September 2000
Volume 41, Issue 10
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Cornea  |   September 2000
Regulation of Collagenase, Stromelysin, and Gelatinase B in Human Conjunctival and Conjunctivochalasis Fibroblasts by Interleukin-1β and Tumor Necrosis Factor-α
Author Affiliations
  • Daniel Meller
    From the Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute; and the
  • De–Quan Li
    From the Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute; and the
  • Scheffer C. G. Tseng
    From the Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute; and the
    Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami.
Investigative Ophthalmology & Visual Science September 2000, Vol.41, 2922-2929. doi:https://doi.org/
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      Daniel Meller, De–Quan Li, Scheffer C. G. Tseng; Regulation of Collagenase, Stromelysin, and Gelatinase B in Human Conjunctival and Conjunctivochalasis Fibroblasts by Interleukin-1β and Tumor Necrosis Factor-α. Invest. Ophthalmol. Vis. Sci. 2000;41(10):2922-2929. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Overexpression and increased activities of matrix metalloproteinases (MMPs) have recently been reported in cultured conjunctival fibroblasts from patients with conjunctivochalasis. The role of inflammatory cytokines in modulating expression of MMPs, their tissue inhibitors (TIMPs), and urokinase plasminogen activator (uPA) as potential contributors to the pathogenesis of conjunctivochalasis was investigated.

methods. Interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α) was added at 10 ng/ml to a serum-free medium. Expression of transcripts and proteins of MMPs, TIMPs, and uPA by cultured normal human conjunctival and conjunctivochalasis fibroblasts was determined by Northern hybridization, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. Gelatin and casein zymographies were performed in serum-free conditioned media with and without the respective enzyme inhibitors.

results. Without challenging the cells, conjunctivochalasis fibroblasts showed mRNA and protein overexpression of MMP-1 and MMP-3 compared with normal conjunctival fibroblasts, which showed minor or no expression of these enzymes. IL-1β markedly and TNF-α to lesser extent increased mRNA and protein expression of MMP-1 and MMP-3 in conjunctivochalasis fibroblasts from 2 subjects when compared with normal conjunctival fibroblasts from 2 subjects and with their nonstimulated counterparts. In conjunctivochalasis fibroblasts and normal conjunctival fibroblasts, TNF-α, but not IL-1β, induced a gelatinolytic activity of MMP-9, which was further confirmed by Western blot analysis and ELISA. Expression of MMP-2, TIMP-1, and TIMP-2 mRNA and protein was not influenced by IL-1β or TNF-α, and no difference was found in the gelatinolytic activity of MMP-2 between both cell types.

conclusions. Inflammatory cytokines such as IL-1β and TNF-α, which can potentially be derived from the ocular surface and tears, may be responsible for increased expression of MMPs in cultured conjunctivochalasis fibroblasts. Ocular inflammation might be one important denominator in the pathogenesis of conjunctivochalasis.

