Accumulating evidence suggests the involvement of TGF-β in the process of corneal opacity, which is one of the serious causes of visual loss. However, whether TGF-β is indeed critical for the pathogenesis remains unknown. We constructed an adenovirus expressing an entire ectodomain of the human type II TGF-β receptor fused to Fc portion of human IgG (AdTβ-ExR): this soluble receptor is secreted from AdTβ-ExR-infected cells, binds to TGF-β and inhibits TGF-β signaling. When AdTβ-ExR was injected into the femoral muscle of Balb/c mice, a high level of the soluble receptor protein (2.0–3.5 × 103 pM) was detectable in the serum and in the ocular fluid for at least 10 days. In the mice subjected to corneal injury with silver nitrate and to intramuscular injection with either saline or a control adenovirus expressing β-galactosidase (AdLacZ), corneal opacification composed of extracellular matrix (ECM) accumulation, of infiltration of neutrophils and monocytes/macrophages, and of angiogenesis were all induced. In contrast, they were markedly reduced in the mice injected with AdTβ-ExR. Immunohistochemical analysis revealed that TGF-β, fibronectin, macrophage chemoattractant protein-1, and vascular endothelial growth factor were densely stained in the edge of wounded cornea, but they were scarcely present in the injured-cornea of AdTβ-ExR-treated mice. Our results demonstrate that TGF-β indeed plays a critical role in the process of cornea opacification, and that adenovirus-mediated expression of a soluble TGF-β receptor can be therapeutically useful.
The cornea is a transparent anterior ocular tissue and protects the eyeball from outside stimuli. Under various conditions such as infection and trauma, the cornea can be damaged and thus becomes cloudy through the process of wound healing, which can therefore cause the serious impairment of vision. An understanding of the molecular basis of this process is considered important for effective therapeutic modalities.
Corneal opacification results from the deposition of ECM, the infiltration of inflammatory cells and the invasion of new vessels, in which the underlying mechanism has yet to be elucidated. Whether various factors are involved or a single molecule induces all these pathological reactions is not known. TGF-β is a multifunctional polypeptide and stimulates the production of ECM in a variety of cells leading to fibrosis, and is also known to modulate inflammation through the up- or down- regulating production of various cytokines depending on conditions.123 Furthermore, TGF-β has also been reported to induce angiogenesis, although these data remain controversial.4567 Three isoforms of TGF-β and their receptors were found to be present in the cornea and corneal stroma and they are thus considered to play an important role in the pathophysiology of cornea.89101112 In fact, TGF-β is present on the edge of wound healing in the cornea.1314 All this information suggests that TGF-β may be a single key molecule in the pathogenesis of corneal opacification. However, the actual role of TGF-β has yet to be elucidated.
A large obstacle to investigate the role of TGF-β in vivo is the lack of any suitable methods to specifically inhibit TGF-β action. TGF-β1- and TGF-β2-deficient mice have been developed by gene targeting. Such mice have already helped to elucidate the role of TGF-β in early stages of life, but they are not so useful for analyzing the pathophysiology in matured adult, since these animals die usually by 3–4 weeks of age.151617 A new method to block TGF-β signaling in a TGF-β-specific manner is needed. We constructed an adenoviral vector (AdTβ-ExR) expressing an ectodomain of the type II TGF-β receptor fused to the Fc portion of human IgG. In this study, we confirmed that this soluble TGF-β receptor is secreted from the infected cells, and inhibits the action of TGF-β both in vitro. We, then, injected AdTβ-ExR into the skeletal muscle of mice which were subjected to corneal injury. The soluble receptor produced in the muscle reaches the cornea by means of normal blood circulation and thus sequesters local TGF-β and inhibits its action in the cornea, into which a direct gene transfer seems difficult. In AdTβ-ExR-injected mice, ECM accumulation, cell infiltration and also angiogenesis were all largely reduced, thus indicating that TGF-β is the single critical molecule in this apparently complicated disease process, and that the adenovirus-mediated expression of the soluble TGF-β receptor in a remote organ may thus have some potential therapeutic value.
