMPS attenuated the inhibition induced by PGE2 and
NO, respectively. In summary, PGE2 and NO appear to function in parallel as autocrine/paracrine mediators of cytokine-driven fibroblast inhibition of the contraction of collagen gels
and may contribute to remodeling during repair and inflammation in lung disorders.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Inflammatory processes are frequently accompanied by alterations in tissue structure. Such alterations may result from tissue damage due to active proteases or toxic moieties released by inflammatory cells. In addition, mediators released during the inflammatory process are capable of directly altering parenchymal cell function, leading to tissue remodeling and repair. Included among these processes are the deposition and organization of extracellular matrix. In this context, both the formation of scar tissue and the development of fibrosis are characterized by tissue contraction.
The ability of fibroblasts to contract gels composed of
native type I collagen has been used as an in vitro model of
the contraction that characterizes both normal wound
healing and the development of fibrosis (1, 2). A number
of mediators released by inflammatory cells, including interleukin (IL)-1, tumor necrosis factor (TNF)-, and interferon (IFN)-, have been reported to inhibit fibroblast-mediated gel contraction (3, 4). After interaction with their
respective receptors, these inflammatory mediators can
initiate biologic effects by a variety of mechanisms. Understanding the mechanisms by which tissue repair is modulated by inflammatory mediators is essential in identifying
potential therapeutic strategies to alter repair in inflammatory settings. Not only would identification of relevant
pathways identify potential targets, but the identification of parallel pathways that may be, to some extent, redundant is essential. The current study, therefore, was undertaken to determine the pathways through which IL-1,
TNF-, and IFN- inhibit fibroblast-mediated collagen gel
contraction. Two parallel pathways, each capable of mediating the inhibition
stimulation of prostaglandin (PG) E
production and stimulation of nitric oxide (NO) productionwere identified. These two pathways act through
distinct signal transduction pathways as the PGE2 pathway
appears to function by increasing intracellular cyclic adenosine monophosphate (cAMP), leading to activation of
protein kinase A (PKA), whereas NO appears to act by
stimulating cyclic guanosine monophosphate (cGMP) formation leading to activation of protein kinase G (PKG).
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Materials
Type I collagen was extracted from rat-tail tendons (RTTC) by a
previously published method (5, 6). Briefly, tendons were excised
from rat tails, and the tendon sheath and other connective tissues
were removed carefully. After repeated washing with Tris-buffered saline and 95% ethanol, type I collagen was extracted in 4 mM acetic acid. Protein concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution. Sodium dodecyl
sulfate polyacrylamide gel electrophoresis demonstrated no detectable proteins other than type I collagen. Human recombinant TNF-,
human recombinant IL-1, and human recombinant IFN-
were purchased from R&D Systems (Minneapolis, MN). PGE2
and indomethacin were purchased from Sigma (St. Louis, MO).
L-NG-monomethyl arginine citrate (L-NMMA), DETA nonoate
(NONO), and 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1) were purchased from Cayman Chemical (Ann Arbor, MI). KT5720 was purchased from Calbiochem (San Diego, CA)
and Rp-8-pCPT-cGMPS was purchased from Biomol (Plymouth
Meeting, PA). Tissue culture supplements and media were purchased from GIBCO BRL (Life Technologies, Grand Island,
NY). Fetal calf serum (FCS) was purchased from Biofluid (Rockville, MD).
Fibroblasts
Human fetal lung fibroblasts (HFL-1) were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in 100-mm tissue culture dishes (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) with Dulbecco's modified Eagle medium (DMEM), which contained 0.4 mM L-arginine supplemented with 10% FCS, 50 µg/ml penicillin, 50 µg/ml streptomycin, and 0.25 µg/ml fungizone. The fibroblasts were passaged every 3 to 5 d. Subconfluent fibroblasts were trypsinized (trypsin-ethylenediaminetetraacetic acid [EDTA]: 0.05% trypsin, 0.53 mM EDTA-4Na) and used for collagen gel culture. Fibroblasts used in these experiments were between cell passages 16 and 20.
