Published online before print June 16, 2003
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* Departments of Biomolecular Recognition and Ophthalmology and
Ocular Pathophysiology, Yamaguchi University School of Medicine, Ube City, Japan
1Correspondence: Department of Biomolecular Recognition and Ophthalmology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi 755-8505, Japan. E-mail: nishida1{at}yamaguchi-u.ac.jp
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Key Words: corneal ulcer • collagen gel • IL-1 receptor antagonist • matrix metalloproteinase
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Corneal ulceration results from the destruction of collagen fibrils in the stroma by proteolytic enzymes. Activated corneal fibroblasts and infiltrated leukocytes are thought to play an important role in the pathobiology of corneal ulceration by contributing to the degradation of stromal collagen. Clinical observations have revealed the presence of many infiltrated cells in corneal tissue affected by ulceration, most of which are thought to be polymorphonuclear leukocytes or neutrophils [8 9 10 ]. Indeed, pathological examination has demonstrated the presence of neutrophils in or surrounding corneal ulcers [11 12 13 14 ]. Given that the corneal stroma comprises mostly type I collagen and resident fibroblasts, it provides a model system for investigation of the interactions among extracellular collagen, resident cells, and infiltrated cells. Neutrophils are activated by extracellular type I collagen [15 ]. The function of neutrophils that have entered the corneal stroma from blood vessels may thus be affected by stromal collagen.
To investigate the roles of interactions between resident fibroblasts and infiltrated neutrophils and between extracellular collagen and these cells in collagen degradation and corneal ulceration, we have now examined the effect of coculture of neutrophils with corneal fibroblasts on collagen degradation. We have also examined the effect of conditioned medium (CM) from neutrophils cultured in three-dimensional collagen gels or as monolayers on fibroblast-mediated collagen breakdown.
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Materials
Eagle’s minimum essential medium (EMEM) was obtained from Gibco-BRL (Osaka, Japan); fetal bovine serum (FBS) was from Flow Laboratories (North Ryde, Australia); native porcine type I collagen (acid-solubilized) and 5 x Dulbecco’s modified Eagle’s medium (DMEM) were from Nitta Gelatin (Osaka, Japan); bovine plasminogen was from Sigma Chemical Co. (St. Louis, MO); dextran was from Nacalai Tesque (Kyoto, Japan); Ficoll-Hypaque and an enhanced chemiluminescence (ECL) kit were from Amersham Pharmacia Biotech (Uppsala, Sweden); ilomastat (GM6001) was from Chemicon (Temecula, CA); Diff-Quick was from Green Cross (Osaka, Japan); and IL-1 receptor antagonist (IL-1ra) was from Anapure Bioscientific (Beijing, China). Immobilon-P membranes were from Millipore (Bedford, MA). An RNeasy mini kit and QuantiTect SYBR Green polymerase chain reaction (PCR) were from Qiagen (Hilden, Germany), and a TaKaRa RNA PCR kit was from Takara Shuzo (Shiga, Japan). Ethidium bromide and DNA molecular size markers (Marker 11) were from Nippongene (Toyama, Japan), and agarose (NuSieve 3:1) was from FMC Bioproducts (Rockland, ME). All media and reagents used for cell culture were endotoxin-minimized.
Cell culture
Rabbit corneal fibroblasts were isolated and cultured as described previously [16
]. In brief, the endothelial layer of the cornea was removed mechanically, and the tissue was then incubated with dispase (2 mg/ml, in EMEM) for 1 h at 37°C. After mechanical removal of the epithelial sheet, the tissue was treated with collagenase (2 mg/ml, in EMEM) at 37°C until a single-cell suspension of corneal fibroblasts was obtained. Isolated corneal fibroblasts were cultured under a humidified atmosphere containing 5% CO2 at 37°C in EMEM supplemented with 10% FBS. They were used for experiments after five or six passages. Rabbit neutrophils were freshly isolated from peripheral blood by density gradient centrifugation with Ficoll-Hypaque after dextran sedimentation. The purity of the neutrophil preparations was >95% as judged on the basis of cell morphology revealed by staining with Diff-Quick; their viability was >96% as determined by trypan blue staining. The neutrophils were maintained on ice until use within 1 h of separation. All procedures for the isolation of neutrophils were performed under sterile conditions.