The soluble receptor secreted from AdTβ-ExR-infected cells inhibits TGF-β1 activity in vitro
A replication-defective E1− and E3− recombinant adenovirus (AdTβ-ExR) expressing an entire ectodomain of the TGF-β type II receptor fused to the human immunoglobulin Fc portion under a CA promoter (composed of cytomegalovirus enhancer and chicken β-actin promoter) was constructed, as previously described.181920 Immunoglobulin Fc portion was used as a tag protein to monitor the secreted protein. In order to see the effect of protein secreted from AdTβ-ExR infected cells, the supernant of adenovirally-transfected COS cells were tested using a mink lung epithelial cell line that had been stably transfected with an expression plasmid containing a plasminogen activator inhibitor (PAI)-1 promoter fused to the firefly luciferase gene.21 The results showed that the luciferase activity increased by TGF-β1 dose dependently in the medium from COS cells transfected with either AdLacZ or no adenovirus. In contrast, the induction of luciferase activity was not apparent in the medium from the COS cells transfected with AdTβ-ExR containing TGF-β1 (Figure 1), thus indicating that TGF-β1 activity was blocked by the supernatant from the AdTβ-ExR-transfected cells.
The soluble TGF-β receptor was detectable in both the serum and ocular fluid in the mice intramuscularly injected with AdTβ-ExR
In the following studies, no difference was observed between the nontreated and BSS-treated animals (data not shown), therefore, the data of BSS-treated animals represented those of nontreated animals. Since the intra-ocular injection of an adenovirus vector impaired the retinal function to a certain extent19 and the extra-ocular muscle of mouse does not have enough space to receive the solution containing AdTβ-ExR sufficient to transfer genes, AdTβ-ExR was injected into the femoral muscle and the soluble protein was intended to reach the eye. The results showed that the soluble receptor was clearly detectable in the serum of the rats following an intramuscular injection with AdTβ-ExR. The tear fluid and/or the aqueous humor seemed most responsible for the corneal wound healing, however, the amount of them from seven mice eyes was too small for the following studies. Hence, the supernant of homogenized eyes was used as an ocular fluid. In this study, we measured the soluble receptor protein in the serum and in the ocular fluid of the AdTβ-ExR-injected mice. As shown in Figure 2, a substantial amount of the soluble receptor protein was detectable not only in the serum, but also in the ocular fluid. The amounts of soluble receptor protein and its temporal changes in the serum and in the ocular fluid were indistinguishable. The soluble receptor reached its peak on the 7th day after gene transfer into the muscle and then declined gradually thereafter. No receptor protein was detectable in the serum of the mice injected with either an AdLacZ or balanced salt solution (BSS, Figure 2).
Corneal opacity, edema and neovascularization were all markedly reduced in the mice intramuscularly injected with AdTβ-ExR
We investigated whether or not the corneal wound healing process is regulated by TGF-β and whether anti-TGF-β intervention can modulate the disease process. For this end, we injected AdTβ-ExR into muscle to secrete the soluble receptor which should sequester TGF-β and thereby inhibit the actions of TGF-β in vivo. With the intramuscular injection of AdTβ-ExR, we induced eye injury and then evaluated the eyes 120 h after cauterization. We observed that the inflammatory cell infiltration starts 3 h after cauterization and that angiogenesis appears at 96 h after cauterization and become prominent at 120 h after cauterization.22 Macroscopically, it is obvious that the eyes of mice treated with AdTβ-ExR looked much clearer than those of mice treated with either AdLacZ or BSS. Representative photographs of the corneas in such animals are shown in Figure 3a, b and c. We tried to evaluate the changes in corneas (opacity and edema) in a semiquantitative manner by grading (see the Materials and methods). The cornea became thickened in both the BSS- and AdLacZ-injected mice, but the thickness of the cornea was significantly less in the AdTβ-ExR-treated mice (Figure 3d and e). A large elevation in the central area of the cornea was often observed in such eyes.
Angiogenesis accompanied corneal injury and was visualized by injecting black ink into the vessels. Vigorous angiogenesis was observed in the cornea of the mice injected with either BSS or AdLacZ (Figure 4a and b). Even hemorrhaging was observed in some restricted areas. No difference was seen in the values of angiogenesis between these control groups. Angiogenesis was also observed in the AdTβ-ExR-treated mice, however, its degree was much less than those of the control mice (Figure 4c). For semiquantification, we measured the length and the area of newly formed vessels in the cornea and found that corneal angiogenesis was strongly inhibited in the AdTβ-ExR-treated mice (Figure 4d).