Conjunctivochalasis, defined as a redundant, loose, nonedematous inferior bulbar conjunctiva interposed between the globe and the lower eyelid, has been shown to cause different forms of ocular surface irritation (see a recent review in Ref. 1) . Information regarding the pathogenesis of conjunctivochalasis is scanty and conflicting. Abnormalities in conjunctival extracellular components in conjunctivochalasis have been suggested. For example, degeneration of elastic fibers has been noted, 2 but no fragmentation or other abnormalities of the elastic fibers were observed using hematoxylin and eosin and Weigert’s elastic-tissue stain. 3 It should be noted that elastotic degeneration, a well-known histopathologic feature of pingueculae, 4 pterygium, 4 and photoaged skin, 5 has been regarded as a hallmark for actinic damage and that conjunctivochalasis tends to be associated with pingueculae. 6  
The looseness of redundant conjunctiva suggests that extracellular matrix–degrading enzymes may contribute to the pathogenesis of conjunctivochalasis. Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) participate in connective tissue degradation and remodeling (for reviews see Refs. 7 8 9 ). These enzymes are synthesized and secreted by a variety of cell types including fibroblasts. 10 At least 22 members of the MMP family have been identified and are normally coexpressed with a family of TIMPs, which inhibit active forms of MMPs. 9 11 A delicate balance among the production, activation, and inhibition of MMPs and TIMPs determines the final outcome of extracellular matrix components, including collagen and elastin (for reviews see Refs. 8 and 9). Overexpression of MMPs is implicated in such diseases as rheumatoid arthritis, corneal ulceration, and tumor invasion and angiogenesis (for reviews see Refs. 12 13 14 15 16 17 ). Other than MMPs and TIMPs, another potential proteolytic cascade leading to tissue degradation and remodeling involves urokinase plasminogen activator (uPA), a serine protease. 18 19 20  
Our laboratory has recently reported that fibroblasts isolated from conjunctivochalasis tissues overexpress MMP-1 and MMP-3 but not MMP-2, MMP-9, TIMP-1, and TIMP-2 compared with normal human conjunctival fibroblasts. 21 Interestingly, upregulation of MMP-1, MMP-3, and MMP-9, but not MMP-2, has also been reported in skin fibroblasts cultured from patients suffering with cutis laxa, 22 a disorder exhibiting loose and sagging skin with reduced elasticity. Based on data summarized in a recent review, 1 we have speculated that conjunctivochalasis may be linked to ocular surface inflammation. We, thus, examine herein whether inflammatory cytokines, such as IL-1 and TNF-α, may play a role in upregulating MMPs. 
Methods
Materials
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin–EDTA, fungizone (amphotericin B), phenol, DNA or RNA size marker, and a random primers DNA labeling kit were purchased from GIBCO–BRL (Grand Island, NY). Cell culture dishes, 6-well plates, and 15-ml centrifuge tubes were from Becton–Dickinson (Lincoln Park, NJ). The protein assay kit using bicinchoninic acid was from Pierce (Rockford, IL). Zymogram-ready gels containing gelatin or casein, 4% to 15% Tris–HCl polyacrylamide gradient-ready gels, sodium dodecyl sulfate (SDS), and the electrophoresis equipment were from Bio–Rad (Hercules, CA). Human MMP-1, MMP-3, and MMP-9 Enzyme Immunoassay (enzyme-linked immunosorbent assay; ELISA) kits and the monoclonal antibodies against human MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 were from Oncogene Research Products of Calbiochem (Cambridge, MA). Vectastain Elite ABC peroxidase kit was from Vector Laboratories (Burlingame, CA). Nitrocellulose membranes were from Schleicher and Schuell (Keene, NH). GeneAmp RNA–PCR kit was from Perkin–Elmer Cetus (Norwalk, CT). Wizard PCR Preps DNA purification kit was from Promega (Madison, WI). [α-32P]–dCTP was from DuPont New England Nuclear (Boston, MA). XAR-5 and BioMax MS-1 films and intensifying screens were from Eastman Kodak (Rochester, NY). All other reagents and chemicals came from Sigma (St. Louis, MO). 
Human Conjunctival and Conjunctivochalasis Fibroblast Cultures
All procedures followed the tenets of the Declaration of Helsinki, and informed consent was obtained from each patient. Conjunctivochalasis specimens were obtained from two patients after the removal of the lesion. Age- and gender-matched specimens of normal human conjunctiva were obtained from the superior bulbar conjunctiva of two patients during cataract surgery. For this study, fibroblasts between the 3rd and 7th passage were used, and the method of growing fibroblasts from the explant culture has been recently reported. 23 In brief, each conjunctival specimen was cut into explants of approximately 2 × 2 mm2 and placed onto 100-mm tissue culture dishes. Ten minutes later, each explant was covered with a drop of FBS, and placed overnight in an incubator at 37°C under 95% humidity with 5% CO2. Ten milliters of D–FBS (i.e., DMEM containing 10% FBS, 50 μg/ml gentamicin and 1.25 μg/ml amphotericin B) was added the next day, and the medium was changed every 2 days thereafter. Fibroblasts were subcultured with 0.05% trypsin and 0.85 mM EDTA in a calcium-free minimal essential medium (MEM) at 80% to 90% confluence with 1:3 to 1:4 split for three passages. 
Fibroblasts thus obtained from normal conjunctiva, abbreviated as HJF, or conjunctivochalasis, abbreviated as ChF, were seeded at a density of 0.5 to 1.0 × 106 cells per 100-mm dish. On confluence cultures were switched to a serum-free medium (D–ITS) containing DMEM supplemented with 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 50 μg/ml gentamicin, and 1.25 μg/ml amphotericin B. After 48 hours of incubation in D–ITS, 10 ng/ml of either interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α) was added to the cultures and compared with the control culture with D–ITS alone. 
Total RNA Isolation
After 5 hours of incubation with the above cytokines, cells were extracted for total RNA after two washes with ice-cold PBS and lysis with 4 M guanidium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarkosyl, and 0.1 M 2-mercaptoethanol. The cell lysate was passed through a syringe with a 20-gauge needle four to six times and transferred to a 15-ml centrifuge tube. Sequentially added to the cell lysate were 0.1 volume of 2 M sodium acetate, pH 4.0, 1 volume of phenol saturated with water, and 0.4 volume of chloroform-isoamyl alcohol mixture (49:1), with thorough mixing by inversion after the addition of each reagent. The final suspension was shaken vigorously for 15 seconds and cooled on ice for 15 minutes. Samples were centrifuged at 3000g at 4°C for 60 minutes. After centrifugation, the aqueous phase was transferred to a fresh tube, mixed with 1 volume of isopropanol, and then placed at −20°C overnight. Centrifugation at 3000g for 60 minutes was again performed, and the resultant RNA pellet was dissolved in 0.7 ml of the aforementioned guanidium solution, transferred into a 1.5 ml microtube, and precipitated with 1 volume of isopropranol at −20°C for 3 hours. After centrifugation at 12,000 rpm for 15 minutes at 4°C, the RNA pellet was washed in 75% ethanol, sedimented, vacuum-dried, and then dissolved in water. Total RNA was quantitated by measuring the absorption at 260 nm; samples were stored at −80°C before use. 
