January 2003
Volume 44, Issue 1
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Immunology and Microbiology  |   January 2003
Effects of Th2 Cytokines on Expression of Collagen, MMP-1, and TIMP-1 in Conjunctival Fibroblasts
Author Affiliations
  • Andrea Leonardi
    From the Department of Neuroscience, Ophthalmology and Ocular Inflammation Unit; the
    Department of Laboratory Medicine, University of Padua, Padua, Italy.
  • Roberta Cortivo
    Department of Histology, Microbiology and Medical Biotechnologies; and the
  • Iva Fregona
    From the Department of Neuroscience, Ophthalmology and Ocular Inflammation Unit; the
  • Mario Plebani
    Department of Laboratory Medicine, University of Padua, Padua, Italy.
  • Antonio G. Secchi
    From the Department of Neuroscience, Ophthalmology and Ocular Inflammation Unit; the
  • Giovanni Abatangelo
    Department of Histology, Microbiology and Medical Biotechnologies; and the
Investigative Ophthalmology & Visual Science January 2003, Vol.44, 183-189. doi:10.1167/iovs.02-0420
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      Andrea Leonardi, Roberta Cortivo, Iva Fregona, Mario Plebani, Antonio G. Secchi, Giovanni Abatangelo; Effects of Th2 Cytokines on Expression of Collagen, MMP-1, and TIMP-1 in Conjunctival Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2003;44(1):183-189. doi: 10.1167/iovs.02-0420.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To determine whether cytokines involved in chronic allergic conjunctival disorders may affect formation of giant papillae and tissue remodeling.

methods. Conjunctival fibroblast cultures were challenged with different concentrations of human recombinant interleukin (IL)-4, IL-13, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Procollagens I (PIP) and III (PIIIP), matrix metalloproteinase (MMP)-1 and -9, and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants, and their respective mRNAs were evaluated by RT-PCR.

results. IL-4 and -13 (10 ng/mL) significantly increased production and expression of PIP compared with nonstimulated cells, whereas IFN-γ elicited the opposite effect, at both the protein and mRNA levels. Both IL-4 and -13 significantly decreased production of MMP-1 and increased that of TIMP-1, whereas TNF-α increased production of MMP-1 and -9. Expression of MMP-1 was reduced by IL-4 and increased by the other tested cytokines, whereas expression of TIMP-1 was increased by all tested cytokines.

conclusions. IL-4 and -13 increased production of collagen and modified the equilibrium between MMP-1 and its inhibitor, TIMP-1. These effects were partially opposed by IFN-γ and TNF-α.

Vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) are severe chronic allergic inflammatory diseases associated with pronounced infiltration of various inflammatory cells including lymphocytes, mainly of the T-helper (Th)2 subtype 1 2 3 ; eosinophils; and mast cells. The Th2-derived cytokines, interleukin (IL)-4 and -13, have been shown to be essential to production of IgE antibodies in tissue eosinophilia and in airway remodeling in asthma. 4 Increased levels of IL-4 have been reported in tears in both VKC and AKC. 5 IL-13 has been shown to be increased in tear cells of patients with seasonal allergic conjunctivitis 6 and in tears of patients with VKC and AKC patients (Leonardi A, manuscript in preparation). Interferon (IFN)-γ, the main product of Th1 lymphocytes, antagonizes Th2-related effects and is less expressed in allergic conjunctivitis, 2 3 7 whereas tumor necrosis factor (TNF)-α is a proinflammatory cytokine that is increased in VKC tears and tissues. 8  
Tissue remodeling and formation of giant papillae are some of the consequences of chronic allergic eye disorders. Remodeling involves both production and deposition of extracellular matrix (ECM) components, as well as degradation and clearance of newly synthesized products. Immunohistochemical studies have shown the increased deposition of collagens in giant papillae in VKC, possibly as a result of increased expression of growth factors, 8 which may stimulate resident fibroblasts to produce extracellular matrix proteins. The predominant collagens present in giant papillae are types I and III. 9 Both IL-4 and -13 have been reported to activate lung and skin fibroblasts to produce collagens I and III, 10 11 whereas IFN-γ may have an inhibitory effect in fibroblasts. 12 Overproduction and deposition of collagens and tissue remodeling may be modulated by matrix metalloproteinases (MMPs) and their physiological inhibitors, tissue inhibitors of metalloproteinases (TIMPs). 13 MMPs are a family of zinc- and calcium-dependent enzymes involved in many physiological and pathologic processes, including progression of tumors, metastasis, inflammatory diseases, and wound healing. Of the 17 members of the human MMP family that have been described, collagenase I (MMP-1), gelatinase A and B (MMP-2 and -9), stromelysin (MMP-3), and matrilysin (MMP-7) are those most involved in anterior segment disease and wound healing. These enzymes are produced and secreted by a variety of cells, including epithelial cells, inflammatory cells, and conjunctival fibroblasts 14 and are regulated by several cytokines, both in physiological conditions such as wound healing and in diseases states. 15 16 MMP-1 can cleave the triple helix of interstitial collagen types I, II, and III. Stromelysin and matrilysin are involved in degrading a variety of ECM components such as proteoglycans, fibronectin, and laminin. Gelatinase A and B are involved in cleaving collagen types IV (the main component of basal membrane), V, VII, and X; fibronectin; laminin; elastin; and collagen degradation products, and TIMP-1 is the natural inhibitor of both collagenase I and gelatinase. 
Because collagen type I is the most important collagen present in VKC conjunctiva, and basal membrane remodeling is also involved in VKC, this study focused on MMP-1, MMP-9, and their inhibitor, TIMP-1. Thus, the purpose of the present study was to determine the effects of Th1- and Th2-type cytokines on conjunctival fibroblasts by measuring the production and expression of collagen and to determine their effects on the expression, production, and activity of MMP-1, MMP-9, and their inhibitor, TIMP-1. 
Materials and Methods
Cell Cultures
Biopsy specimens were obtained from the upper tarsal conjunctiva of two patients with VKC and from the lower tarsal conjunctiva of two age-matched normal subjects who underwent surgery for strabismus. Informed consent was obtained from subjects after explanation of the nature and the possible consequences of the study. Research adhered to the tenets of the Declaration of Helsinki. Fibroblast cultures were obtained as already described. 17 Briefly, specimens were washed, cut in small pieces, seeded in culture plates (Nunclon Multidishes; Nunc, Roskilde, Denmark) containing 100 mL Ham’s F12 medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal calf serum (FCS; Sigma) and antibiotics (penicillin 100 U/mL, streptomycin 100 mg/mL, l-glutamine 2 mM; Sigma), and incubated at 37°C in 5% carbon dioxide in a humidified air atmosphere. Fibroblasts were subcultured with 0.05% trypsin and replated into 24-well plates (>95% vitality). Cells were characterized morphologically and stained positively with vimentin and negatively with cytokeratins. Third- to seventh-passage fibroblasts were used for experiments. 