Cell infiltration was reduced in the cornea of the AdTβ-ExR-treated mice
To obtain some insight into the cellular mechanisms underlying the observed changes, we characterized the infiltrated cells into the cauterized cornea. We enzymatically isolated cells in the cornea and identified them through a flowcytometric analysis. We first counted the total number of cells per cornea. Compared with the cauterized corneas of control mice, the number of infiltrated cells in the corneas of AdTβ-ExR-treated mice was substantially reduced (Figure 5a). No inflammatory cells were observed in the nontreated cornea. An analysis after cell-type specific staining revealed that most infiltrated cells in the cauterized cornea were either neutrophils or macrophages, and a smaller number of both types of cells were found in the cornea of the AdTβ-ExR-infected mice than in those of control mice (Figure 5b, c and d). The fraction of T cells (anti-Thy-1 mAb positive) and B cells (anti-B220 mAb positive) consisted of less than 1% of the total cells in the cornea (data not shown). Very few cells which were not stained by an anti-Mac-1 mAb were detectable and those cells were probably derived from such resident corneal cells as corneal epithelial cells, stromal cells and endothelial cells. However, they did not include bone marrow-derived cells, since they were not stained with an anti-CD45 mAb, a common marker for bone marrow-derived cells (data not shown).
Infiltrated cells secrete VEGF and may induce angiogenesis in the injured cornea
We histopathologically examined the cornea. The cornea had thickened with stroma and numerous inflammatory cells (Figure 6A and B), most of which were neutrophils and macrophages/monocytes based on the findings of a flowcytometric analysis (Figure 5). Many vessels were also observed in the corneal stroma (indicated by arrows). Intracameral inflammatory cell infiltration was also noticed in part. However, those changes were markedly reduced in the cornea of the AdTβ-ExR-treated mice (Figure 6C).
An immunohistostaining analysis showed that TGF-β1 was abundant in the wound edge and around the inflammatory cells in the injured-cornea of control mice (BSS- or AdLacZ-injected mice) (Figure 7a and b, arrows). In contrast, only faint staining of TGF-β1 was found in the AdTβ-ExR-treated mice corneas (Figure 7c). TGF-β2 was also densely noticed in the wound edge (Figure 7d and e, arrows) as well as in the corneal epithelium and the subepithelial area. Although TGF-β2 showed faint staining in the corneal epithelium (Figure 7f). ECM as fibronectin, dense staining was observed in the thickened stroma (Figure 7g and h, arrowheads) and such infiltrating cells (arrows) while fibronectin staining was scarcely observed in the AdTβ-ExR-treated eyes (Figure 7i).
Since we observed that a considerable number of neutrophils and macrophage/monocytes infiltrated into the injured cornea, we hypothesized that the macrophage/monocytes were attracted by MCP-1, which was probably produced by keratocytes and inflammatory cells in the injured cornea in response to TGF-β, and that the attracted macrophage/monocytes in turn secreted VEGF and thus induced rigorous angiogenesis in the cornea. We found that MCP-1 was expressed on the inflammatory cells and keratocytes in the stroma (Figure 7j and k). VEGF was also expressed in the infiltrated macrophage/monocytes (Figure 7m and n). However, neither MCP-1 nor VEGF was detectable in the cornea of the AdTβ-ExR-treated mice (Figure 7l and o, respectively). We stained the sections with nonimmunized rabbit serum to confirm that all positive staining described above was specific to each of the antibodies used (Figure 7p, q, and r). Although no positive staining for a soluble TGF-β-receptor-IgG complex was found in the cornea of AdLacZ-treated mice, a positive staining was observed on corneal surface, and in and around the newly formed vessels in the cornea of AdTβ-ExR-treated mice (Figure 8a, b and c). These findings also correlated with our hypothesis.
We histologically examined various organs including the lungs, livers, kidneys, hearts, brains, and intestines from mice on the 6th and the 14th day after injury and AdTβ-ExR-injection into femoral muscle. We noticed neither any apparent histological changes in those organs nor alterations in the general conditions of the mice (data not shown).