Preparation of Collagen Gels
Collagen gels were prepared by mixing the appropriate amount of RTTC, distilled water, fourfold concentrated DMEM, and cell suspension so that the final mixture resulted in 0.75 mg/ml of collagen, 4.5 × 105 cells/ml, and a physiologic ionic strength and 1× DMEM (6). Fibroblasts were always added last. One-half milliliter of the mixture was cast into each well of 24-well tissue culture plates (Falcon, Becton Dickinson Labware). Gelation occurred in 20 min at room temperature, after which the gels were released and transferred to 60-mm tissue culture dishes containing 5 ml of medium and incubated at 37°C, 5% CO2.
To demonstrate inhibition of collagen gel contraction, fibroblasts were cast into gels as described previously and then cultured in medium containing TNF- (5 ng/ml), IL-1 (5 ng/ml), or
IFN- (10 ng/ml) alone or in combination. To investigate the effects of indomethacin and L-NMMA on the gel contraction, indomethacin (1 µM) or L-NMMA (0.1 mM) was added into the
surrounding medium at the same time. To test the effects of exogenous PGE2 and NO donors on gel contraction, various doses
of PGE2, SIN-1, and NONO were added into the medium. Gel
size was determined daily using an image analysis system (Optomax, Hollis, NH).
Collagen Content in the Gels after Contraction
To quantify the amount of collagen in the gels after contraction, the amount of hydroxyproline, which is directly proportional to type I collagen content, was determined by spectrophotometric assay (7). Briefly, the gels were transferred into glass tubes (KIMAX USA; Fisher Scientific, St. Louis, MO) with 2 ml of 6N HCl. Oxygen in the solution was removed by ventilating nitrogen gas for 30 s. The gels were hydrolyzed at 110°C for 12 h, dried with a vacuum centrifuge, and dissolved in dH2O. Hydroxyproline in the samples was reacted with oxidant (1.4% chloramine T in acetate/ citric acid buffer; Sigma) and Ehrlich's reagents (0.4% p-dimethylamino-benzaldehyde; Sigma) in 60% perchloric acid (Fisher Chemical, Fairlawn, NJ) at 65°C for 25 min, and absorbance was determined by spectrophotometer at 550 nm (model DU 62; Beckman, Fullerton, CA).
DNA Content
DNA content was determined in order to estimate cell number in the gels (8). Briefly, the gels were dissolved in 0.5 ml of DMEM, which contained collagenase (1 mg/ml Sigma), by incubating at 37°C for 2 to 3 h. The cells were collected by centrifugation (2,000 × g, 10 min, 4°C). One-half milliliter of dH2O was added to each sample, and cells were sonicated. DNA content was determined with Hoechst dye 33258 (Sigma) using an excitation wavelength of 356 nm and an emission of 458 nm.
PGE2 Assay
Supernatant fluids were frozen and stored at 
80°C until assay.
PGE2 was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI).
Statistical Evaluation
The results are expressed as the mean of three determinations ± standard error of the mean (SEM) unless described otherwise. Data shown in the figures are from single experiments that were generally confirmed by repeating experiments on at least three separate occasions. Group data were evaluated by analysis of variance. Differences between two groups of data that appeared statistically significant were further analyzed by unpaired Student's t test. Comparisons were considered statistically significant if P < 0.05.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of Cytokines on Collagen Gel Contraction
Under control culture conditions, fibroblasts contracted the
collagen gels over the 5-d culture period. IFN- at 10 ng/ml inhibited the contraction slightly, but significantly; TNF- at 5 ng/ml inhibited the contraction more potently; and IL-1
at 5 ng/ml was the most potent inhibitor. Although the magnitude of the gel contraction varied among experiments performed with different batches of fibroblasts and different
preparations of collagen, the effects of the cytokines were
consistently observed (Table 1). Cytokine inhibition of gel
contraction, moreover, was present throughout the course
of the culture period (Figure 1). The combination of the
three cytokines was no more potent than IL-1 alone.
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The concentration dependence of the cytokines for the
inhibition of gel contraction was tested directly (Figure 2).