Probe Preparation
Five human DNA probes, including a 185-bp fragment of MMP-1, 480 bp of MMP-2, 155 bp of MMP-3, 551 bp of TIMP-1, and 590 bp of TIMP-2, were kindly provided by Velidi H. Rao (University of Nebraska Medical Center, Omaha, NE). Three cDNA probes (640 bp of MMP-9, 519 bp of uPA, and 498 bp of glyceraldehyde-3-phosphate dehydrogenase, GAPDH), were purified from reverse transcription–polymerase chain reaction (PCR) products by electrophoresis through a 1.2% low melting agarose gel using a Promega Wizard PCR Prep DNA purification kit according to the manufacturer’s protocol. The primers used for PCR were 1502 to 1531 (sense) and 2111 to 2140 (anti-sense) for MMP-9 (accession No. J05070), 487 to 506 (sense) and 982 to 1002 (anti-sense) for uPA (accession No. A18397), and 541 to 561 (sense) and 1018 to 1038 (anti-sense) for GAPDH (accession No. M33197). The 32P-labeled cDNA probes (1 to 2 × 109 cpm/μg DNA) were prepared with [γ-32P]–dCTP (3000 Ci/mmol) using a random primers DNA labeling system. 
Northern Hybridization
Total RNA at 20 μg/lane was electrophoresed through 1.2% agarose gels containing formaldehyde, transferred to nitrocellulose membranes, and hybridized with 32P-labeled cDNA probes at 2 to 4 × 106 cpm/3 to 8 ng/ml in the hybridization solution. After visualization of the hybridization product in the x-ray film, the 32P-label on the membrane was stripped by washing the membranes twice at 65°C for 1 hour in 5 mM Tris–HCl, pH 8.0, 0.2 mM EDTA, 0.05% sodium pyrophosphate, and 0.1× Denhardt’s solution, and rehybridized with other 32P-labeled probes. The relative amount of each mRNA of interest was determined by scanning its autoradiogram, analyzed using Gel-Pro imaging software (Media Cybernetics, Silver Spring, MD), and normalized as a ratio to that of the GAPDH mRNA band. 
MMP-1, MMP-3, and MMP-9 ELISAs
HJF and ChF were seeded at a density of 1.0 to 1.5 × 105 cells/well in a 6-well plate, and cultured for 10 days until confluence in D–FBS. They were then switched to the serum-free D–ITS medium for 24 hours, and combined with 10 ng/ml of IL-1β or TNF-α in D–ITS, and compared with the control with D–ITS alone. Each of these treatments was performed in triplicate wells. After 24 hours of incubation, the conditioned media were collected, centrifuged, and stored at −80°C until lysis with a solution containing 50 mM Tris–HCl, pH 7.6, 300 mM NaCl, and 0.5% Triton X-100 for 3 hours. After lysis, the cellular protein was collected, centrifuged, and stored at −80°C until assayed. Human MMP-1, MMP-3, and MMP-9 in each conditioned medium were determined in duplicate using their respective double-sandwiched ELISA kits according to the manufacturer’s protocol. The levels of secreted MMP-1, MMP-3, and MMP-9 were expressed as nanograms per milliter. 
Western Blot Analysis
To identify MMP and TIMP proteins present in each fibroblast-conditioned medium, Western blot analysis was performed using their specific antibodies. Conditioned media from different fibroblast cultures were adjusted to a final volume of 25 to 42 μl to represent the same quantity of cellular protein (53.4–89.7 μg, respectively) and electrophoresed at 4°C in a 4% to 15% gradient polyacrylamide gel. Except for MMP-9, all electrophoreses were performed under reducing conditions. MMP-9 was analyzed under nonreducing methods according to the manufacturer. After electrophoretic transfer to a nitrocellulose membrane at 4°C, the membrane was immersed with 0.1% (vol/vol) Tween-20 in Tris-buffered saline (100 mM Tris, 0.9% NaCl, pH 7.5; TTBS) for 30 minutes with agitation. The primary antibody (i.e., 1 μg/ml of mouse monoclonal antibody against human MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, or TIMP-2) in TTBS containing 1% horse serum was placed on each membrane and incubated at 4°C overnight. The monoclonal antibodies against human MMP-1, MMP-2, and MMP-3 recognized the latent and active form of human MMP-1, MMP-2, and MMP-3, respectively, but the monoclonal antibody against MMP-9 recognized only the latent form of MMP-9. After being washed with 3 to 4 changes of TTBS over 15 minutes, each membrane was transferred to a 1:200 diluted solution of biotinylated second antibody (goat anti-mouse IgG from Vectastain Elite ABC kit) in TTBS containing 1% horse serum and incubated for 30 minutes. After 3 to 4 washes with the same solution, they were incubated with 1:50 diluted Vectastain Elite ABC reagent conjugated with peroxidase for 30 minutes and processed for color development in 0.5 μg/ml of diaminobenzidine in 50 mM Tris–HCl, pH 7.2 containing 0.05% H2O2 for 10 to 20 minutes. 
Zymography of Metalloproteinase Activity
To determine gelatinolytic and caseinolytic activities of the various fibroblast cultures, zymography was performed using a method similar to that previously described. 24 Each conditioned medium (40 μl), after being adjusted to represent the same quantity of cellular protein (85 μg), was treated with sample buffer without boiling under nonreducing conditions. SDS—polyacrylamide gel electrophoresis was performed using a 10% polyacrylamide gel containing 0.1% gelatin or a 12% gel containing 0.1% casein. The gels were soaked in 2.5% Triton X-100 for 30 minutes at room temperature to remove the SDS and incubated in a development buffer (50 mM Tris–HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2 and 0.02% Brij-35) at 37°C overnight to allow proteinase digestion of its substrate. Gels were rinsed again in distilled water, stained with 0.5% Coomassie brilliant blue R-250 in 40% methanol and 10% acetic acid for 1 hour, and destained with 40% methanol and 10% acetic acid. Proteolytic activities appeared as clear bands of lysis against a dark background of stained gelatin or casein. To verify that the detected gelatinolytic and caseinolytic activities were specifically derived from metalloproteinases, the gels were treated with the Triton X-100 solution and the Tris/NaCl/CaCl2 development buffer containing 5 mM phenylmethylsulfonyl fluoride with or without 10 mM EDTA in the parallel experiments. 
Statistical Analysis
Data from Northern hybridization and ELISA were analyzed by paired or unpaired Student’s t-tests, as appropriate. 
Results
Transcript Expression of MMPs and TIMPs in Human Conjunctival Fibroblasts and Conjunctivochalasis Fibroblasts
Northern blot analysis showed that the 2.2-kb MMP-1 transcript was not expressed in D–ITS by two strains of normal human conjunctival fibroblasts (HJF) but was expressed by two strains of conjunctivochalasis fibroblasts (ChF; 40-fold). These results are in line with our recent report. 21 When compared with the control of D–ITS, the MMP-1 transcript expression in HJF was slightly upregulated by IL-1β (1.8-fold) and by TNF-α (1.2-fold; Fig. 1 , row 1). In ChF, however, such MMP-1 expression was more markedly upregulated by IL-1β (4.2-fold) and TNF-α (2.4-fold). 