Procollagens, MMP-1, MMP-9, and TIMP-1
To evaluate the effects of IL-4 and -13 on fibroblast synthesis of procollagens, 6500 cells/well were seeded into 24-well plates in the presence of 10% FCS. At confluence, the medium in each well was replaced with 500 μL of medium containing 0.4% FCS and fresh ascorbic acid (50 μg/mL). After 24 hours, the medium was refreshed with media supplemented with different concentrations (0, 0.1, 1, 10 ng/mL) of human recombinant IL-4, IL-13, IFNγ, and TNF-α (R&D Systems, Abingdon, UK). After incubation times of 24 or 48 hours, the media were removed, centrifuged, and stored at −20°C. Three culture wells were used for each treatment. Experiments were repeated twice for each fibroblast line. To evaluate the production and activity of MMPs, VKC-derived fibroblasts were stimulated with the above-mentioned cytokines (0, 1, 10, and 100 ng/mL) for 24 hours. 
Using antibodies to the respective N-terminal procollagen peptides, procollagen type I (PIP) was determined in culture medium using a radioimmunoassay (RIA; Orion Diagnostica, Espoo, Finland), and PIIIP, by two-stage sandwich assay (Cis Bio International, Bagnois-sur-Cèze, France). Total proteins were measured by microassay (Biorad, Munich, Germany). Procollagens were expressed as PIP and PIIIP over total protein in culture medium. Human pro-MMP-1, pro-MMP-9, and TIMP-1 were measured in duplicate by, using their respective enzyme-linked immunoassay (ELISA) kits (Chemicon International, Temecula, CA), according to the manufacturer’s protocol. 
The sensitivities of the assays were as follow, PIP, 25 μg/L; PIIIP, 0.1 U/mL; pro-MMP-1, 0.11 ng/mL; pro-MMP-9, 3.1 ng/mL; and TIMP-1, 1.2 ng/mL. 
MMP Activity Assay
MMP activity was measured in media of cultures stimulated with cytokines for 24 hours using the MMP-1 and -9 assay systems (Chemicon) according to the manufacturer’s instructions. These colorimetric assays capture MMPs present in culture media and measure their activity in cleaving a modified prodetection enzyme, along with the subsequent cleavage of its chromogenic peptide substrate. Data are expressed as units per nanogram MMP. 
Proliferation Assay
To determine the proliferation rate of human conjunctival fibroblasts in response to cytokines, the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test (Sigma) was performed as previously described. 17 Two thousands cells per well were seeded into 96-well tissue culture plates and treated with different concentrations of IL-4, IL-13, IFN-γ, or TNF-α (0, 0.1, 1, or 10 ng/mL). After an incubation of 24 hours, the MTT test was performed. The optical density of each well was measured by automatic plate reader with a 560-nm test wavelength and a 690-nm reference wavelength. In each experiment, eight wells were used for the same concentration. Experiments were repeated twice for each fibroblast line. 
RNA Isolation and RT-PCR
Total RNA was extracted from cells cultured in the monophasic solution of phenol and guanidine isothiocyanate (TRIzol reagent; Life Technologies-GibcoBRL, Milan, Italy). Briefly, the cells were lysed by addition of 1.0 mL of extraction reagent, and total RNA was subsequently isolated according to the manufacturer’s instructions. Complementary DNA was synthesized from 500 ng total RNA per sample with 50 minutes’ incubation at 37°C, using Moloney murine leukemia virus reverse transcriptase (Life Technologies-Gibco BRL) and oligo (dT) priming. Amplification was performed in a programmable thermal controller (PTC-100; MJ Research Inc., Watertown, MA), with recombinant Taq DNA polymerase (Applied Biosystems, Foster City, CA) and the specific primer pairs reported in Table 1 . The parallel amplification of cDNA for the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. To enable a semiquantitative comparison between samples, serial threefold dilutions of cDNA (corresponding to 100–1.2 ng of total) were subjected to increasing PCR cycles (23–40), to define the linear amplification range for each primer set. All cDNA samples were then amplified at the optimal cycle number for each gene of interest: MMP-1 and TIMP-1, 30 cycles (45 seconds at 94°C, 1 minute at 59°C, 1.5 minutes at 72°C) preceded by a denaturation step at 94°C for 10 minutes, and terminated with an elongation step at 72°C for 5 minutes); collagen I and the housekeeping gene GAPDH, 24 cycles (1 minute at 94°C, 1 minute at 59°C, 1 minute at 72°C) preceded by a denaturation step at 94°C for 10 minutes, and terminated with an elongation step at 72°C for 5 minutes. The resultant bands were visualized on a 1% agarose gel stained with ethidium bromide and compared with HindIII lambda DNA to confirm the predicted size. Results are expressed as a percentage of the signal obtained from a parallel amplification of GAPDH in the same RT product. 