For TNF-, no significant effect on the contraction was
observed at 0.1 ng/ml, but 1 ng/ml produced significant inhibition, and 10 ng/ml had an increased effect. IFN- at 1 ng/ml had no effect. At 100 ng/ml, the inhibition was only
slightly greater than at 10 ng/ml. At 0.1 ng/ml, IL-1 caused
a potent inhibition that increased over the concentration
range tested. The cytomix also inhibited gel contraction in
a concentration-dependent manner (Figure 2). To determine
if cytokines were modulating fibroblast numbers, DNA content was assessed at the end of the 5-d experimental periods. The DNA content in cytokine-treated cultures was no different than that of control cultures, indicating no effect of the
cytokines on fibroblast numbers under the experimental
conditions used (data not shown). Similarly, in order to determine if the cytokines could lead to an alteration in collagen content in the floating gels, the hydroxyproline content of the gels was determined after the 5-d incubation
period. TNF- did cause a significant reduction in collagen
content; IL-1 and cytomix caused a small reduction in
collagen content (Table 2).
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Role of PGE2 in Cytokine Inhibition of Collagen Gel Contraction
To evaluate if IL-1, TNF-, and IFN- were attenuating
gel contraction by stimulating fibroblasts to produce
eicosanoids, two methods were used. First, indomethacin,
a cyclooxygenase inhibitor, was used. At 1 µM, indomethacin alone slightly enhanced fibroblast-mediated gel contraction (34 versus 38% at Day 5; P > 0.05) and significantly (P < 0.05, all comparisons) attenuated the inhibition of gel
contraction caused by TNF-, IL-1, and IFN- (Figure
3). Cytomix inhibition of gel contraction was also reduced
by indomethacin, and this inhibition was concentration-dependent (Figure 4). However, even at the highest concentration tested (10 µM), indomethacin was unable to
completely block the attenuation of contraction induced by cytomix.
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Second, to determine if endogenous PGE2 production
contributes to cytokine attenuation of gel contraction, direct measurement of PGE2 release by fibroblasts in gel
culture in the presence of cytokines was made and compared with the effect of the addition of exogenous PGE2.
PGE2 levels in the media in which the gels were floated
were significantly increased in the presence of TNF- (32-fold over control), IL-1 (34-fold over control), and cytomix (37.5-fold over control) (P < 0.001 for all comparisons) (Figure 5A). IFN- did not induce more PGE2 production than did unsupplemented medium. PGE2 production
induced by cytokines was completely blocked by 1 µM indomethacin (P < 0.001). Exogenous PGE2 added to the culture medium inhibited the gel contraction at a concentration range similar to that produced by the fibroblasts in response to the cytokines (Figure 5B).
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Role of NO in Cytokine Inhibition of Fibroblast-Mediated Gel Contraction
To determine if NO could contribute to cytokine-induced
inhibition of collagen gel contraction, two approaches were
used. First, L-NMMA, an inhibitor of NO synthase, was
used. L-NMMA alone had a minimal effect on gel contraction (Figure 6). In contrast, L-NMMA attenuated the inhibition of collagen gel contraction caused by IFN- completely (P < 0.05) and partially attenuated that induced by
TNF- (P < 0.05). L-NMMA also had a significant but less
pronounced effect on the inhibition induced by IL-1 (P < 0.05; Figure 6). The inhibition of gel contraction caused by
cytomix was also significantly attenuated by L-NMMA,
and this inhibition demonstrated concentration dependence (Figure 7). The combination of indomethacin (1 µM) with L-NMMA (0.1 mM) attenuated the cytomix-induced
inhibition of collagen gel contraction more than either indomethacin or L-NMMA alone (P < 0.05; Figure 8), although even the combination only partially attenuated the
inhibition induced by cytomix.
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Second, the effect of exogenous NO donors on fibroblast-mediated gel contraction was tested using NONO and SIN-1. Both NONO and SIN-1 significantly inhibited gel contraction in a concentration-dependent manner (Figure 9). When either NONO or SIN-1 was added together with PGE2, the inhibition of gel contraction was augmented (P < 0.05).