Three-dimensional cell culture in collagen gels
Collagen gels were prepared as described [5
]. In brief, corneal fibroblasts were harvested by exposure to trypsin, collected by centrifugation, and resuspended in serum-free EMEM. Acid-solubilized type I collagen (3 mg/ml), 5 x DMEM, reconstitution buffer (0.05 M NaOH, 0.26 M NaHCO3, 0.2 M HEPES, pH 7.3), and corneal fibroblast suspension (22x105 cells/ml, in EMEM) or neutrophil suspension (220x105 cells/ml, in EMEM) were mixed on ice at the ratio of 7:2:1:1. The resulting mixture (0.5 ml) was added to each well of a 24-well culture plate and was allowed to solidify in an incubator under 5% CO2 at 37°C for 30 min, after which 0.5 ml serum-free EMEM containing test agent and plasminogen (120 µg/ml) was overlaid. Collagen gels were not detached from the wall of culture wells. The resulting cultures were incubated for 48 h.
Preparation of CM
CM was prepared by culturing neutrophils or fibroblasts, without the addition of plasminogen, in collagen gels or as monolayers for 48 h. The medium was then collected, cells and debris were removed by centrifugation (4000 g for 5 min), and the resulting supernatant was used immediately for experiments or stored at -80°C until use.
Measurement of collagenolytic activity
Measurement of degraded collagen in culture supernatants was performed as described previously [5
]. The medium from collagen-gel incubations was collected, and native collagen fibrils with a molecular size of >100 kDa were removed by ultrafiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that native collagen, but not degraded collagen, was removed by this procedure (Fig. 1
). The filtrate was then subjected to hydrolysis by 6 M HCl in a heat-block for 24 h at 110°C. The amount of hydroxyproline in the hydrolysate was measured spectrophotometrically as described [17
], and the amount of degraded collagen was expressed as µg hydroxyproline per well.
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Figure 1. Effect of ultrafiltration on the composition of culture supernatants of corneal fibroblasts in collagen gels. Corneal fibroblasts were cultured in collagen gels for 48 h, after which the culture medium was collected and subjected to ultrafiltration. Samples of the medium before (lane 2) and after (lane 3) filtration were subjected to SDS-PAGE and staining with Coomassie brilliant blue R-250. Lane 1 contains native type I collagen. The positions of molecular size standards are also indicated in kilodaltons.
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Reverse transcription (RT) and real-time, quantitative PCR analysis
RT and quantitative, real-time PCR analysis determined the abundance of MMP-1 and MMP-3 mRNAs as described previously [18
, 19
]. In brief, total RNA was extracted from fibroblasts and subjected to RT, and the resulting cDNA was then subjected to real-time PCR with a LightCycler instrument (Roche Molecular Biochemicals, Indianapolis, IN). Transcripts of the constitutively expressed gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served to normalize the amounts of MMP mRNAs in each sample. Real-time PCR data were analyzed with LightCycler software 3.01 (Roche Molecular Biochemicals). To verify the specificity of amplification, we also subjected PCR products to electrophoresis on a 3% agarose gel, which was then stained with ethidium bromide (1 µg/ml) and examined with a Nighthawk system (pdi, Huntington Station, NY). The sequences of the PCR primers were as follows: rabbit MMP-1 forward, 5'-TCAGTTCGTCCTCACTCCAG-3', rabbit MMP-1 reverse, 5'-TTGGTCCACCTGTCATCTTC-3'; rabbit MMP-3 forward, 5'-AAGTTCCTTGGCTTGGAGGT-3', rabbit MMP-3 reverse, 5'-ATCTCCATGTTCTCGGACTC-3'; rabbit GAPDH forward, 5'-TCACCATCTTCCAGGAGCGA-3', rabbit GAPDH reverse, 5'-CACAATGCCGAAGTGGTCGT-3'. These primers yielded PCR products of the expected sizes of 649 bp for MMP-1 mRNA [20
], 305 bp for MMP-3 mRNA [21
], and 293 bp for GAPDH mRNA [20
].