The 1.9-kb MMP-3 transcript was not expressed by two strains of HJF in D–ITS but was expressed by two strains of ChF (sixfold), also confirming our recent report. 21 The MMP-3 transcript was negligibly upregulated by IL-1β or TNF-α in HJF (Fig. 1 , row 3). In contrast, expression of MMP-3 transcript by ChF was markedly increased by IL-1β (2.7-fold) and by TNF-α (1.2-fold). Collectively, upregulation of MMP-1 and MMP3 in HJF and ChF was differently regulated. IL-1β markedly and TNF-α to a lesser extent increased mRNA expression of MMP-1 and MMP-3. As will be shown below, Western blot analysis, ELISA and zymography data will confirm a similar pattern of differential regulation of MMP-1 and MMP-3 by IL-1β and TNF-α. 
The 3.1-kb MMP-2 transcript was uniformly expressed by both HJF and ChF without notable variation among different cytokine treatments (Fig. 1 , row 2). Likewise, there was no difference in the expression of 0.9-kb TIMP-1 and 3.5-kb TIMP-2 transcripts between HJF and ChF under these treatments (Fig. 1 , rows 4 and 5, respectively). The 2.3-kb uPA transcript was also uniformly expressed by both HJF and ChF and did not reveal any stimulation by IL-1β or TNF-α (not shown). The 2.8-kb MMP-9 transcript was not detected in either HJF or ChF by Northern hybridization (not shown). 
ELISAs of MMP-1, MMP-3, and MMP-9 Proteins Secreted in Media of HJF and ChF
The protein levels of MMP-1, MMP-3, and MMP-9 were determined by their respective ELISAs in serum-free conditioned media of two different cell strains of HJF and ChF after stimulation with IL-1β or TNF-α. The amount of MMP-1 in the conditioned medium of nonstimulated HJF (in D–ITS) was 10.8 ± 5.4 ng/ml, which was significantly increased by TNF-α (19.9 ± 11.2 ng/ml, a 1.8-fold increase, P = 0.027) but not by the addition of IL-1β (17.9 ± 13.5 ng/ml, a 1.65-fold increase, P = 0.12; Fig. 2A ). However, for ChF, the level of MMP-1 protein was significantly increased by IL-1β (252.5 ± 59.5 ng/ml, a 32-fold increase, P = 0.0014) and to a lesser extent by TNF-α (111.3 ± 13.5 ng/ml, a 1.4-fold increase, P = 0.0002) compared with the baseline of D–ITS (78.9 ± 13.6 ng/ml; Fig. 2A ). When compared with the respective levels of MMP-1 produced by HJF, these levels were more markedly upregulated by IL-1β in ChF (1.65-fold compared with a 3.2-fold increase, respectively). 
A similar pattern was observed with the protein level of MMP-3 protein. In HJF, MMP-3 was significantly upregulated by IL-1β (311.8 ± 161.8 ng/ml, a 51.5-fold increase, P = 0.0053) or by TNF-α (28.5 ± 22.1 ng/ml, an 8.5-fold increase, P = 0.0342) compared with the baseline of D–ITS (6.0 ± 3.4 ng/ml; Fig. 2B ). In ChF, expression of MMP-3 was also significantly upregulated by IL-1β (415.3 ± 19.7 ng/ml, a 22-fold increase, P < 0.0001) or by TNF-α (104.6 ± 33.0 ng/ml, a 6-fold increase, P = 0.0014) compared with the baseline of D–ITS (18.3 ± 3.9 ng/ml; Fig. 2B ). 
The pattern with the protein level of MMP-9 was different from those of MMP-1 and MMP-3. In both HJF and ChF, TNF-α stimulated the secretion of MMP-9 protein. This finding corroborated with those from Western blot analysis and casein zymography (see below). In HJF, the MMP-9 protein was markedly upregulated by TNF-α (2.9 ± 1.1 ng/ml, a 572-fold increase, P < 0.0001) and to a lesser extent by IL-1β (0.21 ± 0.14 ng/ml, a 42-fold increase, P = 0.0151) compared with the baseline of D–ITS (0.005 ± 0.004 ng/ml; Fig. 2C ). In ChF, expression of MMP-9 protein was also significantly upregulated by TNF-α (2.3 ± 1.5 ng/ml, a 119-fold increase, P = 0.0027) and to a lesser extent by IL-1β (0.13 ± 0.1 ng/ml, a 7-fold increase, P = 0.0276) compared with the baseline of D–ITS (0.02 ± 0.024 ng/ml; Fig. 2C ). Collectively, production of MMP-1, MMP3, and MMP-9 proteins in HJF and ChF was differently regulated. IL-1β predominantly increased protein expression of MMP-1 and MMP-3, whereas TNF-α predominantly increased protein expression of MMP-9. These results were further confirmed by Western blot analysis and zymography (see below). 
Protein Expression of MMPs and TIMPs by Western Blot Analysis
Western blot analysis was performed to identify and compare the protein expression of MMPs and TIMPs in serum-free conditioned media of HJF and ChF, using their specific monoclonal antibodies. Except for the antibody against MMP-9, which recognizes only the latent form, all the others recognize both latent and active forms. As shown in Figure 3 , the intensity of the protein band of each MMP and TIMP expressed by these two fibroblasts was consistent with their mRNA expression and ELISA results. The 54-kDa MMP-1 band was not produced in D–ITS by HJF but was expressed by two strains of ChF (Fig. 3A) , confirming our recent report. 21 Compared with the nonstimulated control in D–ITS, secretion of MMP-1 protein by ChF was upregulated by either IL-1β or TNF-α, and these levels were more pronounced than that of HJF in corresponding cultures (Fig. 3A) . IL-1β generally induced a stronger MMP-1 protein secretion than TNF-α in HJF or ChF. 
A similar pattern was noted for the 57-kDa band of MMP-3. MMP-3 was not produced in D–ITS by HJF but was expressed by the two strains of ChF (Fig. 3A) , also confirming our recent report. 21 The level expressed by ChF was more pronounced when stimulated by IL-1β and to a lesser extent by TNF-α, and such levels were higher than those of HJF in corresponding cultures (Fig. 3A) . The protein bands of 72-kDa MMP-2, 28-kDa TIMP-1, and 21-kDa TIMP-2 did not reveal any notable difference in either HJF or ChF under these treatments. These results were consistent with those obtained by Northern blot analysis. The protein band of 92-kDa MMP-9 was not detected in nonstimulated controls (Fig. 3B) or in samples stimulated with IL-1β (not shown) but was selectively increased under stimulation with TNF-α in both HJF and ChF (Fig. 3B) . The positive protein band noted migrated to the same position as that of a purified 92-kDa MMP-9 control (Fig. 3B , control). No significant difference was noted between HJF and ChF. These results were further confirmed by zymography (see below) and ELISA (Fig. 2)
Zymography for Gelatinolytic and Caseinolytic Activities of MMP-2, MMP-3, and MMP-9
Zymography was performed on conditioned media of HJF or ChF cultured for 24 hours in D–ITS alone or with additional IL-1β or TNF-α to verify the gelatinolytic and caseinolytic activities, respectively, of MMP-2, MMP-3, and MMP-9. Gelatin zymography was used to demonstrate the secretion of MMP-2 and MMP-9 proteins. The result showed a clear band at 72 kDa and a minor band at 68 kDa, corresponding to the latent (predominantly) and active forms of MMP-2, respectively (Fig. 4A , upper panel). Both bands were completely abolished by incubating the gel with a solution containing 10 mM EDTA (not shown). No difference in the gelatinolytic activity of 72-kDa MMP-2 was noted between HJF and ChF (Fig. 4A , upper panel). There was a clear band at 92 kDa suggestive of MMP-9 only when cell cultures were stimulated with TNF-α. To verify that this band was indeed MMP-9, the experiment was repeated in two strains of HJF and ChF and compared with a purified MMP-9 control. As shown in Figure 4B , this clear band of gelatinolytic activity upregulated by TNF-α migrated to the same position of the purified MMP-9 control. All these bands were abolished by adding 10 mM EDTA (not shown). 