To clarify the role of TGF-β in the pathophysiological conditions occurring in adults, a conventional gene targeting approach may not be practical, since such animals die shortly after birth.151617 We have proven that the adenovirus-mediated local expression of a dominant-negative TGF-β receptor is useful for this purpose.23 We applied this method in our study to clarify the role of TGF-β in the pathogenesis of corneal injury. Surprisingly, an inhibition of TGF-β resulted in a marked reduction of a wide variety of pathological reactions including the accumulation of ECM, the infiltration of inflammatory cells, and angiogenesis. This is the first clear evidence showing that TGF-β does play a central role in the disease process following corneal injury.
The soluble TGF-β receptor blocks TGF-β signaling most probably by adsorbing TGF-β from the receptor, although there is a possibility that it also works as a dominant-negative receptor (the effect as a dominant-negative receptor has been shown in a case of VEGF receptor2425). Therefore, the soluble receptor should be excessive to the ligand, TGF-β to block TGF-β signaling. Although we did not measure the content of TGF-β in our study, it was reported that the serum level of TGF-β in healthy humans has reported to be less than 5 pM, and that an increased amount of TGF-β is secreted into tear fluid after corneal surgery in human, but it is around 9 ng/ml (210 pM) at maximum.26 We detected the soluble receptor both in the serum and in the ocular fluid at more than 2.5 × 1000 pM, which is at least 15 times higher than TGF-β in the tear fluid following corneal surgery. Immunohistochemical studies showed the presence of a soluble TGF-β receptor–Fc complex in the cornea. It may thus be reasonably assumed that most of TGF-β signaling can be inhibited in the AdTβ-ExR-injected mice in this study. However, it has to be noted that the present ‘ocular fluid’ contained the serum, the vitreous and the corneal fluid. Further detailed study is necessary to confirm this conclusion.
The soluble receptor used in this study is fused to Fc portion of human IgG at its C-terminal. Since we did not observe any changes with either an injection of an adenovirus vector encoding for Fc portion or an intravenous injection of a large amount of human IgG (500 mg per mouse), it is unlikely that the inhibitory effects observed in the AdTβ-ExR-injected mice could be due to Fc portion of IgG.
Corneal transparency is essential for maintaining normal vision. In silver nitrate-injured cornea, edema, opacity, and neovascularization were induced within 5 days after chemical cautery, the pathophysiology of which is very similar to that observed in human corneal injury.27 A histological analysis suggests that these changes are composed of thick stromal ECM deposition, of inflammatory cell infiltration and of new vessel formation. Surprisingly, all these pathological changes were largely diminished by the inhibition of signaling by a single growth factor, TGF-β (through the introduction of the soluble receptor). It is well known that TGF-β is a critical regulator of the production, accumulation, and degradation of ECM, and a potent chemoattractant of monocytes128 by enhancing the expression of MCP-1 in macrophages and osteoblasts,2930 although TGF-β was reported to inhibit the production of MCP-1 in macrophages in one controversial report.31 Our results through histological and immunohistochemical analyses are consistent with the hypothesis that TGF-β acts as a proinflammatory cytokine, induces the expression of MCP-1 thereby inducing infiltration of macrophages/monocytes, and accumulates ECM in the cornea.
An important finding in this study is that the inhibition of angiogenesis was achieved by a soluble TGF-β receptor. Angiogenesis is a crucial biological process in wound healing to remove damaged tissue and to reconstruct normal structures, but this process also hinders the light transparency of the cornea. Inflammatory cells produce a number of angiogenic factors including fibroblast growth factor, VEGF, granulocyte–macrophage colony-stimulating factor, tumor necrosis factor-α, and interleukin-8.32 In fact, a histological analysis revealed that VEGF was expressed in the inflammatory cells (mostly macrophages) in the injured cornea. It can thus be hypothesized that the inhibition of TGF-β signaling by the soluble receptor reduces infiltration of the inflammatory cells leading to a reduction in the production of VEGF, thereby inhibiting corneal angiogenesis. Furthermore, it has been reported that TGF-β directly stimulates the production of VEGF in various cells including fibroblastic epithelial cells,333435 thus suggesting that another mechanism suppresses VEGF production in the injured cornea. There is still another possibility that the circulating TGF-β receptors affect the immunological system of mice, thus resulting in a reduction of corneal inflammation. Although we did not find any systemic effect in this study, this possibility should be studied in the future. In ocular tissues, anterior chamber associated immune deviation (ACAID) and corneal endothelial proliferation are modulated by TGF-β and the blockade of TGF-β might affect them.3637 This is a model for the corneal surface disease not a model for intra-ocular disease and it simulated traumatic damage, where the recruitment of neutrophils and/or macrophages plays a pivotal role. As a result, the blockade of TGF-β might not have a significant effect on the physiology of the eye in this study. However, a detailed study of the effect on ACAID and corneal endothelial proliferation is necessary before clinical application can be started.