Statistics
Data are expressed as means ± SD. Statistical analysis was performed with Dunnett’s multiple comparison test, Scheffe’s test, or Student’s unpaired t-test. A P value of <0.05 was considered statistically significant.
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Figure 2. Effects of cell density and time of incubation on collagen degradation by corneal fibroblasts (A) or neutrophils (B). The indicated numbers of corneal fibroblasts or neutrophils were cultured in collagen gels in the presence of plasminogen for 0–3 days, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are means ± SD of triplicates from an experiment that was repeated a total of three times with similar results. *, P < 0.05; **, P < 0.01 (Dunnett’s test), versus the corresponding value for cell-free control cultures.
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3 x 105 neutrophils, the collagen gel was completely degraded. In the converse experiment, neutrophils (10x105) were cultured for 2 days in collagen gels in the presence of various numbers of corneal fibroblasts. In the experiment shown in Figure 3B , although the extent of collagen degradation increased with cell number in cultures of fibroblasts alone, this effect was not statistically significant. Culture of neutrophils alone also had no effect on collagen degradation. However, the addition of fibroblasts to the neutrophil cultures resulted in an increase in the amount of collagen degraded that was significant and dependent on fibroblast number. Thus, in cultures containing 1 x 105 fibroblasts, the extent of collagen degradation in the presence of neutrophils was about four times that apparent in the absence of neutrophils (Fig. 3B)
. We also examined the time course of collagen degradation by cocultures of corneal fibroblasts (1x105) and neutrophils (10x105); collagen degradation increased with time for up to 3 days (Fig. 4
). Again, these results indicated that neutrophils promote collagen degradation by fibroblasts.
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Figure 3. (A) Effect of neutrophils on collagen degradation in the absence or presence of corneal fibroblasts. The indicated numbers of neutrophils were cultured for 48 h in collagen gels with plasminogen in the absence (open circles) or presence (closed circles) of corneal fibroblasts (1x105 cells), after which the amount of degraded collagen was determined. (B) Effect of corneal fibroblasts on collagen degradation in the absence or presence of neutrophils. The indicated numbers of corneal fibroblasts were cultured for 48 h in collagen gels with plasminogen in the absence (open circles) or presence (closed circles) of neutrophils (10x105 cells), after which the amount of degraded collagen was determined. All data are means ± SD of triplicates from an experiment that was repeated a total of three times with similar results. *, P < 0.05; **, P < 0.001 (Student’s unpaired t-test), versus the value for cell-free control cultures.
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Figure 4. Time course of collagen degradation by cocultures of corneal fibroblasts and neutrophils. Corneal fibroblasts (1x105 cells) and neutrophils (10x105 cells) were cultured separately or together in collagen gels in the presence of plasminogen for the indicated times, after which the amount of degraded collagen was determined. Data are means ± SD of triplicates from an experiment that was repeated a total of three times with similar results.
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Neutrophil–CM from monolayer or collagen-gel cultures alone did not affect the integrity of collagen (Fig. 5A
). CM from monolayer cultures of neutrophils induced an 
2.6-fold increase in the amount of collagen degraded by corneal fibroblasts. However, neutrophil–CM derived from collagen-gel cultures induced an approximate tenfold increase in collagen degradation by corneal fibroblasts. These results thus suggested that neutrophils stimulate collagen degradation by corneal fibroblasts, not through cell–cell contact but rather through the release of a soluble factor, and that the release of such a factor by neutrophils is promoted by extracellular collagen.