Statistics
The effects of cytokines on fibroblast cultures were analyzed by ANOVA with a post hoc analysis (Fisher’s protected least-significant difference [PLSD] test). Data are presented as the mean ± SD. Differences between cell lines were compared with the unpaired Student’s t-test. 
Results
Procollagens, MMP-1, MMP-9, and TIMP-1
Cultures of human conjunctival fibroblasts from patients with VKC or normal subjects spontaneously produced PIP and PIIIP. After stimulation for 24 hours with different concentrations of IL-4 and -13 (0, 0.1, 1, and 10 ng/mL), the production of PIP increased, with a tendency to show a dose-dependent results that were significant only at the higher dose (P < 0.05). Conversely, fibroblasts treated with either 10 ng/mL IFNγ or 10 ng/mL TNF-α significantly reduced PIP production compared with either nontreated, IL-4–, or IL-13-treated cells. Data were consistent among different experiments and different cultures. Experiments performed with 24- and 48-hour incubation times and cytokine concentrations of 10 ng/mL showed a similar tendency (Fig. 1)
In both VKC- and normal subject–derived cultures, PIIIP levels in medium were not modified by IL-4, IL-13, or TNF-α, but were significantly reduced by 1 ng/mL and 10 ng/mL IFNγ at 24 hours (P < 0.01; Fig. 2 ). 
VKC-derived fibroblast cultures were more responsive to cytokines in terms of PIP production than were normal cell cultures (Table 2) . Thus, the subsequent experiments were performed with VKC-derived cells. VKC-derived fibroblast culture medium contained detectable amounts of pro-MMP-1 and TIMP-1. Levels of pro-MMP-1 were significantly reduced by exposure for 24 hours to high levels of IL-4 and -13 (100 ng/mL) or 10 ng/mL IFNγ, whereas 1 and 10 ng/mL TNF-α increased the levels of pro-MMP-1 (Fig. 3A) . The combined treatments with 100 ng/mL IL-4 and 10 ng/mL TNF-α or 100 ng/mL IL-13 and 10 ng/mL TNF-α also induced a significant reduction of MMP-1 levels in culture medium compared with the control (P < 0.01 and P < 0.05, respectively; data not shown). Conversely, the same concentrations of IL-4 and -13 significantly increased production of TIMP-1 in culture medium compared with nontreated fibroblasts (P < 0.05; Fig. 3B ). Both IL-4 and -13 significantly increased the ratio of TIMP-1 to MMP-1 (P < 0.05; Fig. 3C ). 
Pro-MMP-9 was not detected in any sample and increased only when cells were treated with 10 ng/mL TNF-α (Fig. 3D) . Data were consistent among different experiments and in the two different VKC-derived cultures. 
Collagenase and Gelatinase Activity
Collagenase activity was very low in VKC-derived fibroblast cultures treated with only medium for 24 hours. IL-4 at 100 ng/mL induced a 45-fold increase in MMP-1 activity, and TNF-α at 10 ng/mL, a 40-fold increase (Fig. 4A) . After a 24-hour exposure, IL-4 at 100 ng/mL and IL-13 at 100 ng/mL significantly reduced the activity of MMP-9, whereas IFNγ and TNF-α did not (Fig. 4B)
Cell Proliferation
Treatment with various concentrations of IL-4 and -13 (0, 0.1, 1, and 10 ng/mL) for 24 hours showed a significant positive effect on proliferation of VKC-derived fibroblast (Fig. 5) . In contrast, IFNγ (0.1, 1, and 10 ng/mL) significantly reduced cell growth compared with untreated cells (P < 0.05). TNF-α did not affect fibroblast proliferation. At 48 hours, only 10 ng/mL IFNγ maintained its negative effect on cell growth, whereas the other treatments did not show significant effects (data not shown). 
RT-PCR
The effects on the expression of collagen I, MMP-1, and TIMP-1 mRNA in VKC conjunctival fibroblasts were evaluated in cell lysates and quantified by densitometry after a 24-hour exposure to different concentrations of cytokines. Expression of collagen I mRNA was increased by 10 and 100 ng/mL of both IL-4 and -13, reduced by 10 ng/mL IFNγ, and unchanged by TNF-α (Fig. 6A) . Expression of MMP-1 mRNA was increased by 100 ng/mL IL-13, but reduced by IL-4 compared with the control. Both IFNγ and TNF-α (100 ng/mL), increased expression of MMP-1. Expression of TIMP-1 was increased by 10 ng/mL IL-4 (Fig. 6B) and 100 ng/mL IL-13 (Fig. 6C) . Both IFNγ and TNF-α also increased expression of TIMP-1. 
Discussion