Casein zymography demonstrated that secretion of MMP-3 by nonstimulated ChF was more than that by nonstimulated HJF (Fig. 4A , bottom panel), confirming our recent report. 21 This zymography revealed both glycosylated and nonglycosylated forms of MMP-3 (i.e., 59 and 57 kDa, respectively). A marked increase in this caseinolytic activity was elicited by IL-1β, and to a lesser extent by TNF-α, in two strains of ChF compared with HJF. All these bands were abolished by 10 mM EDTA (not shown). 
Discussion
The major finding of this study is that both IL-1β and TNF-α markedly increased the mRNA and protein expression of MMP-1 and MMP-3, but not of MMP-2, in ChF when compared with HJF and with their nonstimulated controls. TNF-α, but not IL-1β, further upregulated MMP-9 in both types of fibroblast. In contrast, the expression of TIMP-1, TIMP-2, and uPA remained unchanged. Production of MMP-1, MMP-3, and MMP-9 over their TIMPs favors degradation of extracellular matrices and supports the hypothesis that inflammation may play an important pathogenic role in promoting progression of conjunctivochalasis. 1  
This finding extends the results of our recent report, which showed that conjunctivochalasis fibroblasts overexpress MMP-1 and MMP-3 compared with normal conjunctival fibroblasts under the nonstimulated situation. 21 MMP-1, an interstitial collagenase, can cleave the triple helix of types I, II, and III collagen molecules; MMP-3, also known as stromelysin, has a broader substrate specificity via degrading collagens III, IV, IX, and X; laminin; proteoglycans; and fibronectin. 9 Therefore, such an overexpression of MMP-1 and MMP-3 helps explain why redundant and loose conjunctiva may develop in conjunctivochalasis. If these in vitro data can be extrapolated to the in vivo situation, our new finding predicts that both IL-1β and/or TNF-α if present might facilitate the progression of conjunctivochalasis. The finding that IL-1β and TNF-α upregulate MMP-1 and MMP-3 has also been reported in fibroblasts isolated from gingiva, 25 synovial membrane, 26 and colon 27 and in endometrial stromal cells. 10 Additionally, IL-1β and TNF-α also upregulate MMP-1 in human skin fibroblasts 28 and MMP-1 and MMP-3 mRNA and protein expression in articular fibrochondrocytes. 29  
Both MMP-2 and MMP-9 (i.e., gelatinases A and B) have a broad spectrum of degrading gelatin, basement membrane collagen, and elastin (see reviews in Refs. 7 and 9). Herein, we noted that the expression of MMP-2 transcript and protein was unchanged, but that of MMP-9 was specifically induced by TNF-α in normal conjunctival and conjunctivochalasis fibroblasts. The latter finding was demonstrated by gelatin zymography, ELISA, and Western blot analysis when experiments were carried out for 24 hours, but not by Northern hybridization when experiments were carried out for 5 hours, suggesting that protein upregulation might take a longer time. No significant difference has been noted between normal conjunctival fibroblasts and conjunctivochalasis fibroblasts in the protein expression of MMP-9. In human corneas, MMP-9 is not found in uninjured tissue but is synthesized by cells of the repairing corneal epithelium and stroma after injury. 30 Likewise, in rabbit corneas, MMP-9 is not produced in normal tissue 31 but is found in corneal epithelial and stromal layers after keratectomy. 32 To the best of our knowledge, the expression of MMP-9 by HJFs has not been reported. In this report, we noted that expression of MMP-9 by both HJF and ChF was apparent only when TNF-α was added. A similar finding has been reported in human endometrial stromal cells, 10 uterine cervical fibroblasts, 33 and dermal fibroblasts. 34 Collectively, these data indicate that TNF-α may play a unique role in MMP-9 expression by these mesenchymal cells. It has been reported that upregulation of MMP-1 and MMP-3 genes by phorbol ester and IL-1 may be via an AP-1 site. 7 Nevertheless, the MMP-9 gene comprises additional promoter sites for NFκB and SP-1, 35 which may allow further induction by TNF-α. For cultured keratinocytes, TNF-α–stimulated MMP-9 and recombinant MMP-9 bind to the cell membrane, gelatin, and type I and IV collagens. 36 This binding may cause the delay in its clearance and may enhance matrix degradation in chronic inflammation. 36 Moreover, enhanced production of MMP-9 has been noted in human foreskin fibroblasts after treatment with basic calcium phosphate crystals, 37 in human uterine cervical fibroblasts after treatment with 12-O-tetradecanoylphorbol, 33 in human dermal skin fibroblasts after UV radiation, 38 in corneal stroma after surgical trauma, 30 and in granulation tissue of oral mucosa wounds. 39 Therefore, we speculate that matrix degradation in conjunctivochalasis may be further promoted when TNF-α is produced. 
Proinflammatory cytokines such as IL-1β and TNF-α can be produced by stromal fibroblasts 40 and such inflammatory cells as macrophages and lymphocytes. 41 42 43 The conjunctival epithelium has been shown to secrete TNF-α in response to lipopolysaccharides. 44 Production of IL-1β, TNF-α, or both is increased by surgical trauma 45 46 and UV irradiation. 40 Furthermore, IL-1β has been found in the normal tear fluid. 47 It has been shown that ocular irritation is aggravated 44 when the tear clearance is delayed, 48 leading to accumulation of proinflammatory cytokines. 49 Because tear clearance is frequently delayed in conjunctivochalasis, 50 one may imagine that the levels of IL-1β and TNF-α in the tears may be elevated. Indeed, preliminary results of a tear analysis of patients suffering of conjunctivochalasis compared with normal human conjunctival fibroblasts showed a higher concentration of IL-1β and IL-1α (Meller, D and Tseng, SCG, unpublished observations, September 1999). Collectively, chronic inflammation of the ocular surface, triggered by UV irradiation, tear deficiency, and microtrauma from climatic or occupational environments, may be one important factor in contributing progression of conjunctivochalasis. Alternatively, other factors such as growth factors or extracellular matrices could be responsible for the upregulation of MMP expression. Further experiments analyzing the source of cytokines and their involvement in the pathogenesis of conjunctivochalasis are needed to support the hypothesis that ocular inflammation as an important denominator might trigger enzyme-linked matrix degradation in conjunctivochalasis. 
We do not know whether such an overexpression of MMP-1 and MMP-3 represents an altered genotype or epigenetic phenotype of conjunctivochalasis fibroblasts. Hours after exposure to UVB radiation, the skin also shows increased mRNAs, proteins, and activities of MMP-1, MMP-3, and MMP 9 but not MMP-2. 22 Additionally, UVB radiation and PUVA treatments increase expression of MMP-2 and MMP-9 in human skin fibroblasts, and this finding has been linked to actinic damages that lead to deposition of elastotic material and degeneration of collagen. 38 As stated above, acute UV-irradiation exposure upregulates production of proinflammatory cytokines in the cornea. 40 Because photoaged skin shows a similar pathologic change of elastotic degeneration, which is also found in pingueculae and pterygium (diseases associated with UV exposure, 4 6 and because pingueculae are frequently associated with conjunctivochalasis, 6 future studies are needed to investigate whether conjunctivochalasis may be causatively linked with UV exposure leading to overexpression of MMP-1, MMP-3, and MMP-9. 
 