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Figure 5. Effects of CM from neutrophils (A) or corneal fibroblasts (B) on collagen degradation. (A) Collagen gels containing (or not) corneal fibroblasts (1x105 cells) were incubated for 48 h with plasminogen in the absence (open bars) or presence of neutrophil–CM derived from monolayer cultures (hatched bars) or collagen-gel cultures (solid bars). (B) Collagen gels containing (or not) neutrophils (10x105 cells) were incubated for 48 h with plasminogen in the absence or presence of fibroblast–CM derived from monolayer cultures or collagen-gel cultures. The amount of degraded collagen was determined at the end of all incubations. Data are means ± SD of quadruplicates from an experiment that was repeated a total of three times with similar results. *, P < 0.01 (Scheffe’s test), versus the corresponding value for cells incubated in the absence of CM; #, P < 0.01 (Scheffe’s test), versus the corresponding value for cells incubated with CM from monolayer cultures; , P < 0.01 (Scheffe’s test), for the indicated comparison. N.S., Not significant.
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Expression of MMP-1 and MMP-3 in fibroblasts induced by neutrophil–CM
To investigate the mechanism by which neutrophils stimulate collagen degradation by corneal fibroblasts, we examined the effects of neutrophil–CM on MMP expression by fibroblasts with the use of immunoblot analysis. Neutrophil–CM derived from collagen-gel cultures did not contain proteins recognized by antibodies to rabbit MMP-1 (Fig. 6A
). In contrast, medium obtained after culture of corneal fibroblasts in collagen gels in the absence of plasminogen yielded 57- and 61-kDa immunoreactive bands (Fig. 6A)
, which corresponded to nonglycosylated and glycosylated forms of proMMP-1, respectively [22
]. Incubation of collagen-gel cultures of fibroblasts in the presence of neutrophil–CM increased the abundance of the 57- and 61-kDa immunoreactive proteins (Fig. 6A)
. Similar immunoblot analysis with antibodies to MMP-3 revealed that neutrophil–CM did not contain proMMP-3, that fibroblasts cultured in collagen gels produced a small amount of the 57-kDa proMMP-3, and that neutrophil–CM increased the expression of proMMP-3 by fibroblasts (Fig. 6A)
. Immunoblot analysis with antibodies to rabbit MMP-1 of the medium from corneal fibroblasts cultured in the presence of plasminogen revealed a small amount of 49- and 45-kDa immunoreactive proteins corresponding to active MMP-1. The further addition of neutrophil–CM to the fibroblast cultures increased the amounts of proMMP-1 and active MMP-1. Immunoblot analysis with antibodies to MMP-3 revealed that in the presence of plasminogen, corneal fibroblasts produced a small amount of 57-kDa and 45-kDa immunoreactive proteins corresponding to proMMP-3 and active MMP-3, respectively. The further addition of neutrophil–CM to the fibroblast cultures increased the abundance of proMMP-3 and active MMP-3 (Fig. 6A)
.
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Figure 6. Effects of neutrophil–CM on the expression of MMPs by corneal fibroblasts. (A) Collagen gels containing (or not) corneal fibroblasts (Fbs) were incubated for 48 h with or without plasminogen as well as in the absence (None) or presence of neutrophil–CM (CM) derived from collagen-gel cultures. The culture supernatants were then subjected to immunoblot analysis with antibodies to MMP-1 (upper panel) or to MMP-3 (lower panel). Data are representative of three independent experiments. The positions of proMMP-1, MMP-1, proMMP-3, and MMP-3 proteins are indicated on the right, and those of molecular size standards are shown on the left. (B) Corneal fibroblasts were incubated in collagen gels for 12 h without plasminogen and in the absence (None) or presence of neutrophil–CM (CM) derived from collagen-gel cultures, after which the amounts of MMP-1 mRNA (left panel) and MMP-3 mRNA (right panel) in the cells were determined by RT and real-time PCR analysis. Data were normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are means ± SD of values from three experiments. *, P < 0.01 (Student’s unpaired t-test), versus the corresponding value for cells incubated in the absence of neutrophil–CM.