It should be noted that we did transfer AdTβ-ExR into the femoral muscle, not into the eye. In previous studies, the direct injection of plasmid cDNA or an adenovirus vector to the periocular tissue could transfer genes to the corneal epithelium.3839 In our preliminary studies and another report, these methods could not effectively transfer genes to the cornea without inflammation.40 Therefore, the direct gene transfer of a dominant-negative TGF-β receptor into the ocular tissue does not help to elucidate the role of TGF-β in the eye. A transduced gene product should be present in the circulating blood and thus reach the cornea, thereby inhibiting TGF-β signaling. In fact, a substantial amount of the soluble receptor was detectable in not only the circulating blood but also in the ocular fluid (Figure 2). The immunohistochemistry for a tag protein (human IgG) also showed a soluble TGF-β protein to be present on the corneal surface and also in and around the vessels (Figure 8). The approach used in the current study based on the expression of soluble protein in the muscle, is thus considered to be very useful. The possible occurrence of problems needs to be carefully examined in a further study, although all mice survived and no apparent side-effects such as inflammation were either macroscopically or histologically observed in the current study.
An adenoviral vector can transfer the gene very efficiently, however, the duration of gene expression is limited (around 1 month).2541 In this study, a large amount of soluble receptor was observed in mice for 2 weeks, but the amount gradually declined thereafter. As a result, the current approach using an adenoviral vector was thus found to help clarify the molecular basis of the pathophysiology in vivo, but this approach may not always be useful for clinical application in chronic or slowly progressive degenerative diseases. A modification of the gene transfer vector or the development of another method may thus be needed. However, the present method might be effective for inhibiting the excessive scarring after traumatic damage or corneal infection. In conclusion, the adenoviral vector-mediated gene transfer of soluble TGF-β receptor is therefore considered to give us a better understanding of the role of TGF-β in vivo while it may also help us to develop some new gene therapeutic methods for corneal diseases.
Materials and methods
A replication-defective E1− and E3− recombinant adenovirus (AdTβ-ExR) expressing an entire ectodomain of the TGF-β type II receptor fused to the human immunoglobulin Fc portion (as a tag protein) under a CA promoter (composed of cytomegalovirus enhancer and chicken β-actin promoter) was constructed, as previously described.181920 Since the intramuscular injection of adenoviral vector encoding for the human immunoglobulin Fc portion or whole human immunoglobulins (500 mg per mouse, Sigma, St Louis, MO, USA) did not have any effect on the corneal wound healing when compared with the BSS-injected mouse in our preliminary study (data not shown), an adenoviral vector expressing bacterial β-galactosidase (AdLacZ) was thus used as a control adenoviral vector. The titer of the virus stock was assessed by a plaque formation assay using 293 cells and expressed in plaque formation units (p.f.u.).
Measurement of the soluble TGF-β-receptor protein
The concentration in the serum and in the ocular fluid of the soluble TGF-β receptor which was fused to Fc of the human IgG was assessed by ELISA.21 Briefly, a 96-well plate was coated with a rabbit polyclonal anti-human IgG (Dakopatts, Glostrup, Denmark) overnight. Next, the samples (100 μl each) were applied and kept at room temperature for 2 h. Peroxidase conjugated anti-human IgG (Fc fragment specific; Dakopatts) was added and incubated for a further hour. Finally, a buffer containing o-phenylenediamine and 0.01% H2O2 was then added and incubated for 2 h in the dark. The absorbance at 492 nm was measured by a multiscan spectrophotometer. A Western blot analysis disclosed that the molecular weight of soluble TGF-β receptor-Fc produced by AdTβ-ExR transfected cells was 60 kDa (data not shown) and the amount of the soluble receptor protein was expressed as a mol/l (M). The two anti-IgG antibodies used did not cross-react with any mouse immunoglobulins (data not shown).