Conjunctival fibroblasts are now known to be involved in the development of Th2-mediated chronic allergic diseases such as VKC and AKC. Giant papillae and subepithelial fibrosis are a result not only of inflammatory cell infiltration, but also of collagen deposition. 8 Previous studies showed that histamine, the main allergic mediator, can increase production of collagen 17 and proinflammatory cytokines by conjunctival fibroblasts. 18 In the present study, results showed that IL-4 and -13, which are produced by Th2 lymphocytes, may be active not only in inflammatory cells but also in fibroblasts, contributing to their transformation into effectors of the fibrotic and remodeling process. Although both IL-4 and -13 increased the production and expression of collagen, IFNγ, which is produced by Th1 lymphocytes, showed an opposite effect. The imbalance between Th1 and Th2 cells, which is pathogenic to the development of allergy, 4 may also be involved in collagen production and distribution in the conjunctiva. 
Both IL-4 and -13 reduced MMP-1 at the protein level but increased its inhibitor, TIMP-1. These data were in line with RT-PCR results, the only difference being that high levels of IL-13 increased expression of both MMP-1 and TIMP-1. This suggests that an imbalance between deposition and degradation of collagen is involved in the formation of giant papillae, conjunctival fibrosis, and tissue remodeling. Conversely, TNF-α augmented the production and expression of MMP-1 without changing TIMP-1 protein levels. Cotreatment with IL-4 or -13 and TNF-α still resulted in decreased production of MMP-1, suggesting that the effect of the Th2-derived cytokines was prevalent in the present model. TNF-α has been reported to upregulate expression of MMP-1 markedly and production in fibroblasts derived from different tissues. 13 19 20 IL-4 has been shown to upregulate the expression of collagen I mRNA by lung and skin fibroblasts. 9 10 Furthermore, in a subpopulation of murine lung fibroblasts, IL-4 was shown to increase production of both collagen I and TIMP-1 without affecting MMP-1. 11 IL-13 has also been suggested to play a relevant role in fibrosis, because its inhibitors were shown to prevent the development of Th2-driven hepatic fibrosis in infected mice. 21 Recently, IL-4 and -13 were shown to upregulate production of TIMP-1 and to inhibit production of MMP-1 and -3 in human skin fibroblast cultures. 10 TIMP-1 is a fundamental and ubiquitous sialoglycoprotein with specific inhibitory effects on interstitial collagenase, gelatinase A and B, and stromelysin, but not on bacterial collagenase and thermolysin. 12 TIMP-1 also has potent growth-promoting effects on a wide range of cells, 22 including corneal epithelial cells. 23 If the increases of TIMP-1 levels and expression are confirmed in patients with chronic ocular allergic diseases, it may be an important growth-promoting factor involved in conjunctival fibrosis. 
In the present study, gelatinase B (MMP-9) was increased in culture medium only after stimulation with TNF-α. Similar results were obtained in conjunctivochalasis fibroblasts 13 and uterine cervical fibroblasts. 20 TNF-α has been found in tears and tissues in VKC- 8 24 and conjunctiva-derived mast cells in vitro. 25 Besides lymphocytes and macrophages, eosinophils and epithelial cells may be a source of TNF-α. 26 27 Gelatinase B, which is implicated in the degradation of basement membrane, in the wound-healing process, and in chronic inflammations, was found increased in sputum and in the bronchoalveolar lavage of asthmatic patients 28 29 in which inflammatory cells, eosinophils, and alveolar macrophages, are probably responsible for its production. Tear levels of MMPs will be evaluated further to study a possible role of these enzymes in the development of chronic ocular allergic disorders. 
That effects of IL-4 and -13 on fibroblasts are supported by the presence of specific high-affinity receptors on human lung and skin fibroblasts. 30 31 Recently, the high-affinity and functional IL-4 receptor complex was also identified in human corneal fibroblast cultures. 32 Both IL-4 and -13 trigger the tyrosine phosphorylation of several proteins; activate specific signal transduction and activator of transcription (STAT)1, -3, and -6; and upregulate the transcription of c-fos, c-jun, and c-myc proto-oncogenes. 30 31 32 The same mechanisms may be activated in the present model, in which the ultimate outcome—that is, the increased production of collagen—was demonstrated. 
In summary, these findings support the theory that the Th2-type cytokines IL-4 and -13 and the imbalance between Th1 and Th2 cytokines characteristic of allergic disorders are involved in tissue remodeling that occurs by stimulation of production of collagen and alteration of the equilibrium between MMP and TIMP. 
 