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Effect of PKA and PKG Inhibitors on PGE2 and NO Inhibition of Gel Contraction
To investigate whether the inhibition of PGE2 and NO was related to PKA and PKG activity, inhibitors of PKA and PKG were used. The PKA inhibitor KT5720 alone slightly stimulated the contraction, but it significantly attenuated the inhibition induced by PGE2 (Figure 10A: 51 versus 60%; P < 0.05). Similarly, the PKG inhibitor Rp-8-pCPT-cGMPS alone did not affect the contraction, but it significantly attenuated NONO inhibition of the gel contraction (Figure 10B: 51 versus 60%; P < 0.05).
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The current study demonstrated that the cytokines IL-1,
TNF-, and IFN-, both alone and in combination, can inhibit human lung fibroblast-mediated contraction of collagen gels. This inhibition is mediated by multiple signal
transduction pathways. Both cytokine-stimulated PGE2
production and cytokine induction of NO production appear to play a role in cytokine attenuation of collagen gel
contraction. Consistent with multiple pathways for cytokine attenuation of collagen gel contraction, both cAMP-
and cGMP-dependent protein kinase pathways appear to
play a role. The current study, therefore, supports the concept that several mechanisms can be initiated by cytokines
that can attenuate fibroblast-mediated collagen gel contraction.
The current study used fibroblasts cultured in three-dimensional collagen gels as a model for tissue remodeling. Fibroblasts cultured in a native collagen gel will generate a traction force on the collagen fibers. If the collagen
gel is allowed to float, this traction force results in contraction of the collagen gel. This process has been used to
model tissue contraction that is characteristic both of normal healing and of the development of fibrosis (1, 2). A
number of profibrotic mediators, including transforming
growth factor- and platelet-derived growth factor, are
characterized by their ability to stimulate fibroblast-mediated collagen gel contraction (9, 10). These same mediators are also able to increase fibroblast numbers and
matrix production. These mediators could, therefore, contribute to the development of fibrosis by stimulating several profibrotic activities. In this regard, the ability of
fibroblasts to contract extracellular matrices should be regarded as a separate activity.
Lung diseases are often associated with the recruitment
and activation of inflammatory cells, together with the
production of a complex mixture of inflammatory mediators. Many of these mediators are believed to drive not
only the inflammatory response but can also regulate repair and remodeling. In this regard, both IL-1 and IFN-
have been demonstrated to inhibit fibroblast-mediated gel
contraction (3, 4). TNF- is a multifunctional cytokine that
can induce a variety of responses. Several studies have
suggested a role for TNF- in the development of fibrosis
in the lung (11, 12). The current study suggests that TNF-
may also initiate pathways that serve to mitigate at least
one aspect of fibrosis, tissue contraction. Varying effects of
TNF- are observed for other responses as well. For example, TNF- can both activate pathways leading to apoptosis
through TNF receptor-associated death domain signaling
and inhibit apoptosis through nuclear factor (NF)-
B-mediated pathways (13, 14). The response in an individual cell,
therefore, depends on the balance between competing pathways. It is likely that TNF induction of inducible NO synthase (iNOS) and cyclooxygenase leading to inhibition of
fibroblast-mediated collagen gel contraction depends on
NF-B signaling. A potential role for TNF- in the development of fibrosis, however, remains a possibility.
The current study extends earlier observations, confirming that the cytokines, acting individually, can inhibit fibroblast-mediated collagen gel contraction, they can interact, and importantly, they can activate multiple signal transduction pathways, leading to inhibition of collagen gel contraction. In vivo, the cytokine milieu is likely to contain multiple active species; the ability of cytokines to both interact and to stimulate multiple signal transduction pathways with similar effects has important therapeutic implications.
Previous studies by Zhang and colleagues (3) demonstrated that IL-1 can inhibit collagen gel contraction, at
least in part, by inducing the production of NO. The results
of the current study are consistent with these observations
and demonstrate that NO synthase inhibitor L-NMMA
partially attenuates collagen gel contraction inhibition induced by IL-1. In the current study, however, NO inhibition could not completely block the effect of IL-1, suggesting other signal transduction pathways were also active.