Figure 1.
 
MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 mRNA expression by two human conjunctival fibroblasts (HJF1 and HJF2) and two conjunctivochalasis fibroblasts (ChF1 and ChF2) treated with different cytokines for 4 hours. HJF and ChF were grown to confluence in D–FBS and switched to serum-free D–ITS for 48 hours before adding 10 ng/ml of IL-1β or TNF-α. Total RNA was isolated and individually hybridized with 32P-labeled specific cDNA probes. The same filter was then hybridized with GAPDH probe as a control.
Figure 1.
 
MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 mRNA expression by two human conjunctival fibroblasts (HJF1 and HJF2) and two conjunctivochalasis fibroblasts (ChF1 and ChF2) treated with different cytokines for 4 hours. HJF and ChF were grown to confluence in D–FBS and switched to serum-free D–ITS for 48 hours before adding 10 ng/ml of IL-1β or TNF-α. Total RNA was isolated and individually hybridized with 32P-labeled specific cDNA probes. The same filter was then hybridized with GAPDH probe as a control.
Figure 2.
 
Levels of secreted MMP-1 (A), MMP-3 (B), or MMP-9 (C) measured by their respective double-sandwiched ELISAs in conditioned media of HJF or ChF collected after 24 hours in D–ITS, and stimulated with IL-1β or TNF-α. (A) *P = 0.0014, **P = 0.0002, ***P = 0.027; (B) *P < 0.005, **P = 0.034, ***P < 0.0001, ****P = 0.0014; (C) *P < 0.0001, **P = 0.015, ***P = 0.0027, ****P = 0.028.
Figure 2.
 