Table 1.
 
Sequences of PCR Primer Sets
Table 1.
 
Sequences of PCR Primer Sets
Factor Primer Sequence Product Size (bp)
MMP-1 5′-CGACTCTAGAAACACAAGAGCAAGA-3′ (sense) 786
5′-AAGGTTAGCTTACTGTCACACGCTT-3′ (antisense)
TIMP-1 5′-ATCCTGTTGTTGCTGTGGCTGATAG-3′ (sense) 667
5′-TGCTGGGTGGTAACTCTTTATTTCA-3′ (antisense)
Collα2(I) 5′-GGTGGTTATGACTTTGGTTAC-3′ (sense) 702
5′-CAGGCGTGATGGCTTATTTGT-3′ (antisense)
Figure 1.
 
PIP levels in culture medium of conjunctival fibroblasts from one of the VKC-derived cultures treated for 24 or 48 hours with 10 ng/mL of the tested cytokines. At 24 hours, both IL-4 and -13 significantly increased PIP, whereas IFNγ and TNF-α significantly reduced it. At 48 hours, only IL-4 and IFNγ significantly modified production of PIP. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 1.
 
PIP levels in culture medium of conjunctival fibroblasts from one of the VKC-derived cultures treated for 24 or 48 hours with 10 ng/mL of the tested cytokines. At 24 hours, both IL-4 and -13 significantly increased PIP, whereas IFNγ and TNF-α significantly reduced it. At 48 hours, only IL-4 and IFNγ significantly modified production of PIP. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 2.
 
PIIIP levels in culture medium of conjunctival fibroblasts derived from one patient with VKC and treated for 24 hours with different concentrations of cytokines (0, 0.1, 1, 10 ng/mL): 1 and 10 ng/mL IFNγ significantly reduced PIIIP. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 2.
 
PIIIP levels in culture medium of conjunctival fibroblasts derived from one patient with VKC and treated for 24 hours with different concentrations of cytokines (0, 0.1, 1, 10 ng/mL): 1 and 10 ng/mL IFNγ significantly reduced PIIIP. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Table 2.
 
Procollagen I Production from VKC-Derived and Normal Subject–Derived Fibroblasts after a 24-Hour Exposure to Different Cytokines
Table 2.
 
Procollagen I Production from VKC-Derived and Normal Subject–Derived Fibroblasts after a 24-Hour Exposure to Different Cytokines
Control IL-4 (10 ng/mL) IL-13 (10 ng/mL) IFN-γ (10 ng/mL) TNFα (10 ng/mL)
VK-F 422 ± 104 550 ± 38 614 ± 28 337 ± 36 309 ± 21
NS-F 309 ± 19 393 ± 11 387 ± 29 105 ± 42 154 ± 6
P NS 0.002 0.001 0.002 0.001
Figure 3.
 
Levels of (A) MMP-1, (B) TIMP-1, and (D) MMP-9 and (C) the TIMP-1-to-MMP-1 ratio in culture medium of VKC conjunctival fibroblasts treated for 24 hours with 100 ng/mL IL-4, 100 ng/mL IL-13, 10 ng/mL IFNγ or 10 ng/mL TNF-α. IL-4 and -13 significantly reduced MMP-1 and increased TIMP-1 and the ratio of TIMP-1 to MMP-1, whereas only TNF-α increased MMP-9. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 3.
 
Levels of (A) MMP-1, (B) TIMP-1, and (D) MMP-9 and (C) the TIMP-1-to-MMP-1 ratio in culture medium of VKC conjunctival fibroblasts treated for 24 hours with 100 ng/mL IL-4, 100 ng/mL IL-13, 10 ng/mL IFNγ or 10 ng/mL TNF-α. IL-4 and -13 significantly reduced MMP-1 and increased TIMP-1 and the ratio of TIMP-1 to MMP-1, whereas only TNF-α increased MMP-9. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 4.
 
Activity of (A) MMP-1 and (B) -9 in VKC fibroblast cultures exposed to different cytokines for 24 hours. The activity of MMP-1 was increased by 100 ng/mL IL-4 and 10 ng/mL of TNF-α. The activity of MMP-9 was reduced by 100 ng/mL IL-4 (*P < 0.05, compared with the control [CT], i.e., cells grown in medium alone).
Figure 4.
 