It may be that the current study in human cells differs in
part from the study by Zhang and colleagues in that their
study was done in rat cells (3). In this regard, rat and human cells are known to differ in several respects regarding
the functional importance of cyclooxygenase and NO pathways. IL-1 is known to induce PGE2 production as well as
inducing NO production (15). The ability of indomethacin to partially block the inhibitory effect of IL-1 supports
a role for PGE2, as well as NO, in mediating the IL-1 effect. That the combination of L-NMMA and indomethacin was more effective than either agent alone suggests that
multiple signal transduction pathways are leading to the
inhibition of contraction. More importantly, the effect of
indomethacin and L-NMMA in combination is unlikely to
be due to a concentration dependence effect as maximal
inhibition could not be achieved by using higher than conventional concentrations of either indomethacin or L-NMMA.
The results of the current study differ from those of Zhang and colleagues in another important respect (3). In rat cells, Zhang and coworkers (3) observed an NO-dependent induction of apoptosis induced by IL-1. In the current study, there was no difference in DNA content in cytokine-treated gels compared with control gels, suggesting that cytokine-driven apoptosis was not taking place. Whether this experimental difference reflects a difference in response between human and rat cells or whether it represents a methodologic difference between these studies remains to be determined.
Tissue contraction is a process that likely involves coordinated cellular activity within a local region. It is, therefore, of interest that all three cytokines evaluated appeared to act through the production of secondary mediators that are capable of diffusing through a tissue and thus acting as paracrine regulators. Through such a mechanism, it may be possible to coordinate the activity of multiple fibroblasts within a tissue.
The current study also evaluated the signal transduction pathways activated by TNF- and IFN-. TNF- resembled IL-1 in that both PGE2-mediated and NO-mediated inhibition appeared to play a prominent role. These
observations are consistent with previous studies demonstrating that TNF-, like IL-1, is a potent inducer of both
iNOS and cyclooxygenase-2 (17, 19). IFN- was less potent at inhibiting collagen gel contraction. Interestingly, L-NMMA was capable of nearly completely blocking the
IFN- effect, whereas indomethacin was not as effective.
This suggests that the effect of the various cytokines evaluated in the current study may not be entirely equivalent.
PGE2 is capable of exerting effects by activating several receptors. A major mechanism for PGE2 signaling, however, is the stimulation of cAMP through activation of adenylate cyclase. cAMP, in turn, can activate cAMP-dependent PKA. Consistent with such a role for PGE2 inhibition of collagen gel contraction in the current study, the PKA inhibitor KT5720 was demonstrated to block the ability of exogenous PGE to inhibit collagen gel contraction. In contrast, NO is believed to act, at least in part, by activating guanylyl cyclase. cGMP, in turn, is believed to activate cGMP-dependent PKG. Consistent with such a role for NO in inhibiting collagen gel contraction, the PKG inhibitor RP-8-pCPT-cGMPS was able to block the ability of the NO donor NONO to block collagen gel contraction. These experiments, therefore, suggest that both PKA- and PKG-mediated processes contribute to the inhibition of fibroblast-mediated collagen gel contraction induced by PGE and NO, respectively. Interestingly, neither the PKA inhibitor nor the PKG inhibitor was completely effective. This raises the interesting possibility that both PGE and NO may be mediating effects through multiple pathways. It is also likely that interactions occur between pathways activated by PGE and NO.
In addition to NONO, SIN-1 was also used as an exogenous NO donor. This reagent is capable of generating peroxynitrite, which can result in protein nitrosylation (20, 21). Recent evidence suggests that endogenous NO generation in vivo may also lead to protein nitrosylation through the formation of peroxynitrite (22, 23). Whether such a pathway plays a role in regulating fibroblast-mediated contraction of collagen gels remains to be determined.