The serum of the mice without ocular injury injected intramuscularly either with AdTβ-ExR, AdLacZ (5 × 108 p.f.u. per mouse) or BSS was collected at 0, 3, 5, 7, 11, 14, and 21 days after injection (n = 6, each day). The eyes were enucleated (six eyes from six mice) and centrifuged at 500 g for 20 min, and thereafter the ocular fluid was collected.
Inhibition of TGF-β activity in vitro
COS cells infected with either AdLacZ or AdTβ-ExR (MOI 10) were incubated in DMEM (GIBCO-BRL, Gaithersburg, MD, USA) containing 0.5% fetal calf serum (FCS) for 24 h. The medium was incubated with AdTβ-ExR contains about 1000 pM TGF-β receptor-Fc protein, which was as much as 50 ng/ml TGF-β1 on a molar basis. The medium was collected and mixed with human recombinant TGF-β1 (Genzyme, Cambridge, MA, USA) at the indicated concentrations. The medium was next cultured with a mink lung epithelial cell line that had been stably transfected with an expression plasmid containing a TGF-β responsible PAI-1 promoter fused to the firefly luciferase gene.21 The luciferase activity was assayed using the enhanced luciferase assay kit (Analytical Luminescience, San Diego, CA, USA). Briefly, the cells were washed twice with PBS and were extractred with cell lysis buffer (Analytical Luminescience) for 20 min at room temperature. Next the lysate (80 μl) was transferred to a 96-well plate (Dynatech Laboratories, Chantilly, VA, USA) and were analyzed by a luminometer with 100-μl injections of substrate.21
Animal model of corneal injury
Balb/c mice (8 weeks old, 25–30 g, Kyudo, Fukuoka, Japan) were used. All animals were treated humanely, including proper institutional approval, and complied with the Association in Research for Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and Declaration of Helsinki. The mice were anesthetized by intraperitoneal injection of 3 mg of ketamine and 0.01 mg of xylazine. Then, each mouse received an intramuscular (femoral muscle) injection of either AdTβ-ExR, AdLacZ (5 × 108 p.f.u. per mouse), or BSS. The corneas on the left side were cauterized with an application stick containing 4 g/ml silver nitrate (for 1 s so that a grayish-white patch of cauterized tissue, 1 mm diameter was made).1722 After cauterization, all mice were kept under a sterile hood to avoid any microbial infection. The corneas were observed every day for signs of inflammation and neovascularization. The eyes of untreated mice (n = 10) were used as controls. On day 14, some mice were histologically examined at sites such as brain, lung, heart, liver, kidney, intestine, and arteries.
Evaluation of corneal opacity, edema and neovascularization
Five days after injury, the corneas were clinically evaluated under a stereoscopic microscope in a masked fashion. The indices of corneal edema and opacity were scored on a scale from 0 to +4 according to the severity of the lesions.224243 For edema: 0, completely normal cornea; +1, no evidence of any ongoing edema, epithelialization was sometimes observed; +2, mild edema, the corneal thickness was less than twice that of the normal cornea, edema was limited to the cauterized area; +3, severe edema, the corneal thickness was more than twice that of the normal cornea, edema was limited to the cauterized area; +4, extensive edema, severe edema extended to the whole cornea and corneal perforation also occurred in some cases. For opacity: 0, completely clear cornea; +1, slight opacity; +2, mild opacity, iris and lens were visible; +3, severe opacity, iris and lens were invisible, opacity was limited to the cauterized area; +4, extensive opacity, the severe opacity was extended to whole cornea. Corneal neovascularization was evaluated, as previously reported.2243 Briefly, the upper body was perfused with a BSS until normal pink color of the ocular fundi disappeared. A mixture of 11% gelatin (Sigma) and 10% Indian ink (Faber-Castell, Lewisberg, TN, USA) in BSS was then perfused. Immediately after that, the gelatin within the corneal vessels was solidified by cooling. The maximum vessel length in the neovascular zone, extending from the limbal vascular plexus toward the cauterized spot, was measured with a linear reticule through a slit lamp. Finally, the central angle of the contiguous circumferential zone of neovascularization was measured with a 360° reticule. When a neovascularization-positive area was found at 50% of the circumferential zone by a slit-lamp examination, it was expressed as 180°.
