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. 2010 Sep 28;107(39):16875-80.
doi: 10.1073/pnas.1012451107. Epub 2010 Sep 13.

Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury

Joo Youn Oh  1 , Gavin W Roddy, Hosoon Choi, Ryang Hwa Lee, Joni H Ylöstalo, Robert H Rosa Jr, Darwin J Prockop
Affiliations

Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury

Joo Youn Oh et al. Proc Natl Acad Sci U S A. .

Abstract

Previous reports demonstrated that adult stem/progenitor cells from bone marrow (multipotent mesenchymal stem cells; MSCs) can repair injured tissues with little evidence of engraftment or differentiation. In exploring this phenomenon, our group has recently discovered that the therapeutic benefits of MSCs are in part explained by the cells being activated by signals from injured tissues to express an anti-inflammatory protein TNF-α-stimulated gene/protein 6 (TSG-6). Therefore, we elected to test the hypothesis that TSG-6 would have therapeutic effects in inflammatory but noninfectious diseases of the corneal surface. We produced a chemical and mechanical injury of the cornea in rats by brief application of 100% ethanol followed by mechanical debridement of corneal and limbal epithelium. Recombinant human TSG-6 or PBS solution was then injected into the anterior chamber of the eye. TSG-6 markedly decreased corneal opacity, neovascularization, and neutrophil infiltration. The levels of proinflammatory cytokines, chemokines, and matrix metalloproteinases were also decreased. The data indicated that TSG-6, a therapeutic protein produced by MSCs in response to injury signals, can protect the corneal surface from the excessive inflammatory response following injury.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
TSG-6 reduced corneal opacity and neovascularization in the cornea following injury. (A) Representative photographs demonstrated the corneal surface on days 3, 7, and 21 after injury. Marked corneal opacity was present by day 3 after injury, and significant neovascularization developed by day 21 after injury in PBS solution-treated cornea. Corneal opacity and neovascularization were significantly decreased by TSG-6 injection. Note clear pupillary margin and well demarcated light reflex in TSG-6–treated cornea. (B) Quantification of corneal opacity following a clinical grading system on a scale from 0 to 4. (C) Quantification of neovascularization. Values are expressed in mean ± SD ratio of the neovascularized area to the whole corneal area.
Fig. 2.
Fig. 2.
TSG-6 reduced inflammation in the cornea following injury. (A) Representative images of H&E staining and immunohistochemistry (IHC) for neutrophil elastase showed severe neutrophil infiltration on day 3 after injury in the PBS solution-treated cornea. Also, thickened fibrovascular corneal stroma was observed on day 21. In contrast, neutrophil infiltration was much decreased in the TSG-6–treated cornea on day 3, and normal-appearing epithelium and stroma were restored by day 21. Note that corneal epithelium was denuded from injury on day 3 in PBS- and TSG-6–treated corneas. (B) The MPO concentration, as quantified by ELISA, was markedly increased in the cornea on day 3 after injury. TSG-6 significantly decreased the levels of MPO on day 3 and day 7. (C) Gelatin zymography demonstrated an increase in the expression of pro-MMP-9 and active MMP-9 on day 3, which was markedly decreased by TSG-6 injection. (D) The levels of total and active MMP-9 in the whole cornea were also significantly decreased by treatment with TSG-6 as assayed by ELISA. Values are mean ± SD.
Fig. 3.
Fig. 3.
TSG-6 reduced protein concentration of proinflammatory cytokines and chemokines. ELISAs showed that the levels of proinflammatory cytokines (IL-6 and IL-1β) and chemokines (CXCL1/CINC-1 and CCL2/MCP-1) were markedly increased on day 3 after injury in the PBS-treated corneas. In contrast, the production of proinflammatory cytokines and chemokines was significantly decreased in the TSG-6–treated corneas. Values are mean ± SD; n = 5 for each group.
Fig. 4.
Fig. 4.
Microarray analysis of the cornea following injury. (A) Microarray heat map of genes from the corneas 4 h and 24 h after injury is shown. (B) Heat map of genes from PBS- and TSG-6–treated corneas at 24 h after injury is shown. Gene ontology categories and the number of genes up-regulated (red) or down-regulated (blue) greater than twofold are indicated. Genes expressed differentially in PBS- versus TSG-6–treated corneas at 24 h are shown in Table S1. (C) Diagrams of genes up-regulated greater than fivefold in the corneas 4 h and 24 h after injury. The number of genes in each ontology category is indicated. Among genes that were temporarily up-regulated after injury (asterisk), secretogranin II showed the largest change: the gene was up-regulated 15.8-fold at 4 h and returned to normal at 24 h. In the TSG-6–treated corneas, up-regulation of the secretogranin II gene was 1.5-fold at 4 h.
Fig. 5.
Fig. 5.
TSG-6 reduced corneal inflammation and opacity in a dose-dependent manner. (A) Representative corneal photographs on day 3 after injury demonstrated that TSG-6 suppressed development of corneal opacity after chemical injury in a dose-dependent manner. (B) The anti-inflammatory effects of TSG-6 were dose-dependent as reflected in clinical grade of corneal opacity and MPO concentration. Values are mean ± SD; n = 3 for each group. (C) Gelatin zymography for pro- and active MMP-9. (D) Total and active MMP-9 concentration in the cornea as quantified by ELISA. Values are mean ± SD; n = 5 for each group. Significant improvements were observed with dose of 0.002 μg but maximal effects were obtained with 2 μg.
Fig. 6.
Fig. 6.
TSG-6 reduced the early inflammatory response. Representative corneal photographs (A), H&E staining (B), and immunohistochemical staining for neutrophil elastase (C) demonstrated the time-dependent infiltration of leukocytes and development of opacity in the cornea following injury. (D) The temporal changes in the expression of MPO, cytokines, and chemokines in the cornea are shown. The MPO concentration increased markedly between 8 h to 24 h. The level of IL-6 increased to a maximum at approximately 4 to 8 h. The levels of IL-1β, CXCL1/CINC-1, and CCL2/MCP-1 reached peak at 24 h. TSG-6 treatment lowered the levels of MPO, cytokines, and chemokines at examined time points. Values are mean ± SD; n = 3 for each group.
Fig. 7.
Fig. 7.
TSG-6 administration within 4 h after injury decreased corneal inflammation. Injection of TSG-6 within 4 h after injury significantly decreased the infiltration of neutrophils as examined by MPO assay. Note that injection of TSG-6 2 h after injury suppressed corneal inflammation as effectively as administration immediately after injury. TSG-6 injection at 8 h was not effective. Values are mean ± SD; n = 3 for each group.
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