Both NO and PGE2 can have effects on fibroblasts in addition to modulation of contraction of collagen gels. PGE2 is a well-described inhibitor of fibroblast proliferation (24), and NO has been suggested to play a role in the induction of fibroblast apoptosis (3). It is likely, therefore, that the production of PGE2 and NO induced by cytokines can affect fibroblast numbers as well as altering their contractile behavior. In the current study, however, DNA content remained unchanged throughout the 5-d culture periods. It is unlikely, therefore, that either alteration of fibroblast proliferation or induction of fibroblast apoptosis is playing a role in modulating fibroblast contraction under the experimental conditions used. In conditions where fibroblast proliferation would be occurring, for example, with high serum concentration, or under conditions where apoptosis could be observed, for example, with longer incubation times, it is possible that such effects could be observed. It seems likely that effects on not only fibroblast numbers and contractility but also on fibroblast production of extracellular matrix and on enzymes capable of degrading extracellular matrix will interact to regulate the tissue remodeling process. It is likely, moreover, that these various processes can interact among themselves. For example, contraction of collagen gels per se can, under some circumstances, induce fibroblast apoptosis (25). Contraction, in turn, is increased in the face of reduction of collagen content in the gels. Although it is likely that complex interactions between collagen production, tissue contraction, and alteration in fibroblast proliferation and apoptosis are crucial in tissue remodeling, such interactions were beyond the experimental scope of the current study.
It is unlikely that the slight degradation of collagen induced by the cytokines accounts for the inhibition of collagen gel contraction observed. In this regard, the ability of fibroblasts to contract collagen gels is inversely related to collagen concentration (26, 27). For this reason, an increase in collagen degradation would likely be associated with augmented contraction rather than with inhibition of contraction. It is likely, however, that degradation of collagen occurring together with contraction plays an important role in the control of tissue remodeling. Therefore, the ability of cytokines to induce matrix metalloproteases suggests multiple mechanisms by which these cytokines can regulate the remodeling process.
The current study focused on the tissue contraction that characterizes both normal healing and fibrotic processes. When contraction of tissues occurs around a hollow tube, such as the airway in chronic bronchitis or a blood vessel in atherosclerosis, significant physiologic abnormalities can result. The ability to alter this tissue contraction could have important therapeutic implications. The current study suggests that therapies designed to block such processes, however, will need to allow for the fact that multiple parallel pathways can be involved. Moreover, it appears that several cytokines likely present in the inflammatory milieu can independently activate these multiple pathways. Thus, it is likely that therapeutic strategies capable of blocking multiple pathways may be required to achieve therapeutic benefit.
In conclusion, the current study demonstrated that the
cytokines IL-1, TNF-, and IFN- can inhibit fibroblast-mediated collagen gel contraction through the activation
of at least two parallel pathways. Both PGE2 and NO function in this regard and may play an important role as paracrine mediators. PGE2 and NO, in turn, act both through
PKA- and PKG-dependent mechanisms. The presence of
multiple parallel pathways by which cytokines can modulate repair and remodeling suggests that therapeutic strategies designed to alter remodeling may also need to act
through multiple mechanisms.
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Footnotes |
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Address correspondence to: Stephen I. Rennard, M.D., University of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, NE 68198-5125. E-mail: srennard{at}unmc.edu
(Received in original form September 27, 2000 and in revised form February 21, 2001).
Abbreviations: cyclic adenosine monophosphate, cAMP; cyclic guanosine monophosphate, cGMP; Dulbecco's modified Eagle medium, DMEM; human fetal lung fibroblast, HFL; interferon, IFN; interleukin, IL; L-NG-monomethyl arginine citrate, L-NMMA; nitric oxide, NO; DETA nonoate, NONO; prostaglandin E2, PGE2; protein kinase A, PKA; protein kinase G, PKG; standard error of the mean, SEM; 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride, SIN-1; tumor necrosis factor, TNF.
Acknowledgments:
The authors wish to acknowledge the thoughtful suggestions and scientific review provided by Dr. Todd Wyatt, the secretarial assistance of Ms. Lillian Richards and the editorial assistance of Ms. Mary Tourek.
This study was supported, in part, by the Larson Endowment, by the University
of Nebraska Medical Center, and by grant RO1 HL64088-01 from the National
Heart, Lung, and Blood Institute.
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References |
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Materials and Methods
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Discussion
References
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