Antibodies and reagents:
Phycoerytherin (PE)-conjugated anti-Mac-1 monoclonal antibody (mAb) (clone name; M1/70.15) was purchased from Caltag (South San Francisco, CA, USA). FITC-conjugated anti-Gr-1 mAb (clone name; RB6–8C5) was purchased from Pharmingen (San Diego, CA, USA). FITC-conjugated anti-F4/80 mAb (clone name; A3–1) was purchased from Caltag. Biotin-conjugated anti-B220 mAb (clone name; RA3–6B2) was purchased from Pharmingen. Biotin-conjugated anti-Thy-1.2 mAb (clone name; 5A-8) was purchased from the Meiji Milk Product (Tokyo, Japan). Biotin-conjugated anti-pan-CD45 mAb (clone name; I3/2) was purchased from Gibco BRL. Streptavidin-Red613 was purchased from Gibco BRL.
The cells which infiltrated into the corneas were isolated using collagenase (Boehringer-Mannheim, Mannheim, Germany), as previously described.22 Corneal infiltrating cells were adjusted to the designated concentrations and then were stained with biotin-conjugated anti-Thy-1.2 mAb and anti-B220 mAb followed by streptoavidin-Red613. Furthermore, the cells were stained with either PE-Mac-1 mAb and FITC-Gr-1 or FITC-F4/80 mAb, and pan-CD45 mAb followed by streptavidin-Red613. Both macrophages and neutrophils have been reported to be Mac-1 surface molecule positive.44 F4/80 mAb stained macrophages45 and Gr-1 mAb stained neutrophils.46 Flow cytometry was performed with a FACScalibur (Becton Dickinson, San Jose, CA, USA). The number of corneal infiltrating neutrophils and macrophages were calculated based on the percentage of each population in live gating and the precounted total number of viable cells.
The cornea and other organs were fixed with 4% paraformaldehyde, cut into 5 μm sections and stained with hematoxylin-eosin for the histological analysis. Some tissue specimens were fixed briefly (20 min) with 4% paraformaldehyde, and then frozen. The frozen sections were subjected to immunohistostaining as the previously described method19 using various antibodies: an anti-mouse fibronectin polyclonal rabbit antibodies (Chemicon, Temecula, CA, USA), an anti-human VEGF monoclonal antibody, an anti-mouse MCP-1 polyclonal goat antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA), anti-human IgG rabbit polyclonal antibody (Dakopatts), rabbit polyclonal antibodies to TGF-β1-LAP, and TGF-β2-LAP (kindly provided by Drs H Yamashita and K Miyazono, Tokyo University, and by C-H Heldin, Ludwig Institute for Cancer Research, Uppsala, Sweden), which were raised against the TGF-β1-LAP corresponding to amino acid residues 262 to 277, the TGF-β2-LAP corresponding to amino acid residues 285 to 300.47 Nonimmunized rabbit serum was used as a negative control. The chromogen used in this study was 0.05% 3,3,-diaminobenzidine tetrahydrochloride (Wako, Tokyo, Japan).
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This work was supported by Grants-in-Aid for Scientific Research from Ministry of Education, Science and Culture of the Japanese Government (to TS and HU), and by the grants from Japan National Society for the Prevention of Blindness (Tokyo, Japan), from the Fukuoka Anti-Cancer Association (Fukuoka, Japan), from Kaibara Morikazu Medical Science Promotion Foundation (Fukuoka, Japan), from The Casio Science Promotion Foundation (Tokyo, Japan) (to TS), and from Takeda Medical Research Foundation and from Tokyo Biochemical Society (HU). We also thanks Drs H Yamashita and K Miyazono for their kind gift of TGF-β antibodies and Drs H Sanui and M Uehara for their financial support.
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Sakamoto, T., Ueno, H., Sonoda, K. et al. Blockade of TGF-β by in vivo gene transfer of a soluble TGF-β type II receptor in the muscle inhibits corneal opacification, edema and angiogenesis. Gene Ther 7, 1915–1924 (2000). https://doi.org/10.1038/sj.gt.3301320
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