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To examine further whether the enhancement by neutrophils of collagen degradation by corneal fibroblasts is mediated by the up-regulation of MMP expression in fibroblasts, we investigated the effect of ilomastat, a synthetic inhibitor of MMPs [23 ], on collagen degradation. The addition of ilomastat (0.1 µM) resulted in significant inhibition of the stimulatory effect of neutrophil–CM on collagen degradation by fibroblasts (69.37±4.91 vs. 18.80±0.63 µg hydroxyproline per well for incubations in the absence or presence of ilomastat, respectively; means±SD of triplicates from an experiment that was repeated a total of three times with similar results; P<0.0001, Student’s unpaired t-test).
Effect of IL-1ra on collagen degradation by cocultures of neutrophils and corneal fibroblasts
We previously showed that IL-1 is a potent stimulator of collagen degradation by corneal fibroblasts in the present experimental system [5
, 6
]. In addition, neutrophils release IL-1 [24
, 25
]. This cytokine was therefore a candidate mediator of the stimulatory effect of neutrophils or neutrophil–CM on collagen degradation by corneal fibroblasts. To investigate this possibility, we added IL-1ra to the medium of neutrophil-fibroblast cocultures. IL-1ra inhibited collagen degradation in cocultures of neutrophils and corneal fibroblasts in a dose-dependent manner (Fig. 7
); this effect of IL-1ra was significant at a concentration of 0.3 ng/ml, and inhibition was almost 100% at a concentration of 30 ng/ml. IL-1ra had no effect on collagen degradation in cell-free cultures or in cultures of corneal fibroblasts or neutrophils alone. It did, however, inhibit in a dose-dependent manner collagen degradation by corneal fibroblasts in the presence of neutrophil–CM (Fig. 7)
. These results suggest that IL-1 participates in the interaction between neutrophils and corneal fibroblasts, which is responsible for stimulation of collagen degradation.
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Figure 7. Effect of IL-1ra on collagen degradation by cocultures of neutrophils and corneal fibroblasts. Corneal fibroblasts (1x105) and neutrophils (10x105) were cultured alone or together for 48 h in collagen gels in the presence of plaminogen and the indicated concentrations of IL-1ra. Collagen gels containing fibroblasts were also incubated for 48 h with plasminogen, IL-1ra, and neutrophil–CM derived from collagen-gel cultures. The amount of degraded collagen was then determined. Data are means ± SD of triplicates from an experiment that was repeated a total of three times with similar results. *, P < 0.001 (Dunnett’s test), versus the corresponding value for cells cultured without IL-1ra.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pathological studies have demonstrated that neutrophils are rapidly recruited to the wounded cornea [11 , 14 ]. Neutrophils have also been shown to release various cytokines, superoxide, and proteolytic enzymes including elastase, collagenase, and gelatinase [26 27 28 ]. These cells have therefore been thought to participate in the breakdown of the ECM in the cornea. In the present study, however, neither neutrophils nor neutrophil–CM alone exhibited collagenolytic activity. In contrast, fibroblasts or fibroblast–CM exhibited a low level of collagenolytic activity, but fibroblast–CM did not increase collagen degradation by neutrophils. Our results therefore suggest that neutrophils might act as regulators, rather than as effectors, of the degradation of extracellular collagen associated with corneal ulceration. The precise role of neutrophils in corneal ulceration in vivo remains to be determined, however. Our data indicate that corneal fibroblasts are the primary mediators of collagen degradation during corneal ulceration and that infiltrated neutrophils serve to stimulate the collagenolytic activity of these resident cells of the corneal stroma.