Activity of (A) MMP-1 and (B) -9 in VKC fibroblast cultures exposed to different cytokines for 24 hours. The activity of MMP-1 was increased by 100 ng/mL IL-4 and 10 ng/mL of TNF-α. The activity of MMP-9 was reduced by 100 ng/mL IL-4 (*P < 0.05, compared with the control [CT], i.e., cells grown in medium alone).
Figure 5.
 
MTT cell proliferation test in a VKC fibroblast culture exposed to different concentrations of cytokines (0, 0.1, 1, and 10 ng/mL) for 24 hours. Cell proliferation was increased by IL-4 and -13, unchanged by TNF-α, and inhibited by IFNγ (*P < 0.05, compared with the control [CT], i.e., cells grown in medium alone).
Figure 5.
 
MTT cell proliferation test in a VKC fibroblast culture exposed to different concentrations of cytokines (0, 0.1, 1, and 10 ng/mL) for 24 hours. Cell proliferation was increased by IL-4 and -13, unchanged by TNF-α, and inhibited by IFNγ (*P < 0.05, compared with the control [CT], i.e., cells grown in medium alone).
Figure 6.
 
Effect of cytokines on expression of collagen I (Collα2I), MMP-1, and TIMP-1 in VKC conjunctival fibroblasts. Cells were incubated in the absence (control) or presence of different concentrations of cytokines before extraction of total mRNA and RT-PCR. Representative RT-PCR gels (A). Diagrams of each type of mRNA after IL-4, IL-13, and control exposure (B).
Figure 6.
 
Effect of cytokines on expression of collagen I (Collα2I), MMP-1, and TIMP-1 in VKC conjunctival fibroblasts. Cells were incubated in the absence (control) or presence of different concentrations of cytokines before extraction of total mRNA and RT-PCR. Representative RT-PCR gels (A). Diagrams of each type of mRNA after IL-4, IL-13, and control exposure (B).
The authors thank Daniele Violato for expert technical assistance. 
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Figure 1.
 
PIP levels in culture medium of conjunctival fibroblasts from one of the VKC-derived cultures treated for 24 or 48 hours with 10 ng/mL of the tested cytokines. At 24 hours, both IL-4 and -13 significantly increased PIP, whereas IFNγ and TNF-α significantly reduced it. At 48 hours, only IL-4 and IFNγ significantly modified production of PIP. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 1.
 
PIP levels in culture medium of conjunctival fibroblasts from one of the VKC-derived cultures treated for 24 or 48 hours with 10 ng/mL of the tested cytokines. At 24 hours, both IL-4 and -13 significantly increased PIP, whereas IFNγ and TNF-α significantly reduced it. At 48 hours, only IL-4 and IFNγ significantly modified production of PIP. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 2.
 
PIIIP levels in culture medium of conjunctival fibroblasts derived from one patient with VKC and treated for 24 hours with different concentrations of cytokines (0, 0.1, 1, 10 ng/mL): 1 and 10 ng/mL IFNγ significantly reduced PIIIP. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 2.
 
PIIIP levels in culture medium of conjunctival fibroblasts derived from one patient with VKC and treated for 24 hours with different concentrations of cytokines (0, 0.1, 1, 10 ng/mL): 1 and 10 ng/mL IFNγ significantly reduced PIIIP. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 3.
 
Levels of (A) MMP-1, (B) TIMP-1, and (D) MMP-9 and (C) the TIMP-1-to-MMP-1 ratio in culture medium of VKC conjunctival fibroblasts treated for 24 hours with 100 ng/mL IL-4, 100 ng/mL IL-13, 10 ng/mL IFNγ or 10 ng/mL TNF-α. IL-4 and -13 significantly reduced MMP-1 and increased TIMP-1 and the ratio of TIMP-1 to MMP-1, whereas only TNF-α increased MMP-9. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 3.
 