Levels of secreted MMP-1 (A), MMP-3 (B), or MMP-9 (C) measured by their respective double-sandwiched ELISAs in conditioned media of HJF or ChF collected after 24 hours in D–ITS, and stimulated with IL-1β or TNF-α. (A) *P = 0.0014, **P = 0.0002, ***P = 0.027; (B) *P < 0.005, **P = 0.034, ***P < 0.0001, ****P = 0.0014; (C) *P < 0.0001, **P = 0.015, ***P = 0.0027, ****P = 0.028.
Figure 3.
 
(A) Western blot analysis of the protein expression of MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 by conjunctival (HJF) and conjunctivochalasis (ChF1 and ChF2) fibroblasts in conditioned media of unstimulated cultures (D–ITS) or after stimulation with IL-1β or TNF-α. (B) Western blot analysis of the protein expression of MMP-9 by two strains of human conjunctival fibroblasts (HJF1 and HJF2) and by two strains of conjunctivochalasis fibroblasts (ChF1 and ChF2) in their conditioned media of unstimulated cultures (D–ITS) or after stimulation with TNF-α. The control of a purified MMP-9 was added in lane 10. STD, standard.
Figure 3.
 
(A) Western blot analysis of the protein expression of MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 by conjunctival (HJF) and conjunctivochalasis (ChF1 and ChF2) fibroblasts in conditioned media of unstimulated cultures (D–ITS) or after stimulation with IL-1β or TNF-α. (B) Western blot analysis of the protein expression of MMP-9 by two strains of human conjunctival fibroblasts (HJF1 and HJF2) and by two strains of conjunctivochalasis fibroblasts (ChF1 and ChF2) in their conditioned media of unstimulated cultures (D–ITS) or after stimulation with TNF-α. The control of a purified MMP-9 was added in lane 10. STD, standard.
Figure 4.
 
(A) Zymograms of MMP-2, MMP-3, and MMP-9 activity in HJF, ChF1, or ChF2. The gelatinolytic activity of MMP-2 and MMP-9 (upper panel) and the caseinolytic activity of MMP-3 (lower panel) is expressed in conditioned media of unstimulated cultures (D–ITS) or after stimulation with IL-1β or TNF-α. (B) Additional zymogram of MMP-9 activity in conditioned media of HJF1, HJF2, ChF1, or ChF2 together with a purified MMP-9 control (lane 2). Unstimulated cultures (D–ITS) do not develop any gelatinolytic activity of MMP-9. After stimulation with TNF-α, all samples show a gelatinolytic activity at the same 92-kDa–positive band as the purified MMP-9 control. STD, standard.
Figure 4.
 
(A) Zymograms of MMP-2, MMP-3, and MMP-9 activity in HJF, ChF1, or ChF2. The gelatinolytic activity of MMP-2 and MMP-9 (upper panel) and the caseinolytic activity of MMP-3 (lower panel) is expressed in conditioned media of unstimulated cultures (D–ITS) or after stimulation with IL-1β or TNF-α. (B) Additional zymogram of MMP-9 activity in conditioned media of HJF1, HJF2, ChF1, or ChF2 together with a purified MMP-9 control (lane 2). Unstimulated cultures (D–ITS) do not develop any gelatinolytic activity of MMP-9. After stimulation with TNF-α, all samples show a gelatinolytic activity at the same 92-kDa–positive band as the purified MMP-9 control. STD, standard.
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Figure 1.
 
MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 mRNA expression by two human conjunctival fibroblasts (HJF1 and HJF2) and two conjunctivochalasis fibroblasts (ChF1 and ChF2) treated with different cytokines for 4 hours. HJF and ChF were grown to confluence in D–FBS and switched to serum-free D–ITS for 48 hours before adding 10 ng/ml of IL-1β or TNF-α. Total RNA was isolated and individually hybridized with 32P-labeled specific cDNA probes. The same filter was then hybridized with GAPDH probe as a control.
Figure 1.
 
MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 mRNA expression by two human conjunctival fibroblasts (HJF1 and HJF2) and two conjunctivochalasis fibroblasts (ChF1 and ChF2) treated with different cytokines for 4 hours. HJF and ChF were grown to confluence in D–FBS and switched to serum-free D–ITS for 48 hours before adding 10 ng/ml of IL-1β or TNF-α. Total RNA was isolated and individually hybridized with 32P-labeled specific cDNA probes. The same filter was then hybridized with GAPDH probe as a control.
Figure 2.
 
Levels of secreted MMP-1 (A), MMP-3 (B), or MMP-9 (C) measured by their respective double-sandwiched ELISAs in conditioned media of HJF or ChF collected after 24 hours in D–ITS, and stimulated with IL-1β or TNF-α. (A) *P = 0.0014, **P = 0.0002, ***P = 0.027; (B) *P < 0.005, **P = 0.034, ***P < 0.0001, ****P = 0.0014; (C) *P < 0.0001, **P = 0.015, ***P = 0.0027, ****P = 0.028.
Figure 2.
 
Levels of secreted MMP-1 (A), MMP-3 (B), or MMP-9 (C) measured by their respective double-sandwiched ELISAs in conditioned media of HJF or ChF collected after 24 hours in D–ITS, and stimulated with IL-1β or TNF-α. (A) *P = 0.0014, **P = 0.0002, ***P = 0.027; (B) *P < 0.005, **P = 0.034, ***P < 0.0001, ****P = 0.0014; (C) *P < 0.0001, **P = 0.015, ***P = 0.0027, ****P = 0.028.
Figure 3.
 
(A) Western blot analysis of the protein expression of MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 by conjunctival (HJF) and conjunctivochalasis (ChF1 and ChF2) fibroblasts in conditioned media of unstimulated cultures (D–ITS) or after stimulation with IL-1β or TNF-α. (B) Western blot analysis of the protein expression of MMP-9 by two strains of human conjunctival fibroblasts (HJF1 and HJF2) and by two strains of conjunctivochalasis fibroblasts (ChF1 and ChF2) in their conditioned media of unstimulated cultures (D–ITS) or after stimulation with TNF-α. The control of a purified MMP-9 was added in lane 10. STD, standard.
Figure 3.
 
(A) Western blot analysis of the protein expression of MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 by conjunctival (HJF) and conjunctivochalasis (ChF1 and ChF2) fibroblasts in conditioned media of unstimulated cultures (D–ITS) or after stimulation with IL-1β or TNF-α. (B) Western blot analysis of the protein expression of MMP-9 by two strains of human conjunctival fibroblasts (HJF1 and HJF2) and by two strains of conjunctivochalasis fibroblasts (ChF1 and ChF2) in their conditioned media of unstimulated cultures (D–ITS) or after stimulation with TNF-α. The control of a purified MMP-9 was added in lane 10. STD, standard.
Figure 4.
 
(A) Zymograms of MMP-2, MMP-3, and MMP-9 activity in HJF, ChF1, or ChF2. The gelatinolytic activity of MMP-2 and MMP-9 (upper panel) and the caseinolytic activity of MMP-3 (lower panel) is expressed in conditioned media of unstimulated cultures (D–ITS) or after stimulation with IL-1β or TNF-α. (B) Additional zymogram of MMP-9 activity in conditioned media of HJF1, HJF2, ChF1, or ChF2 together with a purified MMP-9 control (lane 2). Unstimulated cultures (D–ITS) do not develop any gelatinolytic activity of MMP-9. After stimulation with TNF-α, all samples show a gelatinolytic activity at the same 92-kDa–positive band as the purified MMP-9 control. STD, standard.
Figure 4.
 
(A) Zymograms of MMP-2, MMP-3, and MMP-9 activity in HJF, ChF1, or ChF2. The gelatinolytic activity of MMP-2 and MMP-9 (upper panel) and the caseinolytic activity of MMP-3 (lower panel) is expressed in conditioned media of unstimulated cultures (D–ITS) or after stimulation with IL-1β or TNF-α. (B) Additional zymogram of MMP-9 activity in conditioned media of HJF1, HJF2, ChF1, or ChF2 together with a purified MMP-9 control (lane 2). Unstimulated cultures (D–ITS) do not develop any gelatinolytic activity of MMP-9. After stimulation with TNF-α, all samples show a gelatinolytic activity at the same 92-kDa–positive band as the purified MMP-9 control. STD, standard.
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