Type I collagen promotes the adhesion of a variety of cell types, including fibroblasts, as well as regulates many other cell functions, such as proliferation, differentiation, migration, and changes in cell shape [2 ]. Consistent with previous observations [3 ], culture of corneal fibroblasts in type I collagen gels enhanced the collagenolytic activity of these cells. Certain types of hematopoietic cells, including neutrophils [29 , 30 ] and monocytes [31 ], also interact with collagens. The binding of neutrophils to collagen has been shown to stimulate various activities of these cells, including the extension of pseudopodia, the secretion of lytic enzymes, superoxide release, and the respiratory burst [15 ]. We have now shown that neutrophil–CM stimulated collagen degradation by corneal fibroblasts and that this effect was enhanced by culture of the neutrophils in the presence of collagen. The activation of neutrophils by collagen thus appears to be an important element in the stimulatory effect of these cells on collagen breakdown by corneal fibroblasts.
MMPs play an important role in the normal turnover of ECM proteins and in the exaggerated breakdown of the ECM associated with pathological conditions such as corneal ulcer. Corneal fibroblasts produce proMMPs [32 ]. Plasminogen, which is converted to plasmin by plasminogen activator, is also present in the cornea [33 ]. Plasmin, in turn, mediates the proteolytic activation of proMMPs. This system might thus initiate and perpetuate the collagen degradation associated with corneal ulceration [33 ]. Human tear fluid contains plasminogen activator and plasmin activity [34 , 35 ], and we have previously shown that plasminogen is important for collagen degradation by corneal fibroblasts in our assay system [6 ]. In the present study, neutrophil–CM alone did not exhibit collagenolytic activity and did not contain MMP-1 or MMP-3. However, neutrophil–CM stimulated collagen degradation by corneal fibroblasts as well as increased the synthesis of MMP-1 and MMP-3 by fibroblasts.
Various cytokines and other soluble mediators contribute to the pathogenesis of corneal inflammation. Among these factors, the inflammatory cytokine IL-1 is produced by resident corneal fibroblasts [36 ] and infiltrated neutrophils [24 ]. Previous studies have shown that IL-1 released from rabbit neutrophils promotes the proliferation of lymphocytes [37 , 38 ]. We have previously shown that recombinant IL-1 stimulates collagen degradation by corneal fibroblasts and that this effect is mediated by an increased production of proMMPs [5 , 6 ]. We were not able to measure the concentration of IL-1 in neutrophil–CM because of the lack of appropriate antibodies to the rabbit protein. We thus chose to block the action of endogenous IL-1 with IL-1ra, given that this protein was previously shown to antagonize IL-1 activity in rabbit fibroblasts including those from the cornea [39 ]. IL-1ra is a member of the IL-1 family of proteins [40 ], shares 30% amino acid sequence identity with IL-1ß, and binds to IL-1 receptor types I and II without inducing apparent cellular activation. Exogenous IL-1ra exhibits anti-inflammatory effects in corneal transplantation [41 ], corneal alkali injury [42 ], and corneal ulcer [43 ]; topical application of IL-1ra thus markedly reduced the severity of a bacterial corneal ulcer in an animal model [43 ]. Together with previous observations, our present results, showing that the stimulatory effect of neutrophils on collagen degradation by corneal fibroblasts was blocked by IL-1ra, suggest that this action of neutrophils is mediated, at least in part, by IL-1 released from neutrophils.
Received January 16, 2003; revised April 25, 2003; accepted April 28, 2003.
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and IL-4 on the expression of thymus- and activation-regulated chemokine in human corneal fibroblasts Biochem. Biophys. Res. Commun. 279,1-5[CrossRef][Medline]
Int. Arch. Allergy Immunol. 129,138-144[CrossRef][Medline]xß2 integrin (CD11c-CD18, gp150–95) J. Immunol. 164,5928-5934 mediates collagenase synthesis stimulated by phorbol 12-myristate 13-acetate J. Biol. Chem. 269,11291-11298This article has been cited by other articles:
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