Levels of (A) MMP-1, (B) TIMP-1, and (D) MMP-9 and (C) the TIMP-1-to-MMP-1 ratio in culture medium of VKC conjunctival fibroblasts treated for 24 hours with 100 ng/mL IL-4, 100 ng/mL IL-13, 10 ng/mL IFNγ or 10 ng/mL TNF-α. IL-4 and -13 significantly reduced MMP-1 and increased TIMP-1 and the ratio of TIMP-1 to MMP-1, whereas only TNF-α increased MMP-9. (*P < 0.05; **P < 0.01, compared with the control [CT], i.e., cells grown in medium alone).
Figure 4.
 
Activity of (A) MMP-1 and (B) -9 in VKC fibroblast cultures exposed to different cytokines for 24 hours. The activity of MMP-1 was increased by 100 ng/mL IL-4 and 10 ng/mL of TNF-α. The activity of MMP-9 was reduced by 100 ng/mL IL-4 (*P < 0.05, compared with the control [CT], i.e., cells grown in medium alone).
Figure 4.
 
Activity of (A) MMP-1 and (B) -9 in VKC fibroblast cultures exposed to different cytokines for 24 hours. The activity of MMP-1 was increased by 100 ng/mL IL-4 and 10 ng/mL of TNF-α. The activity of MMP-9 was reduced by 100 ng/mL IL-4 (*P < 0.05, compared with the control [CT], i.e., cells grown in medium alone).
Figure 5.
 
MTT cell proliferation test in a VKC fibroblast culture exposed to different concentrations of cytokines (0, 0.1, 1, and 10 ng/mL) for 24 hours. Cell proliferation was increased by IL-4 and -13, unchanged by TNF-α, and inhibited by IFNγ (*P < 0.05, compared with the control [CT], i.e., cells grown in medium alone).
Figure 5.
 
MTT cell proliferation test in a VKC fibroblast culture exposed to different concentrations of cytokines (0, 0.1, 1, and 10 ng/mL) for 24 hours. Cell proliferation was increased by IL-4 and -13, unchanged by TNF-α, and inhibited by IFNγ (*P < 0.05, compared with the control [CT], i.e., cells grown in medium alone).
Figure 6.
 
Effect of cytokines on expression of collagen I (Collα2I), MMP-1, and TIMP-1 in VKC conjunctival fibroblasts. Cells were incubated in the absence (control) or presence of different concentrations of cytokines before extraction of total mRNA and RT-PCR. Representative RT-PCR gels (A). Diagrams of each type of mRNA after IL-4, IL-13, and control exposure (B).
Figure 6.
 
Effect of cytokines on expression of collagen I (Collα2I), MMP-1, and TIMP-1 in VKC conjunctival fibroblasts. Cells were incubated in the absence (control) or presence of different concentrations of cytokines before extraction of total mRNA and RT-PCR. Representative RT-PCR gels (A). Diagrams of each type of mRNA after IL-4, IL-13, and control exposure (B).
Table 1.
 
Sequences of PCR Primer Sets
Table 1.
 
Sequences of PCR Primer Sets
Factor Primer Sequence Product Size (bp)
MMP-1 5′-CGACTCTAGAAACACAAGAGCAAGA-3′ (sense) 786
5′-AAGGTTAGCTTACTGTCACACGCTT-3′ (antisense)
TIMP-1 5′-ATCCTGTTGTTGCTGTGGCTGATAG-3′ (sense) 667
5′-TGCTGGGTGGTAACTCTTTATTTCA-3′ (antisense)
Collα2(I) 5′-GGTGGTTATGACTTTGGTTAC-3′ (sense) 702
5′-CAGGCGTGATGGCTTATTTGT-3′ (antisense)
Table 2.
 
Procollagen I Production from VKC-Derived and Normal Subject–Derived Fibroblasts after a 24-Hour Exposure to Different Cytokines
Table 2.
 
Procollagen I Production from VKC-Derived and Normal Subject–Derived Fibroblasts after a 24-Hour Exposure to Different Cytokines
Control IL-4 (10 ng/mL) IL-13 (10 ng/mL) IFN-γ (10 ng/mL) TNFα (10 ng/mL)
VK-F 422 ± 104 550 ± 38 614 ± 28 337 ± 36 309 ± 21
NS-F 309 ± 19 393 ± 11 387 ± 29 105 ± 42 154 ± 6
P NS 0.002 0.001 0.002 0.001
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